Cancer: Oxidative Stress and Dietary Antioxidants [2 ed.] 9780128195475, 0128195479

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Cancer Oxidative Stress and Dietary Antioxidants

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Cancer

Oxidative Stress and Dietary Antioxidants

Second Edition Edited by

Victor R. Preedy Department of Nutrition and Dietetics, King’s College London, London, United Kingdom Department of Clinical Biochemistry, King’s College Hospital, London, United Kingdom

Vinood B. Patel University of Westminster, London, United Kingdom

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom © 2021 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-819547-5 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisitions Editor: Rafael E. Teixeira Editorial Project Manager: Timothy Bennett Production Project Manager: Selvaraj Raviraj Cover Designer: Matthew Limbert Typeset by SPi Global, India

Preface The book Cancer: Oxidative Stress and Dietary Antioxidants, Second Edition bridges the transdisciplinary divide and covers the science of oxidative stress in cancer and the therapeutic use of antioxidants in food matrix in a single volume. The second edition covers new investigations used to determine the comprehensive properties of antioxidants, food items, and extracts, as well as any adverse properties they may have. It has been updated to include new clinical human trials and studies dedicated to models of cancer. Furthermore, studies showing the beneficial effects of plant or natural extracts provide the foundation for further rigorous studies in clinical trials. Throughout the book the processes within the science of oxidative stress are described in concert with other processes, such as apoptosis, cell signaling, and receptor-mediated responses. This approach recognizes that diseases are often multifactorial, and oxidative stress is a single component of this. The book Cancer: Oxidative Stress and Dietary Antioxidants, Second Edition contains two sections. Section A covers oxidative stress and cancer in breast, prostate, lung, stomach, bladder, ovarian, cervical, and colorectal. In Section B the focus is on antioxidants covering vitamins such as folic acid, vitamin C, vegetarian diets, fruit juices; caffeine analogues, omega-3 fatty acids, Manuka honey; natural antioxidants such as lycopene, cinnamon, selenium, zinc, as well as plant-derived products including anthocyanins, polyphenols, ginger root, Lycium barbarum, and fern extracts, where models of cancer are also discussed. Each chapter has Summary Points and a section on Applications to Other Areas of Cancer. Finally we conclude with a chapter on resources and further reading, coverage includes: Key books and further reading Key societies Key research organizations Analytical platforms Governmental bodies Journals covering cancer and oxidative stress Thus, this text is relevant to biologists, biochemists, nutritionists, dieticians, and nutrition researchers as cancer is a multifaceted process covering disease processes, clinical research, and treatment. Vinood B. Patel, Editor University of Westminster, London, United Kingdom

Victor R. Preedy, Editor King’s College London, London, United Kingdom

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Contents Contributors

xix

Section A Oxidative stress and cancer 1. Paraoxonases, oxidative stress, and breast cancer Fatma Ceyla Eraldemir and Tug˘can Korak Introduction Functions and physiological roles of PON1 PON1 and cancer: Focusing on breast cancer PON1 signaling pathways PON1 polymorphisms Function and physiological roles of PON2 and PON3 PON2/3 and cancer: Focusing on breast cancer PON2 signaling pathways PON3 signaling pathways PON2/3 polymorphisms The paradox: PON activities and BC development Concluding remarks Application to other cancers Summary points References

4 4 5 5 5 6 7 9 10 10 10 11 11 12 12

2. Oxidative stress and prostate cancer Masaki Shiota Introduction Causes of oxidative stress in prostate cancer Increased ROS production Impaired antioxidant defenses Role of oxidative stress in the pathogenesis of prostate cancer

15 16 16 17 19

Oxidative stress in carcinogenesis and cancer progression Oxidative stress in castration resistance Oxidative stress in resistance to other therapeutics Prostate cancer risk factors and their links to oxidative stress Aging Genetic background (race and family history) Androgens Inflammation Diet Lifestyle Conclusions Applications to other cancers or conditions Summary points References

19 20 20 21 21 21 22 22 23 23 23 23 24 24

3. Oxidative stress in lung cancer Amir Mousapasandi, Wei Sheng Joshua Loke, Cristan A. Herbert, and Paul S. Thomas Introduction Lung cancer Etiology of lung cancer Tobacco smoking Air pollution Infection and inflammation Radon Genetics Tobacco smoking and oxidants Silica and oxidants Asbestos and oxidants MicroRNAs and oxidative stress Radon and oxidative stress Inflammation and oxidative stress Oxidative stress leads to DNA mutations and lung cancer Lipid and protein peroxidation Antioxidants and lung cancer prevention

28 28 28 28 29 29 29 29 29 30 31 32 32 32 33 34 35 vii

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Conclusion Summary points References

35 35 35

4. Endogenous antioxidants in the prognosis and treatment of lung cancer Laurie Freire Boullosa, Jinthe Van Loenhout, and Christophe Deben Double-edged sword of antioxidants in cancer Nrf2: Transcriptional regulator of the redox balance Role of Nrf2 Nrf2 in NSCLC Nrf2-mediated chemoresistance and therapeutic strategies Glutathione and thioredoxin antioxidant systems GSH system Trx system Dual targeting of the Trx and GSH systems Application to other cancers Summary points References

40 41 41 41 42 42 42 44 46 46 46 47

5. Oxidative stress in stomach cancer Hitoshi Tsugawa and Hidekazu Suzuki Introduction Oxidative stress and host cell Oxidative stress and H. pylori Host damage by oxidative stress and gastric carcinogenesis Oxidative stress and CD44v9-positive gastric cancer stem cells Applications to other areas of toxicology Summary points Acknowledgment References

49 49 50

57 58 58 58 58 60 60 61 62 63 63 63

7. Oxidative stress, epigenetics, and bladder cancer Chanchai Boonla Introduction Urothelial carcinoma Oxidative stress in bladder cancer Epigenetics in cancer Epigenetic alterations in urothelial carcinoma ROS alters DNA methylation in urothelial carcinoma ROS causes alteration of histone modification in bladder cancer Oxidative stress and epigenetic change in other diseased conditions Summary points References

67 68 69 69 70 71 72 72 73 73

51 51 52 52 53 53

6. Oxidative stress and oral cavity cancer Ayca Ant Introduction Oral cancer Etiology Epidemiology

Anatomy of the oral cavity Pathology Premalignant lesions Oral squamous cell carcinoma and treatment Oxidative stress The causes of oxidative stress in the oral cavity Endogenous causes Exogenous causes Oxidative stress in oral precancer and cancer Applications to other cancers or conditions Summary points References

56 56 56 57

8. Linking oxidative stress and ovarian cancers Tsukuru Amano and Tokuhiro Chano Introduction Oxidative stress promotes carcinogenesis from endometriosis to OCCCs Attempts to prevent development of OCCCs from endometriosis Antioxidative pathway in OCCC Therapeutic targets for OCCC Conclusion Applications to other cancers or conditions Summary points References

77 78 78 80 80 82 82 83 83

Contents

9. Redox-dependent mechanisms of carcinogenesis in human papillomavirus infection Cesira Foppoli and Marzia Perluigi Introduction Cervical cancer HPV structure E6 oncoprotein E7 oncoprotein E5 oncoprotein HPV oncoproteins and transcription factors Oxidative stress and cervical cancer Oxidative/nitrosative stress markers Antioxidant systems Protein oxidation Redox control and adaptive mechanisms in HPV-infected cells Modulation of stress response markers Modulation of antioxidant response: Peroxiredoxins Suppression of oxidative stress-induced apoptosis Conclusion Summary points Acknowledgments References

87 88 88 88 90 91 91 92 92 93 94 96 96 96 97 98 98 98 98

10. Polymorphisms, antioxidant genes, and cancer Mazhar Al Zoubi and Alaa Aljabali Introduction Breast cancer MnSOD CAT MPO GPXs Prostate cancer Gastric, colorectal, and colon cancer Lung cancer Other cancers Summary points References

101 102 103 103 104 104 104 105 106 106 107 107

11. The interconnection of high-fat diets, oxidative stress, the heart, and carcinogenesis

111 113

114 114 115 115 115 118 118 118 119

12. Cancer during pregnancy. Maternal, placenta, and fetal damage. Nutrition, antioxidant defenses, and adult offspring tumor-bearing Carla de Moraes Salgado, Natalia Angelo da Silva Miyaguti, Sarah Christine Pereira de Oliveira, Bianca Cristine Favero-Santos, Laı´s Rosa Viana, Melina de Moraes Santos Oliveira, and Maria Cristina Cintra Gomes-Marcondes Introduction: Cancer and pregnancy Cancer during pregnancy incidence Cancer-induced metabolic changes in pregnancy evolution Carbohydrate metabolic changes Lipid metabolic changes Protein metabolic changes Oxidative stress in pregnancy associated with cancer Placental and fetal changes and viability associated with cancer Nutritional supplementation and positive effects in pregnancy and oxidant and antioxidant responses Maternal diet influence in cancer evolution and host responses Maternal antioxidant diet could affect the defenses of the adult offspring tumor-bearing hosts Conclusion Summary points Acknowledgments References

13. Inflammation and oxidatively induced DNA damage: A synergy leading to cancer development

Bianka Bojkova´, Natalia Kurhaluk, and Pawel J. Winklewski Oxidative stress: General introduction Obesity, oxidative stress, and inflammation

Diet-induced oxidative stress in relation to noncommunicable diseases Dietary fat in relation to cancer and CVD risk Total fat Saturated fat Unsaturated fat TFAs and cancer risk Cancer and heart disease Summary points References

ix

Ioanna Tremi, Somaira Nowsheen, Khaled Aziz, Shankar Siva, Jessica Ventura, Vasiliki I. Hatzi, Olga A. Martin, and Alexandros G. Georgakilas

121 121 121 121 122 122 123 123

124 125

125 126 127 127 127

x Contents

Introduction Oxidative DNA damage Mechanisms of induction Pathways of repair Role of inflammation in the induction of oxidative stress and DNA damage leading to cancer Extrinsic pathway of carcinogenesis Intrinsic pathway The link between extrinsic and intrinsic pathways Soluble mediators and cellular components Tissue injury Nontargeted effects, inflammation, oxidative stress, and DNA damage Bystander and abscopal effects Bystander signaling in vitro Role of cytokines for bystander signaling Radiation-induced inflammation Local tumor environment and radiation Radiation exposure and the immunogenic effect Conclusion Summary points References

132 132 132 133

133 134 137 137 138 138 139 139 139 140 142 142 142 143 143 143

14. Ferroptosis, free radicals, and cancer Rui Kang and Daolin Tang Introduction The discovery of ferroptotic cancer cell death The central biochemical event of ferroptotic cancer cell death The core molecular machinery of ferroptotic cancer cell death System xc
GPX4 TP53 NFE2L2 ACSL4 Lipoxygenase The relationship between ferroptosis and autophagy Conclusions and perspectives Applications to other cancers or conditions Summary points Acknowledgments References

149 150 150 151 151 152 153 153 154 155 155 155 156 157 157 157

15. Nrf2, YAP, antioxidant potential, and cancer Giuseppina Barrera, Marie Angele Cucci, Margherita Grattarola, and Stefania Pizzimenti Introduction Nrf2 transcription factor Nrf2 activity in cancer prevention Nrf2 in cancer progression and chemoresistance Hippo pathway and YAP regulation YAP in cancer progression and chemoresistance YAP and antioxidant regulation YAP-Nrf2 cooperation and cross talk Applications to other cancers or conditions Summary points References

160 160 160 162 163 165 165 167 168 168 169

16. Cancer, NFkappaB, and oxidative stress-dependent phenotypes Daniela Sorriento, Jessica Gambardella, and Guido Iaccarino Introduction NFkB and cancer The crosstalk between NFkB and oxidative stress in cancer NFkΒ and oxidative stress-dependent phenotypes in cancer Cell proliferation Inflammation Tumor angiogenesis Apoptosis escape: Molecular bases of chemoresistance Tumor metabolism Application to other cancers or conditions Summary points References

171 171 172 172 172 173 173 173 174 174 174 176

17. 8-Hydroxydeoxyguanosine: A valuable predictor of oxidative DNA damage in cancer and diabetes mellitus Anmar Al-Taie, Mesut Sancar, and Fikret Vehbi Izzettin Introduction Free radicals and oxidative DNA damage

179 180

Contents

Biomarkers of oxidative repair products Measurement of 8-OHdG Practical impact of 8-OHdG in carcinogenesis and cancer therapy Impact of oxidative stress in diabetes mellitus Practical impact of 8-OHdG in diabetes mellitus and diabetic complications Applications to other cancers or conditions Summary points References

181 181 181 182 183 183 183 184

Section B Antioxidants and cancer

197 198 199 199 200 200

19. Prostate cancer and food-based antioxidants in India as plausible therapeutics Ranjana Bhandari, Garima Khanna, and Anurag Kuhad

18. Molecular approaches toward targeted cancer therapy with some food plant products: On the role of antioxidants and immune microenvironment Anisur Rahman Khuda-Bukhsh, Santu Kumar Saha, Sreemanti Das, and Sweta Sharma Saha Introduction Oxidative stress, genomic instability, and cancer: Role of dietary antioxidants Carotenoids (beta carotene and lycopene) Grapes Ginger Spinach ROS-related signaling pathways for targeted cancer therapy Regulation of MAPK signaling pathways by ROS Regulation of PI3K signaling pathways by ROS Nrf2 and Ref-1-mediated redox cellular signaling Regulation of p66shc, mitochondrial oxidative stress Regulation of IRE-IRP system and iron homeostasis by ROS ROS and DNA damage response Oxidative stress-mediated DDR pathway inhibitors for cancer therapy Base excision repair (BER) Nonhomologous end-joining (NHEJ) Nucleotide excision repair (NER) Tumor immune microenvironment (TIME) and the role of antioxidants as immune modulators

Components of TIME and their functions Oxidative stress and the impact of dietary products on the TIME Cross talk between DDR and the tumor immune microenvironment Future perspectives Summary points References

xi

191 192 192 195 195 195 195 195 195 196 196 196 196 196 197 197 197

197

Introduction Cancer and its pathogenesis Genetic factors Environmental factors Prostate cancer Introduction Types Pathogenesis Current therapy and its limitations Functional food as therapeutics for prostate cancer Lycopene Curcumin Quercetin Genistein Resveratrol Epigallocatechin Beta-carotene Omega-3-fatty acids Future perspectives Conclusion Summary points Author’s disclosure References

203 203 204 205 205 205 205 205 206 208 208 208 209 210 210 211 212 212 214 214 214 214 215

20. Linking nonenzymatic antioxidants in the diet and colorectal cancer Esther Molina-Montes, Bel
en Garcı´a-Villanova, Eduardo Jesu´s Guerra-Herna´ndez, and Pilar Amiano Introduction Epidemiology of colorectal cancer CRC risk and prevention factors with a focus on dietary factors Molecular colorectal carcinogenesis Dietary antioxidants: Their health benefits and dietary sources

219 219 219 220 221

xii Contents

Role of nonenzymatic antioxidants in the prevention of colorectal cancer Molecular basis of dietary antioxidants in CRC etiology Levels of evidence of anti-CRC effects of antioxidants from human studies Insights into oxidative stress modulators and colorectal cancer OS implications in CRC Gut microbiome and the antioxidantoxidant balance Conclusions and applications to other cancers or conditions Summary points References

221 221 223 229 229 230 230 231 231

21. Fruit and vegetable juices and breast cancer

235 236 236 237 237 237 237 238 238 239 239 239 239 241 241 241 242 242

22. Oxidative stress and cancer: Role of n-3 PUFAs Concetta Finocchiaro, Maurizio Fadda, Valentina D’Onofrio, Mirko Ippolito, Costanza Pira, and Andrea Devecchi Introduction Oxidative stress Cancer and n-3 PUFAs Guidelines: Supplementation with omega-3 in cancer Omega-3 and cancer cachexia

250 250 250 251 251 251

23. Statins, cancer, and oxidative stress Tahoora Shomali and Mahboobeh Ashrafi Applications to other cancers or conditions Summary points References

260 260 260

24. Role of anthocyanins in oxidative stress and the prevention of cancer in the digestive system

Cı´ntia Ferreira-P^ ego, Bojana B. Vidovi
c, Nuno G. Oliveira, Ana S. Fernandes, and Joa˜o G. Costa Introduction In vitro and in vivo studies Berries juice Grape juice Pomegranate juice Citrus juice Apple juice Noni juice Cruciferous juice Beetroot juice Other fruit and vegetable extracts and juices Epidemiological data FVJ in BC FVJ in combination with chemotherapy Conclusion Fruit and vegetables juices in other cancers Summary points References

Role of omega-3 PUFAs in chemoresistant cancers Application to other cancers and conditions Conclusions Summary points Acknowledgments References

245 245 246 247 248

Elvira Gonzalez de Mejia, Miguel Rebollo-Hernanz, Yolanda Aguilera, and Maria A. Martı´n-Cabrejas Introduction Applications to other cancers Oxidative stress and gastrointestinal cancer Oxidative stress Oxidative stress in gastric cancer Oxidative stress in liver cancer Oxidative stress in colorectal cancer Oxidative stress in pancreatic cancer Anthocyanins: Properties and dietary sources Chemistry of anthocyanins Food sources Bioavailability and metabolism Role of anthocyanins in the prevention of oxidative stress Direct chemical mechanisms Indirect molecular mechanisms Role of anthocyanins in the prevention of gastrointestinal cancer Conclusions Summary points References

265 266 267 267 267 267 268 269 269 269 269 269 271 271 272 272 277 277 278

25. Caffeic Acid targets metabolism of cervical squamous cell carcinoma Malgorzata Tyszka-Czochara Introduction Metabolic reprogramming confers an adaptive advantage to cancer cells

281 282

Contents

CA hampers glucose uptake and glucose catabolism to lactate in cervical cancer cells CA induces oxidative stress in mitochondria and elucidates metabolic-dependent apoptotic death in epithelial cervical cancer cells CA impairs energy generation in cervical cancer cells Energetic stress caused by CA in cervical cancer cells activates adenosine 50 -monophosphate AMP-activated protein kinase CA affects the cervical cancer cells phenotype and migration properties under implementation of the Epithelialto-Mesenchymal Transition process CA has the potency to regulate cell cycle progress in cervical cancer cells with an epithelial phenotype Applications to other conditions Cervical cancer treatment in humans using Cisplatin Co-treatment of cervical cancer cells with CA and the antidiabetic drug, Metformin, augments the toxic action of Cisplatin via regulation of the cell cycle—In vitro study CA and Met hamper proliferation and enhance cell death in cervical cancer cells but not in normal cells CA alleviates lactic acidosis caused by Metformin—In vitro study Bioavailability of CA and perspectives of use in humans Summary points References

282

295 295 295 296 297 297

283 284

27. Oxidative stress and cancer: Antioxidative role of Ayurvedic plants Sahdeo Prasad and Sanjay K. Srivastava

284

285

286 287 287

287

287 288 288 288 288

26. Effects of caffeic acid on oxidative balance and cancer Beatriz da Silva Rosa Bonadiman, Grazielle Castagna Cezimbra Weis, J
essica Righi da Rosa, Charles Elias Assmann, Audrei de Oliveira Alves, P^ amela Longhi, and Margarete Dulce Bagatini Coffee Coffee and oxidative balance Coffee and cancer Berries Raspberry Blueberry Propolis Propolis and oxidative balance Propolis and cancer Apple

Apple and oxidative balance Apple and cancer Grape and wine Grape/wine and cancer Summary points References

xiii

291 291 291 292 292 293 294 294 294 295

Introduction Oxidative stress and cancer Ayurvedic plants with antioxidative nature Emblica officinalis Glycyrrhiza glabra Aloe vera Ocimum sanctum Tinospora cordifolia Other Ayurvedic plants Conclusion Summary points Acknowledgments Conflict of interest References

301 302 302 302 303 304 304 305 305 307 307 307 307 308

28. Polyphenol chlorogenic acid, antioxidant profile, and breast cancer Onur Bender and Arzu Atalay Introduction Chlorogenic acid Antioxidant profile of chlorogenic acid Antioxidant capacity of chlorogenic acid isomers with conventional in vitro tests Antioxidant properties of chlorogenic acid in cellular level Antioxidant effects of chlorogenic acid in vivo Computational evaluations for antioxidant potential of chlorogenic acid Chlorogenic acid and breast cancer Cytotoxic/antiproliferative effects of chlorogenic acid on breast cancer cells Effects of chlorogenic acid on cell cycle distribution in breast cancer Apoptotic effects of chlorogenic acid on breast cancer Effects of chlorogenic acid on mitochondrial membrane potential in breast cancer

312 312 313

313 313 314 316 316 316 318 318

319

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Molecular simulations and validations of chlorogenic acid effects on protein kinase C Effects of chlorogenic acid on breast cancer in vivo Applications to other cancers or conditions Summary points References

319 319 320 320 320

29. Cinnamomum cassia, apoptosis, STAT3 inactivation and reactive oxygen species in cancer studies Yae Jin Yoon and Byoung-Mog Kwon Introduction Oxidative stress-mediated apoptosis induced by cinnamaldehyde and its derivatives Regulation of intrinsic and extrinsic apoptotic pathways Regulation of antioxidant defense system Apoptotic cell death via STAT3 inactivation Direct binding targets of cinnamaldehyde and its derivatives Proteasome subunits Signal transducer and activator of transcription 3 (STAT3) and pyruvate kinase M2 (PKM2) Proviral insertion in murine lymphomas-1 (Pim-1) Low-density lipoprotein receptor-related protein 1 (LRP1) Thioredoxin reductase (TrxR) Applications to other cancers or conditions Summary points References

323

326 326 328 329 330 330

330 333 333 333 334 334 334

30. Antioxidative stress actions of cocoa in colonic cancer: Revisited Sonia Ramos, Luis Goya, and Maria Angeles Martı´n Introduction Chemopreventive mechanism of cocoa polyphenols in cultured colon cancer cells Antioxidant effects Effects on apoptosis and proliferation Antiinflammatory effects Chemopreventive mechanism of cocoa in animal models of colon cancer

337

339 340 341 343 343

Cocoa prevented AOM-induced oxidative stress in colon tissues Cocoa prevented cell proliferation in AOM treated animals Cocoa prevented AOM-induced inflammation in colon tissues Cocoa-induced apoptosis in AOM-treated animals Human studies Epidemiologic studies Intervention studies Summary points Acknowledgments Conflict of interest References

344 344 345 345 345 345 346 346 346 347 347

31. Medicinal plants, antioxidant potential, and cancer Emmanuel Mfotie Njoya Introduction Applications to other cancers or conditions Oxidative stress resulting from the overproduction of free radicals Free radicals and their implication in oxidative stress-related diseases Antioxidant mechanisms of free radical scavengers Methods used for the evaluation of antiradical activity Ferric reducing ability of plasma (FRAP) assay ABTS (2,20 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) assay DPPH (2,2-diphenyl-1-picrylhydrazyl) assay ORAC (oxygen radical absorbance capacity) assay Superoxide anion scavenging assay Hydroxyl radical scavenging assay Free radical scavenger potency versus polyphenolic contents of plants Summary points References

349 350 350 351 352 352 352

353 353 353 353 353 354 355 355

32. Curcumin, oxidative stress, and breast cancer Gloria M. Calaf Introduction Estrogens (17b-estradiol) and oxidative stress Oxidative stress Curcumin as an antioxidant

359 360 360 361

Contents xv

Curcumin and a multifunctional nuclear transcription factor and the enzyme manganese superoxide dismutase protein expression Curcumin and lipid peroxidation Curcumin and epithelial-mesenchymal transition Curcumin and genomic instability Curcumin and specific biomarkers for cancer Summary points Acknowledgments References

362 364 364 365 366 368 368 369

33. Curcumin analogs, oxidative stress, and prostate cancer Marco Bisoffi and Justin M. O’Neill Introduction Prostate cancer and oxidative stress Prostate cancer: A brief introduction Prostate cancer and oxidative stress: Possible factors Reactive oxygen species: A paradox in (prostate) cancer Curcumin, curcuminoids, and curcumin analogs Chemistry and biochemistry of curcumin, curcuminoids, and curcumin analogs Antioxidant versus prooxidant activities of curcumin, curcuminoids, and curcumin analogs The potential of curcumin, curcuminoids, and curcumin analogs as oxidant agents in prostate cancer Molecular targets of curcumin, curcuminoids, and curcumin analogs in prostate cancer Curcumin, curcuminoids, and curcumin analogs as antioxidants in prostate cancer Curcumin, curcuminoids, and curcumin analogs as prooxidants in prostate cancer Summary points References

372 372 372 373 373 374 374

376

380

380

380

381 384 384

34. Fern extract, oxidative stress, and skin cancer Concepcio´n Parrado, Yolanda Gilaberte, Neena Philips, Angeles Juarranz, and Salvador Gonzalez Introduction Ultraviolet radiation and oxidative stress

388 388

Infrared radiation (IR) and visible light (VIS) and oxidative stress Fernblock, oxidative stress, and photoprotection Photoprotective agents Polypodium leucotomos. Origen and composition Composition Molecular, cellular, and clinical evidence of the photoprotective properties of Fernblock Fernblock in DNA photodamage and repair Fernblock effect on free radicals during inflammation Fernblock prevents UV radiation-mediated immunosuppression Fernblock, an anti-UV-induced tumor progression agent Fernblock and malignant melanoma Fernblock prevention of matrix remodeling and other cellular effects Fernblock preventions of photodamage induced by visible light and infrared radiation Potential use of Fernblock in the treatment of other pathological skin conditions Idiopathic photodermatosis Pigmentary disorders Applications to Fernblock to skin cancers or other conditions Summary points Acknowledgments References

388 389 389 389 389

389 389 390 391 391 393 393

394

395 395 395 396 396 396 396

35. Lycium barbarum (goji berry), human breast cancer, and antioxidant profile Anna Wawruszak, Marta Halasa, and Karolina Okla Introduction Natural compounds in cancer therapy and chemoprevention Characteristics of Lycium barbarum (goji berry) Anticancer properties of L. barbarum in breast cancer Antioxidative properties of L. barbarum in breast cancer Applications to other cancers or conditions Summary points References

399 400 400 401 402 403 404 404

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36. Manuka honey, oxidative stress, 5-fluorouracil treatment, and colon cancer cells Sadia Afrin, Tamara Y. Forbes-Herna´ndez, Francesca Giampieri, and Maurizio Battino Overview of etiology and risk factors of colorectal cancer (CRC) Oxidative stress and CRC Management and treatment of 5-FU in CRC Manuka honey (MH) Nutritional composition of MH MH as a source of natural antioxidant Chemopreventive effect of MH in colon cancer cells Antiproliferative effect Apoptosis induction Alteration of oxidative stress Antimetastatic effects Effect of MH on other cancer cells Conclusions Summary points References

408 409 410 410 410 410 411 411 412 412 413 413 413 414 414

37. Piplartine (piperlongumine), oxidative stress, and use in cancer Daniel Pereira Bezerra Introduction Oxidative stress induction Cancer cell death induction Antitumor, antiangiogenic, and antimetastatic effects Conclusion References

417 417 418 420 422 422

38. Antioxidant of Pleurotus ostreatus (Jacq.) P. Kumn and lymphoid cancer cells

432 433 433 434 435 435

39. “Skin cancer, polyphenols, and oxidative stress” or Counteraction of oxidative stress, inflammation, signal transduction pathways, and extracellular matrix remodeling that mediate skin carcinogenesis by polyphenols Neena Philips, Richard Richardson, Halyna Siomyk, David Bynum, and Salvador Gonzalez Introduction Oxidative stress, inflammation, and associated signal transduction pathways: Fundamental biology, the alteration, and counteraction by polyphenols ECM remodeling and associated growth factors: Fundamental biology, the alteration, and counteraction by polyphenols Conclusion Summary points Acknowledgment References

439

440

443 447 448 448 448

40. Pterostilbene and cancer chemoprevention Rong-Jane Chen and Ying-Jan Wang

Md. Moyen Uddin Pk, Jane O’Sullivan, Rumana Pervin, and Matiar Rahman Introduction Applications to other cancers or conditions Cancer Oxidative stress (OxS) Biomarkers of OxS OxS and cell proliferation OxS and apoptosis ROS and mtDNA damage

Antioxidant defense in cancer development P. ostreatus, oyster mushroom Extraction and purification of polysaccharides Treatment of cancer Summary points References

427 428 428 429 430 431 431 432

Introduction Applications to other cancers or conditions Main text Pharmacokinetics of pterostilbene Oxidative stress and inflammation in cancer development Antioxidant and anti-inflammatory effects of pterostilbene Chemopreventive mechanisms of pterostilbene in preclinical studies Inhibiting inflammatory responses Inducing apoptosis in cancer cells

452 453 453 454 454 455 455 456 456

Contents xvii

Pterostilbene induces autophagy in cancer cells Pterostilbene induces cell cycle arrest in cancer cells Pterostilbene induces senescence in cancer cells Pterostilbene inhibits invasion and metastasis in cancer cells Chemopreventive effects of pterostilbene by regulation of microRNAs Summary points References

457 459 459 460 460 461 461

41. Resveratrol, reactive oxygen species, and mesothelioma Saime Batırel Introduction Applications to other cancers or conditions Resveratrol Antioxidant effects of resveratrol Malignant pleural mesothelioma Asbestos and malignant pleural mesothelioma Antiapoptotic effects of resveratrol on MPM cells Chemoprotective effects of resveratrol on MPM cells Effects of resveratrol on cell cycle of MPM cells Signaling pathways in anticancer effects of resveratrol Chemopreventive properties of resveratrol Dual effects of resveratrol on cancer cells Conclusion Summary points References

465 466 466 467 468 468 469 469 470

480 480 480

43. Silybum marianum, antioxidant activity, and cancer patients Sepideh Elyasi Introduction Skin cancer Larynx and lung cancer Breast cancer Hepatic and pancreatic cancers Ovarian cancer Prostate cancer Colorectal cancer Kidney and bladder cancer Cervical cancer Leukemia Antimetastatic effect Radiotherapy- and chemotherapy-induced adverse reaction management Hepatoprotectant Kidney protectant Cardioprotectant Mucocutaneous protection Silymarin administration and dosing Silymarin adverse reactions and drug interactions Summary points References

483 484 485 485 486 486 486 487 488 488 488 489 489 489 490 490 490 490 491 491 491

471 471 472 472 472 472

42. Exercise, selenium, and cancer cells Mahdieh Molanouri Shamsi and Zuhair Mohammad Hassan Introduction Selenium and human health Selenium and cancer Physical exercise and cancer Exercise and selenium: Possible metabolic reprogramming in cancer cells

Applications to other cancers or conditions Summary points References

475 476 476 477 478

44. Plants of the genus Terminalia: Phytochemical and antioxidant profiles, proliferation, and cancer Ian Edwin Cock and Matthew Cheesman Introduction Applications to cancers or other conditions Antioxidant content The relationship between oxidative stress and cancer Phytochemistry of the genus Terminalia Tannins Stilbenes Other compounds with anticancer activities Summary points References

495 495 496 496 497 497 498 498 501 501

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45. Uncaria tomentosa: A promising source of therapeutic agents for prevention and treatment of oxidative stress and cancer

Interaction between genetic polymorphism and antioxidant vitamin on breast cancer risk One carbon metabolism-related gene polymorphisms and dietary factors on breast cancer risk Oxidative stress-related gene polymorphisms and dietary factors on breast cancer risk Research priorities for gene-diet interaction approach Conclusion Summary points References

Francesca Ciani, Natascia Cocchia, Viola Calabro`, Alessandra Pollice, Lucianna Maruccio, Domenico Carotenuto, Luigi Esposito, Luigi Avallone, and Simona Tafuri Introduction Applications to other cancers or conditions Uncaria tomentosa Botanical classification of Uncaria genus Chemical composition of U. tomentosa Oxidative stress Oxidative stress and U. tomentosa Cancer and U. tomentosa Conclusions Summary points References

505 506 506 508 508 508 510 511 512 513 513

518 519 519 520 520 520

Introduction Antioxidant compounds Oxidative stress, antioxidant vitamins, and ALL The antioxidant mechanisms Vitamin C Vitamin A Vitamin E Conclusion Summary points References

536 536 537 537

539 540 540 542 542 542 542 542 543 543

Section C Online resources 49. Recommended resources on cancer: Oxidative stress and dietary antioxidants Rajkumar Rajendram, Vinood B. Patel, and Victor R. Preedy

47. Antioxidant vitamins and genetic polymorphisms in breast cancer

Introduction Resources Summary points Acknowledgments References

Daehee Kang, Sang-Ah Lee, and Woo-Kyoung Shin Introduction Effect of antioxidant vitamin on breast cancer incidence Antioxidant vitamins and genomic integrity: Developmental and degenerative correlates

530

Behnaz Abiri and Mohammadreza Vafa

Rory S. Carroll, Garry R. Buettner, and Joseph J. Cullen 515 515 516 517 518

530

48. Antioxidant vitamins in acute lymphoblastic leukemia

46. Pharmacological ascorbate and use in pancreatic cancer

Introduction Ascorbate biochemistry Selective toxicity to cancer cells P-AscH
as chemosensitizer P-AscH
as radiosensitizer P-AscH
as a protector of normal tissue during chemoradiation Conclusions Applications in other cancers Summary points Acknowledgment References

529

523

547 547 547 551 551

524 Index 527

553

Contributors Numbers in parenthesis indicate the pages on which the authors’ contributions begin.

Arzu Atalay (311), Biotechnology Institute, Ankara University, Ankara, Turkey

Behnaz Abiri (539), Department of Nutrition, Faculty of Paramedicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

Luigi Avallone (505), Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy

Sadia Afrin (407), Department of Gynecology and Obstetrics, Johns Hopkins University, School of Medicine, Baltimore, MD, United States

Khaled Aziz (131), Medical Scientist Training Program, Mayo Graduate School, Mayo Clinic, College of Medicine, Rochester, MN, United States

Yolanda Aguilera (265), Institute of Food Science Research, CIAL (UAM-CSIC), Department of Agricultural Chemistry and Food Science, Universidad Auto´noma de Madrid, Madrid, Spain

Margarete Dulce Bagatini (291), Academic Coordination, Campus Chapeco´, Federal University of Fronteira Sul, Chapeco´, SC, Brazil

Mazhar Al Zoubi (101), Department of Basic Medical Sciences, Faculty of Medicine, Yarmouk University, Irbid, Jordan Alaa Aljabali (101), Department of Pharmaceutical Sciences, Faculty of Pharmacy, Yarmouk University, Irbid, Jordan Anmar Al-Taie (179), Head of Clinical Pharmacy Department, Faculty of Pharmacy, Girne American University, Kyrenia, Turkey Tsukuru Amano (77), Department of Obstetrics & Gynecology, Shiga University of Medical Science, Ostu, Japan Pilar Amiano (219), Ministry of Health of the Basque Government, Public Health Division of Gipuzkoa, Biodonostia Health Research Institute, Donostia-San Sebastian; CIBERESP (Consortium for Biomedical Research in Epidemiology and Public Health), Madrid, Spain Ayca Ant (55), Department of Otorhinolaryngology, Head and Neck Surgery, University of Health Sciences Ankara A.Y. Oncology Education and Research Hospital, Yenimahalle/Ankara, Turkey Mahboobeh Ashrafi (255), Division of Biochemistry, Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran Charles Elias Assmann (291), Department of Biochemistry and Molecular Biology, PPGBTox, CCNE, Federal University of Santa Maria, Santa Maria, RS, Brazil

Giuseppina Barrera (159), Department of Clinical and Biological Sciences, University of Turin, Torino, Italy Saime Batırel (465), Faculty of Medicine, Department of Medical Biochemistry, Marmara University, Istanbul, Turkey Maurizio Battino (407), Department of Analytical and Food Chemistry, Nutrition and Food Science Group, CITACA, CACTI, University of Vigo, Vigo Campus, Vigo, Spain; Department of Clinical Sciences, Universita` Politecnica delle Marche, Ancona, Italy; International Research Center for Food Nutrition and Safety, Jiangsu University, Zhenjiang, China Onur Bender (311), Biotechnology Institute, Ankara University, Ankara, Turkey Daniel Pereira Bezerra (417), Gonc¸alo Moniz Institute, Oswaldo Cruz Foundation (IGM-FIOCRUZ/BA), Salvador, Bahia, Brazil Ranjana Bhandari (203), Pharmacology Research Laboratory, University Institute of Pharmaceutical Sciences, UGC-Centre of Advanced Study, Panjab University, Chandigarh, India Marco Bisoffi (371), Chemistry and Biochemistry, Schmid College of Science and Technology, Chapman University, Orange, CA, United States Bianka Bojkova´ (111), Department of Animal Physiology, Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Sˇafa´rik University in Kosˇice, Kosˇice, Slovak Republic

xix

xx Contributors

Chanchai Boonla (67), Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand Garry R. Buettner (515), Free Radical and Radiation Biology Program, Departments of Surgery and Radiation Oncology, Holden Comprehensive Cancer Center, The University of Iowa Hospitals and Clinics, The University of Iowa College of Medicine, and the Iowa City Veterans Affairs, Iowa City, IA, United States David Bynum (439), School of Natural Sciences, University College, Fairleigh Dickinson University, Teaneck, NJ, United States Viola Calabro` (505), Department of Biology, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Naples, Italy Gloria M. Calaf (359), Instituto de Alta Investigacio´n, Universidad de Tarapaca, Arica, Chile; Columbia University Medical Center, New York, NY, United States Domenico Carotenuto (505), UNMSM, Universidad Nacional Mayor San Marcos, Lima, Peru Rory S. Carroll (515), Free Radical and Radiation Biology Program, Departments of Surgery and Radiation Oncology, Holden Comprehensive Cancer Center, The University of Iowa Hospitals and Clinics, The University of Iowa College of Medicine, and the Iowa City Veterans Affairs, Iowa City, IA, United States Tokuhiro Chano (77), Department of Clinical Laboratory Medicine and Medical Genetics, Shiga University of Medical Science, Ostu, Japan Matthew Cheesman (495), School of Pharmacy and Pharmacology, Griffith University; Menzies Health Institute Queensland, Quality Use of Medicines Network, Southport, QLD, Australia Rong-Jane Chen (451), Department of Food Safety/ Hygiene and Risk Management, College of Medicine, National Cheng Kung University, Tainan, Taiwan Francesca Ciani (505), Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy Natascia Cocchia (505), Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy Ian Edwin Cock (495), School of Environment and Science; Environmental Futures Research Institute, Griffith University, Nathan, QLD, Australia Joa˜o G. Costa (235), CBIOS, Research Center for Biosciences & Health Technologies – School of Health Sciences and Technologies, Luso´fona University, Lisbon, Portugal

Marie Angele Cucci (159), Department of Clinical and Biological Sciences, University of Turin, Torino, Italy Joseph J. Cullen (515), Free Radical and Radiation Biology Program, Departments of Surgery and Radiation Oncology, Holden Comprehensive Cancer Center, The University of Iowa Hospitals and Clinics, The University of Iowa College of Medicine, and the Iowa City Veterans Affairs, Iowa City, IA, United States J
essica Righi da Rosa (291), Department of Technology and Food Science, Rural Science Center, Federal University of Santa Maria, Santa Maria, RS, Brazil Beatriz da Silva Rosa Bonadiman (291), Department of Biological Science: Biochemistry, Federal University of Santa Catarina, Floriano´polis, SC, Brazil Sreemanti Das (191), Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani, India Christophe Deben (39), Center for Oncological Research, University of Antwerp, Wilrijk, Belgium Andrea Devecchi (245), Department of Clinical Nutrition, AOU Citta` della Salute e della Scienza, Turin, Italy Valentina D’Onofrio (245), Department of Clinical Nutrition, AOU Citta` della Salute e della Scienza, Turin, Italy Sepideh Elyasi (483), Department of Clinical Pharmacy, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Fatma Ceyla Eraldemir (3), Department of Biochemistry, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey Luigi Esposito (505), Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy Maurizio Fadda (245), Department of Clinical Nutrition, AOU Citta` della Salute e della Scienza, Turin, Italy Bianca Cristine Favero-Santos (121), Obesity and Comorbidities Research Centre, Department of Structural and Functional Biology, Institute of Biology, University of Campinas, Campinas, SP, Brazil Ana S. Fernandes (235), CBIOS, Research Center for Biosciences & Health Technologies – School of Health Sciences and Technologies, Luso´fona University, Lisbon, Portugal Cı´ntia Ferreira-P^ego (235), CBIOS, Research Center for Biosciences & Health Technologies – School of Health Sciences and Technologies, Luso´fona University, Lisbon, Portugal Concetta Finocchiaro (245), Department of Clinical Nutrition, AOU Citta` della Salute e della Scienza, Turin, Italy

Contributors

xxi

Cesira Foppoli (87), CNR Institute of Molecular Biology and Pathology, Sapienza University of Rome, Rome, Italy

Zuhair Mohammad Hassan (475), Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Tamara Y. Forbes-Herna´ndez (407), Department of Analytical and Food Chemistry, Nutrition and Food Science Group, CITACA, CACTI, University of Vigo, Vigo Campus, Vigo, Spain

Vasiliki I. Hatzi (131), Laboratory of Health Physics & Environmental Health, Institute of Nuclear Technology & Radiation Protection, National Center for Scientific Research “Demokritos”, Athens, Greece

Laurie Freire Boullosa (39), Center for Oncological Research, University of Antwerp, Wilrijk, Belgium

Cristan A. Herbert (27), Mechanisms of Disease and Translational Research, School of Medical Sciences, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia

Jessica Gambardella (171), Department of Advanced Biomedical Sciences, Federico II University, Napoli, Italy Bel
en Garcı´a-Villanova (219), Department of Nutrition and Bromatology, Faculty of Pharmacy, University of Granada, Granada, Spain Alexandros G. Georgakilas (131), DNA Damage Laboratory, Physics Department, School of Applied Mathematics and Physical Sciences, National Technical University of Athens (NTUA), Athens, Greece Francesca Giampieri (407), Department of Analytical and Food Chemistry, Nutrition and Food Science Group, CITACA, CACTI, University of Vigo, Vigo Campus, Vigo, Spain; Department of Clinical Sciences, Universita` Politecnica delle Marche, Ancona, Italy; College of Food Science and Technology, Northwest University, Xi’an, Shaanxi, China Yolanda Gilaberte (387), Dermatology Service, Miguel Servet Hospital, Zaragoza, Spain Maria Cristina Cintra Gomes-Marcondes (121), Laboratory of Nutrition and Cancer, Department of Structural and Functional Biology, Institute of Biology, University of Campinas, Campinas, SP, Brazil Salvador Gonzalez (387, 439), Dermatology Service, Memorial Sloan-Kettering Cancer Center, New York, NY, United States; Medicine and Medical Specialties Department, Alcala University, Madrid, Spain Elvira Gonzalez de Mejia (265), Department of Food Science and Human Nutrition, Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Champaign, IL, United States Luis Goya (337), Department of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Ciudad Universitaria, Madrid, Spain Margherita Grattarola (159), Department of Clinical and Biological Sciences, University of Turin, Torino, Italy

Guido Iaccarino (171), Department of Advanced Biomedical Sciences, Federico II University, Napoli, Italy Mirko Ippolito (245), Department of Clinical Nutrition, AOU Citta` della Salute e della Scienza, Turin, Italy Fikret Vehbi Izzettin (179), Head of Clinical Pharmacy Department, Faculty of Pharmacy, Bezmialem Vakif University, Istanbul, Turkey Angeles Juarranz (387), Biology Department, Sciences School, Universidad Auto´noma de Madrid, Madrid, Spain Daehee Kang (523), Department of Preventive Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea Rui Kang (149), Department of Surgery, UT Southwestern Medical Center, Dallas, TX, United States Garima Khanna (203), Pharmacology Research Laboratory, University Institute of Pharmaceutical Sciences, UGC-Centre of Advanced Study, Panjab University, Chandigarh, India Anisur Rahman Khuda-Bukhsh (191), Emeritus of University Grants Commission at University of Kalyani, Kalyani, India Tug˘can Korak (3), Department of Medical Biology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey Anurag Kuhad (203), Pharmacology Research Laboratory, University Institute of Pharmaceutical Sciences, UGC-Centre of Advanced Study, Panjab University, Chandigarh, India Natalia Kurhaluk (111), Department of Zoology and Animal Physiology, Institute of Biology and Earth Sciences, Pomeranian University, Slupsk, Poland

Eduardo Jesu´s Guerra-Herna´ndez (219), Department of Nutrition and Bromatology, Faculty of Pharmacy, University of Granada, Granada, Spain

Byoung-Mog Kwon (323), Laboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea

Marta Halasa (399), Department of Biochemistry and Molecular Biology, Medical University of Lublin, Lublin, Poland

Sang-Ah Lee (523), Department of Preventive Medicine, Kangwon National University School of Medicine, Chuncheon-si, Gangwon-do, Republic of Korea

xxii Contributors

Jinthe Van Loenhout (39), Center for Oncological Research, University of Antwerp, Wilrijk, Belgium Wei Sheng Joshua Loke (27), Department of Respiratory Medicine, Prince of Wales Hospital, Randwick; Prince of Wales’ Clinical School and Mechanisms of Disease and Translational Research, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia P^ amela Longhi (291), Department Life Science and Health, University of the West of Santa Catarina, Xanxer^e, SC, Brazil Olga A. Martin (131), Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, VIC, Australia Maria Angeles Martı´n (337), Department of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Ciudad Universitaria, Madrid, Spain Maria A. Martı´n-Cabrejas (265), Institute of Food Science Research, CIAL (UAM-CSIC), Department of Agricultural Chemistry and Food Science, Universidad Auto´noma de Madrid, Madrid, Spain Lucianna Maruccio (505), Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy Emmanuel Mfotie Njoya (349), Institute of Pharmacy, Martin-Luther University of Halle-Wittenberg, Halle (Saale), Germany; Department of Biochemistry, Faculty of Science, University of Yaound
e I, Yaound
e, Cameroon Natalia Angelo da Silva Miyaguti (121), Laboratory of Nutrition and Cancer, Department of Structural and Functional Biology, Institute of Biology, University of Campinas, Campinas, SP, Brazil

Justin M. O’Neill (371), Chemistry and Biochemistry, Schmid College of Science and Technology, Chapman University, Orange, CA, United States Jane O’Sullivan (427), Department of Anaesthesiology and Critical Care, Tallaght University Hospital, Dublin, Ireland Karolina Okla (399), The First Department of Gynecological Oncology and Gynecology, Medical University of Lublin, Lublin, Poland Melina de Moraes Santos Oliveira (121), Laboratory of Nutrition and Cancer, Department of Structural and Functional Biology, Institute of Biology, University of Campinas, Campinas, SP, Brazil Nuno G. Oliveira (235), Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal Sarah Christine Pereira de Oliveira (121), Laboratory of Nutrition and Cancer, Department of Structural and Functional Biology, Institute of Biology, University of Campinas, Campinas, SP, Brazil Audrei de Oliveira Alves (291), Department of Physiology and Pharmacology, Federal University of Santa Maria, Santa Maria, RS, Brazil Concepcio´n Parrado (387), Department of Histology and Pathology, Faculty of Medicine, University of Ma´laga, Ma´laga, Spain Vinood B. Patel (547), University of Westminster, School of Life Sciences, London, United Kingdom Marzia Perluigi (87), Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy Rumana Pervin (427), Biochemistry & Molecular Biology, University of Rajshahi, Rajshahi, Bangladesh

Mahdieh Molanouri Shamsi (475), Physical Education & Sport Sciences Department, Faculty of Humanities, Tarbiat Modares University, Tehran, Iran

Neena Philips (387, 439), School of Natural Sciences, University College, Fairleigh Dickinson University, Teaneck, NJ, United States

Esther Molina-Montes (219), Genetic and Molecular Epidemiology Group, Spanish National Cancer Research Center (CNIO), Madrid; Department of Nutrition and Bromatology, Faculty of Pharmacy, University of Granada, Granada, Spain

Costanza Pira (245), Department of Clinical Nutrition, AOU Citta` della Salute e della Scienza, Turin, Italy Stefania Pizzimenti (159), Department of Clinical and Biological Sciences, University of Turin, Torino, Italy

Amir Mousapasandi (27), Prince of Wales’ Clinical School and Mechanisms of Disease and Translational Research, School of Medical Sciences, Faculty of Medicine, UNSW Sydney; Department of Respiratory Medicine, Prince of Wales Hospital, Randwick, Sydney, NSW, Australia

Md. Moyen Uddin Pk (427), Institute of Biological Science, University of Rajshahi, Rajshahi; Biochemistry, Primeasia University; Independent University of Bangladesh; Clinical Biochemistry (Diagnostic), Anwer Khan Modern Medical College & Hospital, Dhaka; Biochemistry & Molecular Biology, University of Rajshahi, Rajshahi, Bangladesh

Somaira Nowsheen (131), Medical Scientist Training Program, Mayo Graduate School, Mayo Clinic, College of Medicine, Rochester, MN, United States

Alessandra Pollice (505), Department of Biology, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Naples, Italy

Contributors

Sahdeo Prasad (301), Department of Immunotherapeutics and Biotechnology and Center for Tumor Immunology and Targeted Cancer Therapy, Texas Tech University Health Sciences Center, Abilene, TX, United States Victor R. Preedy (547), Diabetes and Nutritional Sciences Research Division, Faculty of Life Science and Medicine, King’s College London, London, United Kingdom Matiar Rahman (427), Biochemistry & Molecular Biology, University of Rajshahi, Rajshahi, Bangladesh Rajkumar Rajendram (547), College of Medicine, King Saud bin Abdulaziz University for Health Sciences; Department of Medicine, King Abdulaziz Medical City, Ministry of National Guard Health Affairs, Riyadh, Saudi Arabia; Diabetes and Nutritional Sciences Research Division, Faculty of Life Science and Medicine, King’s College London, London, United Kingdom Sonia Ramos (337), Department of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Ciudad Universitaria, Madrid, Spain Miguel Rebollo-Hernanz (265), Institute of Food Science Research, CIAL (UAM-CSIC), Department of Agricultural Chemistry and Food Science, Universidad Auto´noma de Madrid, Madrid, Spain Richard Richardson (439), School of Natural Sciences, University College, Fairleigh Dickinson University, Teaneck, NJ, United States Santu Kumar Saha (191), Newcastle University Centre for Cancer, Translational and Clinical Research Unit, Newcastle University, Newcastle upon Tyne, United Kingdom Sweta Sharma Saha (191), Newcastle University Centre for Cancer, Translational and Clinical Research Unit, Newcastle University, Newcastle upon Tyne, United Kingdom Carla de Moraes Salgado (121), Laboratory of Nutrition and Cancer, Department of Structural and Functional Biology, Institute of Biology, University of Campinas, Campinas, SP, Brazil Mesut Sancar (179), Head of the Clinical Pharmacy Department, Faculty of Pharmacy, Marmara University, Istanbul, Turkey Woo-Kyoung Shin (523), Department of Preventive Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea Masaki Shiota (15), Department of Urology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

xxiii

Tahoora Shomali (255), Division of Pharmacology and Toxicology, Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran Halyna Siomyk (439), School of Natural Sciences, University College, Fairleigh Dickinson University, Teaneck, NJ, United States Shankar Siva (131), Department of Radiation Oncology, Peter MacCallum Cancer Centre; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, VIC, Australia Daniela Sorriento (171), Department of Advanced Biomedical Sciences, Federico II University, Napoli, Italy Sanjay K. Srivastava (301), Department of Immunotherapeutics and Biotechnology and Center for Tumor Immunology and Targeted Cancer Therapy, Texas Tech University Health Sciences Center, Abilene, TX, United States Hidekazu Suzuki (49), Division of Gastroenterology and Hepatology, Department of Internal Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan Simona Tafuri (505), Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy Daolin Tang (149), Department of Surgery, UT Southwestern Medical Center, Dallas, TX, United States Paul S. Thomas (27), Department of Respiratory Medicine, Prince of Wales Hospital, Randwick; Prince of Wales’ Clinical School and Mechanisms of Disease and Translational Research, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia Ioanna Tremi (131), DNA Damage Laboratory, Physics Department, School of Applied Mathematics and Physical Sciences, National Technical University of Athens (NTUA), Athens, Greece Hitoshi Tsugawa (49), Department of Biochemistry, Keio University School of Medicine, Tokyo, Japan Malgorzata Tyszka-Czochara (281), Jagiellonian University Medical College, Faculty of Pharmacy, Department of Food Chemistry and Nutrition, Krakow, Poland Mohammadreza Vafa (539), Department of Nutrition, School of Public Health; Pediatric Growth and Development Research Center, Institute of Endocrinology and Metabolism, Iran University of Medical Sciences, Tehran, Iran Jessica Ventura (131), Department of Obstetrics & Gynaecology, The University of Melbourne and Royal Women’s Hospital, Melbourne, VIC, Australia

xxiv

Contributors

Laı´s Rosa Viana (121), Laboratory of Nutrition and Cancer, Department of Structural and Functional Biology, Institute of Biology, University of Campinas, Campinas, SP, Brazil

Grazielle Castagna Cezimbra Weis (291), Department of Technology and Food Science, Rural Science Center, Federal University of Santa Maria, Santa Maria, RS, Brazil

Bojana B. Vidovi
c (235), Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia

Pawel J. Winklewski (111), Department of Human Physiology, Medical University of Gdansk, Gdansk, Poland

Ying-Jan Wang (451), Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan Anna Wawruszak (399), Department of Biochemistry and Molecular Biology, Medical University of Lublin, Lublin, Poland

Yae Jin Yoon (323), Laboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea

Section A

Oxidative stress and cancer

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Chapter 1

Paraoxonases, oxidative stress, and breast cancer Fatma Ceyla Eraldemira and Tug˘can Korakb a

Department of Biochemistry, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey, b Department of Medical Biology, Faculty of Medicine,

Kocaeli University, Kocaeli, Turkey

List of abbreviations OH AhR AP-1 ARNT BC BRCA CHOP CoQ10 CREB cytC E2 ER ERK FXR GR GRE H2O2 HDL IGF-1 IL IMM IRS-1 JAK JNK LDL MAPK MICAL1 NF-kB Nrf2 O2 O2 OMM PDGFR-b PI3K/Akt PKC PKD PON PPAR PPRE PTEN

hydroxyl radical aryl hydrocarbon receptor activator protein 1 Aryl hydrocarbon receptor nuclear translocator Breast cancer breast cancer susceptibility gene CCAAT/enhancer-binding protein homologous protein coenzyme Q10 cAMP response element binding cytochrome C Estradiol endoplasmic reticulum extracellular-regulated kinase Farnesol X receptor glucocorticoid receptor glucocorticoid response element hydrogen peroxide high-density lipoprotein insulin like growth factor-1 interleukin inner mitochondrial membrane insulin Receptor Substrate-1 janus kinase c-Jun N-terminal kinase low-density lipoprotein mitogen-activated protein kinase molecule interacting with CasL 1 nuclear factor-kB nuclear factor erythroid 2-related factor 2 oxygen superoxide outer mitochondrial membrane platelet-derived growth factor receptors b phosphatidylinositol-3-kinase protein kinase C protein kinase D paraoxonase peroxisome proliferator-activated receptor PPAR response element phosphatase and tensin homolog

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00001-8 © 2021 Elsevier Inc. All rights reserved.

3

4 SECTION

PTP1B PXR PXRE ROS RXR SP-1 SREBP STAT TNF-a uPA

A Oxidative stress and cancer

protein tyrosine phosphatase 1B pregnane X receptor pregnane X receptor response element reactive oxygen species retinoic acid X receptor specificity protein 1 sterol regulatory element-binding protein signal transducer of activation tumor necrosis factor a urokinase plasminogen activator

Introduction Breast cancer (BC) is a frequently encountered and is a leading cause of death in women worldwide. Oxidative stress is one of the precipitating factors in the development of BC and can be due to a variety of causes including genetic factors, environmental factors, advanced age, and long-term exposure to estrogen.1–3 Reactive oxygen species (ROS) may occur at pathological levels and/or insufficient antioxidants could disturb the normal balance between oxidants, which are essential elements in many cellular functions, and antioxidants leading to oxidative stress. The end result of oxidative stress is the overproduction of ROS, which are extremely reactive molecules that can damage and thus modify the function of macromolecules, such as nucleic acids, proteins, lipids, and carbohydrates. Antioxidant systems are critical for the neutralization of ROS. Oxidative stress may lead to various pathological conditions and diseases including cancers.4 Oxidative stress has been implicated in BC, and oxidative stress crucially affects cell proliferation and malignancy as the BC develops.3 In this regard, paraoxonases (PONs) are essential with respect to their capacity to inhibit oxidative stress based on their antioxidant properties.5 The paraoxonase (PON) gene family is composed of three genes PON1, PON2, and PON3, which are localized on chromosome 7.2 All three PON enzymes are calcium-dependent hydrolases6 and are involved in hydrolyzing a variety of substrates, such as organophosphorus, lactones, aryl esters, and estrogen esters. Although some of the substrates are hydrolyzed by all PONs, specificity of each enzyme to substrates could be different.6, 7 Based on their antioxidative effects, all three enzymes warrant further investigation in terms of their effect on the development and progress of BC. Some polymorphisms of PON1 were reported to contribute BC risk, through polymorphic variation in PON1 activity.2 On the other hand, PON2 and PON3 decrease the formation of superoxide by reducing the formation of intracellular ROS.8 Thus, higher concentrations of PON2 and PON3 in healthy breast cells are beneficial for cellular homeostasis. However, the antiapoptotic effect of these two PONs, by decreasing ROS concentrations in the cancerous cell, makes the treatment of BC more difficult. Therefore, an evaluation of the available data, in terms of the relationship with ROS and possible PONs-dependent molecular mechanisms of cancers, especially in BC, is merited.

Functions and physiological roles of PON1 PON1 enzyme is synthesized predominantly in the liver, transported in the circulation, and then delivered to target tissues through binding with HDL.9, 10 It acts as an antioxidant and plays a role in the hydrolysis of a wide range of substrates, including estrogen esters.6 Although the exact mechanism has not yet been elucidated, there is evidence that PON1 provides effective protection against oxidative damage through two pathways: hydrolysis of lipid hydroperoxides and prevention of oxidative alteration of low-density lipoproteins (LDL).11 Despite the fact that increasing age and long-term exposure to estrogen increases the risk of BC, estrogen has been shown, at certain concentrations, to have an inductive effect on PON1 activity. One of the studies, conducted on human hepatoma cell line Huh7 and rat hepatocytes showed that in vitro estradiol treatment of both cell types resulted in a two to threefold rise in catalytic activity of cell-associated PON1.12 Along similar lines, estradiol treatment of postmenopausal women has been shown to increase serum PON1 activity.13 While PON1 is capable of hydrolyzing estrogen esters, it has been suggested that there is an interaction between them, as at some concentrations of estradiol, PON1 activity has been shown to increase.12 HDL binds to estrogen esters with PON1 in circulation. If the estrogen is esterified at the third position, PON1 can hydrolyze fatty acids thereby contributing to an antioxidant effect of the estrogens.6 This mechanism is thought to be contributory to the enhanced antioxidative roles of HDL in inhibiting LDL oxidation when associated with PON1. Studies have shown that PON1-inhibited LDL oxidation was facilitated in the presence of hydrolysable estrogen esters. Therefore, PON1 may hydrolyze estrogen esters bound to HDL through hydrophobic interaction and may contribute to uncover their antioxidant activities.6, 14 Furthermore, it has

Paraoxonases and breast cancer Chapter

1

5

been shown that both PON1 concentrations and activity is greater in women than in men.15 Apart from gender, differences in serum PON1 activity may depend on age, genetic factors, polymorphisms, diet, and lifestyle.12

PON1 and cancer: Focusing on breast cancer Lipid metabolism is one possible mechanism to explain the relationship between PON1 and cancer. PON1 has lactonase activity which leads to hydrolysis of oxidized lipids. It also has peroxidase activity, providing neutralization of fatty acids, cholesteryl ester hydroperoxides, other hydroperoxides, and hydrogen peroxide. As lipid peroxidation products, such as 4-hydroxynonenal and oxidized LDL, increase in abundance, pro-inflammatory molecule production is stimulated and consequently oxidative stress-associated cancer signaling might be induced.2 In this scenario, since PON1 prevents peroxidation of cell membrane lipids and HDL, it may guard against cancer including BC development. Additional evidence for this was provided by a study on BC patients which correlated lower PON1 activity with the stage and grade of the BC.2, 3, 5 Several potent molecular pathways have been associated with decreased serum PON1 activity which may contribute to the development of cancer. One of them is the inflammatory response in which a decrease in PON1 production has been reported. Other mechanisms which may lead to lower PON1 activity include production of acute phase proteins, which may displace PON1 from HDL, and oxidized phospholipids, which may inhibit PON1 activity. Another mechanism leading to cancer is related to distribution of PON1 to the cells. Finally, since PON1 is transported into cells by HDL, cancer cells can also receive PON1, which would subsequently inhibit oxidative stress in the cancerous cell through the antioxidant activity of PON1. Apart from these mechanisms, posttranslational modification patterns of PON1 have been suggested as a possible contributor to cancer. For example, the glycosylation pattern of PON1 was shown to change as cancer progresses so that may affect cellular responsiveness to oxidative stress.2, 3, 5

PON1 signaling pathways Low serum concentrations of PON1, due to a reduced antioxidant effect, may contribute to systemic oxidative stress.5 As previously discussed, two possible mechanisms, an increased level of oxidative stress and insufficient elimination of ROS, which may occur concurrently, may lead to cancer development. ROS have been shown to stimulate cancer-related pathways and messengers. These include nuclear factor-kB (NF-kB), mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase 1/2 (ERK1/2), Janus kinase (JAK)/signal transducer of activation (STAT), phosphatidylinositol-3kinase (PI3K/Akt), phospholipase C-g1, protein kinase C (PKC), protein kinase D (PKD), phosphatase and tensin homolog (PTEN), and protein tyrosine phosphatase 1B (PTP1B).2, 16, 17 Additionally, ROS contribute to the regulation of transcription factors, including nuclear factor erythroid 2-related factor 2 (Nrf2) and activator protein 1 (AP-1) in addition to tumor suppressors such as p53.2, 17 PON1 may reduce the formation of ROS and exert its inhibitory effect indirectly through the deactivation of these cancer-related ROS pathways. The potential factors and pathways that may regulate PON1 are summarized in Fig. 1.2, 18

PON1 polymorphisms Gene polymorphisms may be important due to their potential effects on enzyme concentration and specific activity. It has been reported that genetic factors including polymorphisms are the basis of more than 60% of phenotypic differences in PON1 activity. In comparison, environmental and metabolic factors account for only 1%–6% and 4%–19% of the variation, respectively. PON1 polymorphism differences were also thought to affect the rates of hydrolysis of estrogen esters.14 The majority of the research into PON1 has focused on the association of PON1 gene polymorphisms with cancer, including BC.19 PON1 has two common polymorphisms in coding region: Q192R (rs662), glutamine to arginine substitution at position 192, and L55M (rs854560), leucine to methionine substitution at position 55 that have been found to affect hydrolytic activity with lipid peroxides and the concentration of the enzyme, respectively.5, 19 Therefore, these polymorphisms are critical to an understanding of the protective effects of PON1 in oxidative stress and its association with cell proliferation and malignancy in BC development.20 To the best of our knowledge there are seven meta-analysis on PON1 polymorphisms (Q192R/L55M) and BC risk19–25 and consideration of these studies may provide a wider perspective of this association. One meta-analysis showed that although Q192R was not demonstrated to increase BC risk, L55M polymorphism was significantly correlated with cancer development, especially for BC.21 These results were in line with four of the other meta-analyses.19, 22, 24, 25 It was reported that the M allele leads to both a decrease in concentration and activity of PON1 enzyme, which in turn might lead to less effective detoxification of oxidants and carcinogens, and finally breast tissue becomes more prone to genetic damage.22 In

6 SECTION

A Oxidative stress and cancer

FIG. 1 The regulation of PON1 expression. With respect to illustrated signaling molecules, TNFa and IL-1b decrease PON1 expression, whereas other factors increase it.2, 18 (The full name of molecules are given in the list of abbreviations.)

the meta-analysis of Saadat, similar associations were reported for the L55M polymorphism but not for Q192R.23 In this meta-analysis, the R allele was significantly associated with decreased BC risk while Q allele was associated with an increased risk. Similarly, Zhang et al. concluded that the presence of the R allele indicates a reduced risk for cancers, including BC.20 It was suggested that the R allele may elevate the detoxification activity of PON1 and thus may be protective in terms of lipid peroxidation and the production of oxidative stress-based carcinogens (Fig. 2).23

Function and physiological roles of PON2 and PON3 PON2 and PON3 are involved in the regulation of cellular oxidative stress and blocking of ROS production in the cell.26 In addition to these antioxidative effects, they express enzymatic activity of varying effectiveness and specificity to different substrates. They play a role in hydrolyzing some drugs and substrates such as lactones, paraoxon, and esters, including estrogenic and aromatic esters.5, 6 PON3 and PON1 are detectable in serum whereas PON2 is not.5, 27, 28 Although serum PON3 concentrations are lower than those of PON1, PON3 much more strongly hydrolyzes estradiol esters due to its higher specificity compared to PON1 and PON2.5, 6 PON2 is localized in the plasma membrane, inner mitochondrial membrane (IMM), endoplasmic reticulum (ER), and nuclear membrane.28 It expresses antioxidant activity by blocking superoxide production from Complex I and Complex III at the IMM, through interaction with Coenzyme Q10 (CoQ10).29 Similarly, PON3 has been detected on the IMM and ER and its intracellular activity resembles that of PON2.2 PON2 and PON3 may reduce ROS formation during estrogen metabolism in the mitochondria of breast cells.29–31 Also, serum PON3 as an HDL-dependent enzyme has protective effects on LDL.5 Furthermore, overexpression of PON2 and PON3 genes was shown to inhibit ER stress and thus ER stress-mediated mitochondrial dysfunction.26

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FIG. 2 Possible rationales behind a significant association between PON1 polymorphisms (Q192R/L55M) and BC risk. The significant association of PON1 polymorphisms with BC risk could be explained by oxidative stress related effects of the R and M alleles on PON1 enzyme activity.

PON2/3 and cancer: Focusing on breast cancer PON2 and PON3 have been shown to be expressed in BC cells32–34 and their activities in the prevention of superoxide production are essential, since superoxide formation is the starting point for ROS production. Low activity of these enzymes leads to the generation of more superoxide and superoxide-based ROS, and eventually an increase in the ROS concentration in cells. Given this mechanism, PON2 and PON3 may play a role in the modulation of ROS-dependent cancer signaling pathways described earlier. The relationship between estrogen and ROS production during BC development should also be considered.35 Estrogen metabolites have been shown to regulate BC stem cell functionality as well as its BC progression.36 Modified estradiols can act as genotoxic metabolites in breast tissue.35 This could result in mutations in DNA directly from an estrogen effect or indirectly through ROS on its metabolic substrates.35 Mitochondria are the main source of ROS and were thought to be essential for estrogen metabolism.30, 37 Estrogen-dependent accumulation of high concentrations of ROS are among the most important risk factors for BC development because of increased genomic instability and stimulation of redox-related signaling pathways.37 Antioxidants have been shown to be effective in the prevention of estrogen stimulated cell growth.30, 38 Estrogens or their metabolites interact with the mitochondria of mammary epithelial cells which leads to the generation of ROS. In turn these ROS trigger the phosphorylation of kinases, which stimulate redox-sensitive transcription factors and finally may induce cancer development (Fig. 3).37 Due to their antioxidant effects, mitochondrial PON2 and PON36, 26 may protect breast cells against estrogen-induced ROS accumulation and decrease the risk of BC development. Interestingly, ROS could also play a role in cell apoptosis pathways and apoptosis stimulation, via ROS, has been suggested as one of the therapeutic targets for BC.39 However, if PON2 and PON3 concentrations are high in cancerous cells, they will decrease ROS production and may serve to suppress apoptosis. To sum up, while the expression of PON2 and PON3 might be protective against estrogen-dependent ROS production during BC development, suppression of PON2 and PON3 might be a significant target for BC therapy by promoting apoptosis of cancer cells.

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FIG. 3 The effect of Estradiol on cell cycle through ROS generation. The ROS generated by mitochondrial estradiol metabolism induces cell cycle genes.

Although cancer-associated molecular mechanism studies on both PON2 and PON3 are scarce, more research has been published concerning PON2. Nrf2 which plays a role in regulation of redox balance has been shown to be activated in tumor cells and enhanced the generation of antioxidant proteins, including PON2.40 The expression and activity of PON2 were also found to be increased during oxidative stress.5 However, there are no studies concerning the signaling pathways of PON2 in BC cells. Mutations in the BC susceptibility genes (BRCA1 and BRCA2), which are tumor suppressor genes, are known to increase susceptibility to BC development.36 It has been reported that mutations in these genes impair the balance of ROS concentrations in the cell through Nrfs.36 The BRCA1 gene normally stimulates Nrf1 and Nrf2 transcription factors and the synthesis of antioxidant and/or xenobiotic detoxifying phase II enzymes and reduces ROS production in the breast cell.36 When some mutations occurs in the BRCA1 gene, the cell will be more exposed to ROS since it cannot adequately stimulate the synthesis of intracellular antioxidants such as PON2. In summary, PON2 may be involved in BC development through BRCA- and Nrf2-associated pathways. PON2 and PON3 also inhibit ER stress-associated ROS production. The ER is involved in both protein folding and protein maturation, as well as redox balance. PON2 and PON 3 have antiapoptotic effects which reduce ROS formation in both mitochondria and ER as illustrated in Fig. 4.2, 39 In a study demonstrating the importance of induction of apoptosis in the treatment of BC, an anticancer molecule, chrysophanol, was found to be effective in promoting ROS production, acting through PI3K/Akt and MAPK signaling pathways which had both antiproliferative and increased apoptotic effects in two BC cell lines, BT-474 and MCF-7.39 Consequently, ROS generation and apoptosis induction might be valuable targets for the development of BC treatment and PON2 is one of the candidates that may regulate these cellular events. Moreover, another study reported that increased PON2 expression was associated with a poor prognosis for patients with different types of solid tumors, including BC.28 The study included more than 10,000 patients with 31 types of malignancies and the expression of PON2 levels was compared between all malignancies which included invasive breast carcinoma. It was concluded that PON2 might protect cancer cells from damaging environmental conditions including chemotherapy. PON3 also could be considered as protective against the development of BC, due to its preventative effect on the development of obesity, which is a known risk factor for BC. PON3, as an HDL-associated enzyme in circulation, has been

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FIG. 4 Antiapoptotic mechanism of action of PON2 and PON3. PON2 and PON3 decrease the release of mitochondrial cytC through reduced ROS generation in ER and mitochondria.

shown to affect cholesterol metabolism and act as a protective factor against obesity. In addition, PON3 deficiency was correlated with enhanced mitochondrial superoxide levels and disrupted mitochondrial respiration.41 Thus, PON3 as a serum enzyme might be protective against BC, may also act through its intracellular antioxidant effects in a manner similar to PON2, via its antiapoptotic activities, and finally through its effect on cholesterol and estrogen metabolism.

PON2 signaling pathways The expression of PON2 was shown to be enhanced by ER stress and a number of cellular systems including the urokinase plasminogen activator (uPA) system, PI3K/platelet-derived growth factor receptors (PDGFR)-b and the Wnt/b-catenin, and AP-1/JNK signaling pathways. The role of some of these pathways in BC progression has been reported and these molecules may be prospective targets for novel BC treatment development.42, 43 During oxidative stress, the expression of PON2 was also found to be increased and was mediated by peroxisome proliferator-activated receptor (PPAR)g and AP1 signaling in some cells.2 AP-1 signaling is known to stimulate cell invasion in BC.43 Moreover, mutations in BRCA1 and BRCA2 have been shown to be associated with both BC and ovarian cancer development. Thus these cancers share a common etiology and these mutations are the basis for nearly 10% of these cancers. They lead to elevated estrogen levels, which may promote BC and ovarian cancers and may also explain the high penetrance of these cancers in breast and ovary.44 The ovary is the major source of estrogen synthesis in the reproductive period. In a study conducted in a mouse xenograft model of ovarian cancer, PON2 overexpression was shown to prevent tumor formation in ovarian tumors (Fig. 5). Davarajan et al. suggested the following mechanism. PON2 expression leads to a decrease in insulin like growth factor-1 (IGF-1) gene expression. PON2 expression also leads to an increase in the activity of the electron transport chain, cholesterol levels decrease and subsequently IGF-1 receptor structure is impaired. This results in reduced cell proliferation and tumor growth diminishes.45 In addition, the PON2-dependent decrease in cholesterol reduces the available substrate for estrogen production in ovarian cells and thus reduces cellular and systemic exposure to estrogen. It could be proposed that PON2 may be indirectly protective against BC through its effect on the ovaries. However, low levels of IGF-1 may be protective against BC development. In other words, the increased levels of IGF-1 in ovaries could cause the development

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FIG. 5 Possible mechanisms of PON2 in carcinogenesis. PON2 may decrease carcinogenesis by reducing the expression of IGF1.

of cancer in breast cells. It has been proposed that increased IGF-1 levels give rise to tumor promoting effects in breast malignancy, while reduced levels are considered to have an anticancer effect. The signaling pathways of IGF-1 could be summarized as follows: after IGF-1 binds to the IGF-1 receptor, IRS-1 is activated and then two signaling cascades, PI3K/AKT and RAF kinase/MAPK, are stimulated which results in proliferative and antiapoptotic effects on breast cells.46 Finally, IGF-1 signaling pathway may lead to development, progression, and metastasis of BC.47

PON3 signaling pathways Similar to PON2, PON3-related molecular mechanisms in BC are not fully understood at present. However, there are several studies investigating the role of PON3 in cancer which may shed some light on these mechanisms.48, 49 The PI3K/Akt signaling pathway is one of the essential mechanisms involved in cell survival, proliferation, and differentiation as well as metastasis and PON3 has been shown to promote cell proliferation and metastasis through this pathway in oral squamous cell carcinoma.48 After the activation of this pathway, Akt is transferred to the nucleus and modulates a number of regulatory factors that result in increased expression of antiapoptotic genes. In addition, molecule interacting with CasL 1 (MICAL1) was found to activate the PI3K/Akt pathway and regulate the invasion of BC cells as well as ROS production.49 MICAL1-dependent BC cell proliferation is mediated by maintaining cyclin D expression, which in turn is mediated by the ROS-sensitive PI3K/Akt/ERK pathway. In the absence of MICAL1, the proliferation of BC cell lines was found to decrease significantly. Based on these results, the PI3K/Akt has been proposed as a target for the development of novel cancer therapies,49 therefore a deeper understanding of the regulation of PON3 by this pathway may shed further light on the mechanisms by which BC develops. Apart from PI3K/Akt signaling, the expression of PON3 is modulated by the transcription factor AP-1, which is responsible for transducing proliferative signals. It was shown in the MCF7 BC cell line that AP-1 was required for the proliferation of the cells and inhibition of AP-1 resulted in the suppression of tumor cell growth. It has therefore been proposed that AP-1 may be a potential target for BC treatment approaches48, 50 and PON3 may have a significant role to play in the manipulation of this pathway (Fig. 6).

PON2/3 polymorphisms To the best of our knowledge, there are no studies examining the association between PON2/3 polymorphisms and BC susceptibility. However, several studies have analyzed the correlation between PON2/3 polymorphisms and diseases associated with oxidative stress. Two polymorphisms in PON2 (A148G, S311C) and PON3 (S311T, G324A) have been investigated. PON2 polymorphisms were correlated with changes in plasma lipid levels in diabetic patients although no correlation was found with PON3 polymorphisms and protein activity.2 Additionally, PON2(A148G) were found to be associated with elevated plasma HDL and glucose, and decreased levels of plasma LDL cholesterol. Moreover, it was suggested that PON2 (S311C) resulted in modified lactonase activity of PON2.8

The paradox: PON activities and BC development The suppression of ROS production by PON2 and PON3 in healthy cells is seen as beneficial. In contrast, the antiapoptotic activity of PON2 and PON3 in cancer cells, including BC, because of this negative effect on ROS production, has been

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FIG. 6 Possible mechanisms of PON3 in carcinogenesis. PON3 may increase cell cycle, proliferation, migration, and invasion through PI3K/Akt and AP-1.

perceived as problematic because enhanced ROS levels promote apoptosis in cancer cells. Therefore, low PON2 and PON3 activity would be beneficial for cancer therapy. Similarly, lower PON activities in BC cells are necessary during anticancer therapy when estrogen is used in the treatment protocol. Thus, lower activity of PON2 and PON3 would promote sustainability of estrogen treatment and increased ROS production which should synergize to enhance apoptosis of cancer cells. So, although PON2 and PON3 are beneficial to healthy cells by restricting the production of ROS, in cancer low PON activities should be maintained to promote effective treatment.8, 51 While oxidative stress causes inactivation of PON1 and PON3, the expression of PON2 is enhanced under oxidative stress.2 Nrf2 enhances both PON1 and PON2 expression. However, it should be noted that synthesis of the PON1 enzyme takes place only in the liver, whereas the PON2 enzyme is found in almost all cells. In this case, although the transcription of both enzymes is increased by Nrf2, PON1 enzyme is mainly measured in serum, so under conditions of oxidative stress lower levels might be detected. Since PON2 is localized within the cell, it appears that Nrf2-mediated expression increases in response to increased intracellular oxidative stress.

Concluding remarks Although there are various studies on the association of PON1 polymorphisms with BC susceptibility, most of them are limited by small populations and inadequate matching of cases and controls.10 Apart from association studies, there is scarce research into the molecular mechanisms of PON1-related cancer development and its effects on cell death regulation.52 Thus, future studies should remedy these limitations with well-defined larger populations with detailed clinical data which should result in more robust results. This may then suggest further experimental studies into the exact mechanisms underlying the correlation between PON1 and BC. Since the PON1 enzyme is localized in serum, there are more studies on PON1 compared with those on PON2 and PON3, which are found intracellularly. Therefore, there is a need for more cell- and tissue-based studies, in order to elucidate molecular mechanisms and effects of PON2 and PON3. These enzymes show particular promise as novel therapeutic targets in a variety of cancers, especially for BC because of their effects on estrogen metabolism, apoptosis, and inhibition of intracellular superoxide production. Finally, further studies should investigate the expression and function of intracellular PON2 and PON3 and their associated signaling pathways, in order to suggest novel therapeutic approaches for BC.

Application to other cancers Since PONs are involved in many cellular events, the potential mechanisms and signaling pathways indicated for BC may also be the cause of a number of other cancers. Similar to BC, lower PON1 serum activity was detected in various cancer types such as lung cancer, prostate cancer, cervix cancer, non-Hodgkin lymphoma, acute lymphoblastic lymphoma,53 central nervous system tumors,54 bladder cancer,55 pancreatic cancer,56 colorectal cancer,57 and hepatocelluar carcinoma.58 As in BC, it has been stated that the increase in oxidative stress in these cancers is accompanied by PON1 enzyme activity.2

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Also, PON2 has been shown to increase in some tumors such as glioblastoma multiforme, hepatocelluar, renal, bladder, and prostate.2 Moreover, PON3 upregulation has also been shown in different type of tumors including stomach, liver, thyroid, lung, prostate, pancreas, colon, kidney, lymphoid, urinary, bladder, testis, and endometrium/uterus.33 Due to the antioxidant and antiapoptotic effects of PON2 and PON3, their upregulation has been associated with poor prognosis in cancers by reducing intracellular ROS formation.2

Summary points l l

l l l

l

Oxidative stress plays a significant role in the development, progression, and treatment of breast cancer (BC). Reactive oxygen species, generated by mitochondrial estradiol metabolism, have widespread effects on all BC processes. Paraoxonases (PONs) might prevent cancer development in breast cells due to their antioxidant effects. Serum PON1 levels are generally observed to be low in BC patients. The current association data suggest that while contradictory results are present for the PON1 Q192R polymorphism, the PON1 L55M polymorphism might be a risk factor for BC. PON2 and PON3 have the potential to be novel therapeutic targets in BC treatment, due to their antioxidant and antiapoptotic effects.

References 1. Okoh V, Deoraj A, Roy D. Estrogen-induced reactive oxygen species-mediated signalings contribute to breast cancer. Biochim Biophys Acta 2011;1815:115–33. 2. Bacchetti T, Ferretti G, Sahebkar A. The role of paraoxonase in cancer. Semin Cancer Biol 2019;56:72–86. 3. Wu J, Fang M, Zhou X, Zhu B, Yang Z. Paraoxonase 1 gene polymorphisms are associated with an increased risk of breast cancer in a population of Chinese women. Oncotarget 2017;8: https://doi.org/10.18632/oncotarget.15911. 4. Birben E, Sahiner U, Sackesen C, Erzurum S, Kalayci O. Oxidative stress and antioxidant defense. World Allergy Organ J 2012;5:9–19. 5. Pr
ecourt L, Amre D, Denis M, Lavoie JC, Delvin E, Seidman E, et al. The three-gene paraoxonase family: physiologic roles, actions and regulation. Atherosclerosis 2011;214:20–36. 6. Teiber J, Billecke S, La Du B, Draganov D. Estrogen esters as substrates for human paraoxonases. Arch Biochem Biophys 2007;461:24–9. 7. Draganov D, Teiber J, Speelman A, Osawa Y, Sunahara R, La Du B. Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities. J Lipid Res 2005;46:1239–47. 8. Witte I, Foerstermann U, Devarajan A, Reddy S, Horke S. Protectors or traitors: the roles of PON2 and PON3 in atherosclerosis and cancer. J Lipids 2012;2012:1–12. 9. NCBI. PON1 paraoxonase 1 [Homo sapiens (human)]: Gene: NCBI. Ncbi.nlm.nih.gov (web archive link, 14 March 2019); 2019 https://www.ncbi. nlm.nih.gov/gene/5444 (accessed 14 March 2019). 10. Mackness M, Mackness B. Human paraoxonase-1 (PON1): gene structure and expression, promiscuous activities and multiple physiological roles. Gene 2015;567:12–21. 11. Levy E, Trudel K, Bendayan M, Seidman E, Delvin E, Elchebly M, et al. Biological role, protein expression, subcellular localization, and oxidative stress response of paraoxonase 2 in the intestine of humans and rats. Am J Physiol Gastrointest Liver Physiol 2007;293:1252–61. 12. Ahmad S, Scott J. Estradiol enhances cell-associated paraoxonase 1 (PON1) activity in vitro without altering PON1 expression. Biochem Biophys Res Commun 2010;397:441–6. 13. Kumru S, Aydin S, Aras A, Gursu MF, Gulcu F. Effects of surgical menopause and estrogen replacement therapy on serum paraoxonase activity and plasma malondialdehyde concentration. Gynecol Obstet Invest 2005;59:108–12. 14. Tikkanen M, Vihma V, Jauhiainen M, H€ockerstedt A, Helisten H, Kaamanen M. Lipoprotein-associated estrogens. Cardiovasc Res 2002;56:184–8. 15. Trentini A, Bellini T, Bonaccorsi G, Cavicchio C, Hanau S, Passaro A, et al. Sex difference: an important issue to consider in epidemiological and clinical studies dealing with serum paraoxonase-1. J Clin Biochem Nutr 2019;64:250–6. 16. Barrera G. Oxidative stress and lipid peroxidation products in cancer progression and therapy. ISRN Oncol 2012;2012:1–21. 17. Moloney JN, Cotter TG. ROS signalling in the biology of cancer. Semin Cell Dev Biol 2018;80:50–64. 18. Ponce-Ruiz N, Murillo-Gonza´lez F, Rojas-Garcı´a A, Mackness M, Bernal-Herna´ndez YY, Barro´n-Vivanco BS, et al. Transcriptional regulation of human Paraoxonase 1 by nuclear receptors. Chem Biol Interact 2017;268:77–84. 19. Wen Y, Huang Z, Zhang X, Gao B, He Y. Correlation between PON1 gene polymorphisms and breast cancer risk: a meta-analysis. Int J Clin Exp Med 2015;8:20343–8. 20. Zhang M, Xiong H, Fang L, Lu W, Wu X, Huang ZS, et al. Paraoxonase 1 (PON1) Q192R gene polymorphism and cancer risk: a meta-analysis based on 30 publications. Asian Pac J Cancer Prev 2015;16:4457–63. 21. Fang D-H, Fan C-H, Ji Q, Qi B-X, Li J, Wang L. Differential effects of paraoxonase 1 (PON1) polymorphisms on cancer risk: evidence from 25 published studies. Mol Biol Rep 2012;39:6801–9.

Paraoxonases and breast cancer Chapter

1

13

22. Liu C, Liu L. Polymorphisms in three obesity-related genes (LEP, LEPR, and PON1) and breast cancer risk: a meta-analysis. Tumor Biol 2011;32:1233–40. 23. Saadat M. Paraoxonase 1 genetic polymorphisms and susceptibility to breast cancer: a meta-analysis. Cancer Epidemiol 2012;36. 24. Hu P, Ma Y, Zhang L, Ma S. PON1 L55M polymorphism might contribute to the risk of cancer. Panminerva Med 2017;59:107–13. 25. Chen L, Lu W, Fang L, Xiong H, Wu X, Zhang M, et al. Association between L55M polymorphism in Paraoxonase 1 and cancer risk: a meta-analysis based on 21 studies. Onco Targets Ther 2016;9:1151–8. 26. Devarajan A, Shih D, Reddy ST. Inflammation, infection, cancer and all that… the role of paraoxonases. Adv Exp Med Biol 2014;824:33–41. 27. Marsillach J, Mackness B, Mackness M, Riu F, Beltra´n R, Joven J, et al. Immunohistochemical analysis of paraoxonases-1, 2, and 3 expression in normal mouse tissues. Free Radic Biol Med 2008;45:146–57. 28. Shakhparonov MI, Antipova NV, Shender VO, Shnaider PV, Arapidi GP, Pestov NB, et al. Expression and intracellular localization of paraoxonase 2 in different types of malignancies. Acta Nat 2018;10:92–9. 29. Altenh€ ofer S, Witte I, Teiber JF, Wilgenbus P, Pautz A, Li H, et al. One enzyme, two functions. J Biol Chem 2010;285:24398–403. 30. Felty Q, Roy D. Mitochondrial signals to nucleus regulate estrogen-induced cell growth. Med Hypotheses 2005;64:133–41. 31. Yue W, Yager JD, Wang J-P, Jupe ER, Santen RJ. Estrogen receptor-dependent and independent mechanisms of breast cancer carcinogenesis. Steroids 2013;78:161–70. 32. Wang R, Li J, Zhao Y, Li Y, Yin L. Investigating the therapeutic potential and mechanism of curcumin in breast cancer based on RNA sequencing and bioinformatics analysis. Breast Cancer 2017;25:206–12. 33. Schweikert E-M, Devarajan A, Witte I, Wilgenbus P, Amort J, F€orstermann U, et al. PON3 is upregulated in cancer tissues and protects against mitochondrial superoxide-mediated cell death. Cell Death Differ 2012;19:1549–60. 34. Expression of PON3 in cancer. Summary: the human protein atlas. https://www.proteinatlas.org/ENSG00000105852-PON3/pathology; 2019 (accessed 1 July 2019). 35. Santen RJ, Yue W, Wang JP. Estrogen metabolites and breast cancer. Steroids 2015;99:61–6. 36. Gurer-Orhan H, Ince E, Konyar D, Saso L, Suzen S. The role of oxidative stress modulators in breast cancer. Curr Med Chem 2018;25:4084–101. 37. Wen C, Wu L, Fu L, Wang B, Zhou H. Unifying mechanism in the initiation of breast cancer by metabolism of estrogen. Mol Med Rep 2017;16:1001–6. 38. Maruyama T, Sachi Y, Furuke K, Kitaoka Y, Kanzaki H, Yoshimura Y, et al. Induction of thioredoxin, a redox-active protein, by ovarian steroid hormones during growth and differentiation of endometrial stromal cells in vitro. Endocrinology 1999;140:365–72. 39. Park S, Lim W, Song G. Chrysophanol selectively represses breast cancer cell growth by inducing reactive oxygen species production and endoplasmic reticulum stress via AKT and mitogen-activated protein kinase signal pathways. Toxicol Appl Pharmacol 2018;360:201–11. 40. Zhang X, Wang M, Teng S, Wang D, Li X, Wang X, et al. Indolyl-chalcone derivatives induce hepatocellular carcinoma cells apoptosis through oxidative stress related mitochondrial pathway in vitro and in vivo. Chem Biol Interact 2018;293:61–9. 41. Shih DM, Yu JM, Vergnes L, Dali-Youcef N, Champion MD, Devarajan A, et al. PON3 knockout mice are susceptible to obesity, gallstone formation, and atherosclerosis. FASEB J 2015;29:1185–97. 42. Prosperi JR, Goss KH. A Wnt-ow of opportunity: targeting the Wnt/beta-catenin pathway in breast cancer. Curr Drug Targets 2010;11:1074–88. 43. Zhao C, Qiao Y, Jonsson P, Wang J, Xu L, Rouhi P, et al. Genome-wide profiling of AP-1-regulated transcription provides insights into the invasiveness of triple-negative breast cancer. Cancer Res 2014;74:3983–94. 44. Mungenast F, Thalhammer T. Estrogen biosynthesis and action in ovarian cancer. Front Endocrinol 2014;5:192. 45. Devarajan A, Su F, Grijalva V, Yalamanchi M, Yalamanchi A, Gao F, et al. Paraoxonase 2 overexpression inhibits tumor development in a mouse model of ovarian cancer. Cell Death Dis 2018;9:392. 46. Christopoulos PF, Corthay A, Koutsilieris M. Aiming for the insulin-like growth Factor-1 system in breast cancer therapeutics. Cancer Treat Rev 2018;63:79–95. 47. Christopoulos PF, Msaouel P, Koutsilieris M. The role of the insulin-like growth factor-1 system in breast cancer. Mol Cancer 2015;14:43. 48. Zhu L, Shen Y, Sun W. Paraoxonase 3 promotes cell proliferation and metastasis by PI3K/Akt in oral squamous cell carcinoma. Biomed Pharmacother 2017;85:712–7. 49. Deng W, Wang Y, Zhao S, Zhang Y, Chen Y, Zhao X, et al. MICAL1 facilitates breast cancer cell proliferation via ROS-sensitive ERK/cyclin D pathway. J Cell Mol Med 2018;22:3108–18. 50. Ludes-Meyers JH, Liu Y, Mun˜oz-Medellin D, Hilsenbeck SG, Brown PH. AP-1 blockade inhibits the growth of normal and malignant breast cells. Oncogene 2001;20:2771–80. 51. Galadari S, Rahman A, Pallichankandy S, Thayyullathil F. Reactive oxygen species and cancer paradox: To promote or to suppress? Free Radic Biol Med 2017;104:144–64. 52. Aldonza MBD, Son YS, Sung H-J, Ahn JM, Choi YJ, Kim YI, et al. Paraoxonase-1 (PON1) induces metastatic potential and apoptosis escape via its antioxidative function in lung cancer cells. Oncotarget 2017;8. 53. Samra ZQ, Pervaiz S, Shaheen S, Dar N, Athar MA. Determination of oxygen derived free radicals producer (xanthine oxidase) and scavenger (paraoxonase1) enzymes and lipid parameters in different cancer patients. Clin Lab 2011;57:741–7. 54. Kafadar AM, Ergen A, Zeybek U, Agachan B, Kuday C, Isbir T. Paraoxonase 192 gene polymorphism and serum paraoxonase activity in high grade gliomas and meningiomas. Cell Biochem Funct 2006;24:455–60. 55. Utang˘ac¸ MM, Yeni E, Savas¸ M, Altunkol A, C¸iftc¸i H, G€um€u¸s K, et al. Paraoxonase and arylesterase activity in bladder cancer. Turk J Urol 2017;43:147–51.

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56. Vecka M, Ja´chymova´ M, Va´vrova´ L, Kodydkova´ J, Maca´sˇek J, Urba´nek M, et al. Paraoxonase-1 (PON1) status in pancreatic cancer: relation to clinical parameters. Folia Biol (Praha) 2012;58:231–7. 57. Ahmed NS, Shafik NM, Elraheem OA, Abou-Elnoeman SE. Association of paraoxonase-1(Q192R and L55M) gene polymorphisms and activity with colorectal cancer and effect of surgical intervention. Asian Pac J Cancer Prev 2015;16:803–9. 58. Yu Z, Ou Q, Chen F, Bi J, Li W, Ma J, et al. Evaluation of the prognostic value of paraoxonase 1 in the recurrence and metastasis of hepatocellular carcinoma and establishment of a liver-specific predictive model of survival. J Transl Med 2018;16:327.

Chapter 2

Oxidative stress and prostate cancer Masaki Shiota Department of Urology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

List of abbreviations 4-HNE 8-OHdG AP-1 AR COX Duox GSTP1 HIF-1a MAPK mtDNA NFkB Nox Nrf2 NSAID PI3K PIA PIN ROS SNP SOD STAT3 TRAMP

4-hydroxy-2-nonenal 8-hydroxydeoxyguanosine activator protein 1 androgen receptor cyclooxygenase dual oxidase glutathione S-transferase p hypoxia-inducible factor-1a mitogen-activated protein kinase mitochondrial DNA nuclear factor-kB NAPDH oxidase NF-E2-related factor 2 nonsteroidal antiinflammatory drug phosphoinositide-3 kinase proliferative inflammatory atrophy prostatic intraepithelial neoplasia reactive oxygen species single-nucleotide polymorphism superoxide dismutase signal transducer and activator of transcription 3 transgenic adenocarcinoma of the mouse model of prostate cancer

Introduction Prostate cancer is a major cause of death among men in developed countries, affecting mainly older men. Under both physiological and pathological conditions, metabolic processes generate oxidative stress by hydroxyl radicals, hydrogen peroxide, and superoxides defined as reactive oxygen species (ROS) as well as reactive nitrogen species (RNS) such as nitrogen dioxide, dinitrogen trioxide, and nitrogen peroxide. Increased ROS/RNS have traditionally been thought to cause tissue injury and/or damage to intracellular components, but they also participate in a wide range of crucial physiological as well as pathological processes, including cell proliferation, cell cycle progression, antiapoptotic mechanisms, invasion, metastasis, and angiogenesis, which contribute to prostate cancer pathogenesis such as prostate carcinogenesis and prostate cancer progression.1 So far, several impairments of prooxidant and antioxidant systems resulting in increased oxidative stress and oxidation damage have been identified in prostate cancer. Oxidative stress has thus been proposed to contribute to the pathogenesis of prostate cancer. Definitive risk factors for the development of prostate cancer include age, race, and family history, while androgens, inflammation, diet, and lifestyle have been suggested as potential risk factors. These risk factors may be linked to oxidative stress and close relationships between oxidative stress and prostate cancer risk factors have been suggested. Here, I summarize the findings regarding functional links between oxidative stress and prostate cancer.

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00002-X © 2021 Elsevier Inc. All rights reserved.

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Causes of oxidative stress in prostate cancer ROS are generated endogenously from mitochondrial bioenergetics, the NADPH oxidase (Nox) complex including Nox1–5 and dual oxidase (Duox)1/2, xanthine oxidase, cyclooxygenase (COX), and exogenously from the cellular microenvironment under conditions such as hypoxia.2 RNS are produced by reacting nitric oxide (NO), which is produced by nitric oxide synthase (NOS), with ROS and metals.3 To protect against elevated ROS/RNS and oxidative stress, cells are equipped with antioxidant defense systems,4 including low molecular weight antioxidant compounds and reducing buffer systems, as well as antioxidant enzymes such as superoxide dismutases (SODs), catalase, glutathione peroxidase, and peroxiredoxins.4 Transcription factors regulating the reduction-oxidation (redox) state in response to oxidative stress, such as NF-E2-related factor 2 (Nrf2), nuclear factor-kB (NF-kB), and activator protein 1 (AP-1), are also important regulators of the antioxidant defense system. These proteins maintain the intracellular homeostasis of redox states and the balance of reducing/oxidizing equivalents. However, impairments of the redox systems can result in a redox imbalance because of increased ROS/RNS production and/or decreased antioxidant defenses, as shown in Fig. 1. This section describes the deregulation of ROS/RNS production and antioxidant systems in prostate cancer.

Increased ROS production Altered mitochondrial bioenergetics Altered mitochondrial bioenergetics is thought to be one of the major sources of increased ROS generation. Metabolic transformation is a common feature of tumors including prostate cancer. Mutation rates in mitochondrial DNA (mtDNA) are high compared with nuclear DNA presumably because of the high concentrations of ROS around mtDNA, the lack of histone protection, and inadequate DNA repair systems. Accelerated mtDNA mutations are thought to increase ROS production and oxidative stress. Petros et al. demonstrated the role of mtDNA mutation-induced oxidative stress in prostate tumorigenesis using an mtDNA mutation (Thr8993Gly) known to cause increased mitochondrial ROS production.5 Indeed, frequent mtDNA mutations have been identified in clinically relevant human prostate cancer.6 In addition to mtDNA mutations, alterations in mitochondrial enzymes are also thought to contribute to prostate carcinogenesis, as suggested by the fact that ROS generation is elevated by increased expression of mitochondrial glycerophosphate dehydrogenase during prostate cancer progression.7 According to these alterations, dysregulation in the mitochondrion is suggested to contribute prostate carcinogenesis and progression.

Increased ROS productions 䞉Mitochondrial bioenergetics 䞉Up regulation of NADPH oxidases 䞉Up regulation of xanthine oxidases 䞉Up regulation of cyclooxygenase 䞉Hypoxia 䞉Nitric oxide synthase

Impaired antioxidant defenses 䞉Antioxidant enzymes 䞉Transcription factors 䞉Antioxidant substances

Increased ROS/oxidative stress FIG. 1 Dysregulated balance between pro- and antioxidant properties. Increased ROS production and impaired antioxidant defenses contribute to increased oxidative stress.

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Upregulation of Nox enzymes Upregulation of membrane-bound Nox is a potential source of increased intracellular ROS production. The Nox enzyme family consists of Nox1–5 and Duox1/2, which catalyzes the production of superoxides using oxygen as a substrate and NADPH as a cofactor.7 Nox1 overexpression has been reported to increase superoxide production and cause malignant transformation.8 Nox1, Nox2, and Nox4 are expressed and further upregulated in the rat prostate after castration, indicating a possible link between androgens and oxidative stress.9 Similarly, Nox1 and Nox2 are expressed in the human prostate, and increased Nox1 correlates with elevated hydrogen peroxide levels.8 In addition, Nox2, Nox4, and Nox5 levels are elevated in human prostate cancer cells, whereas the Nox inhibitor diphenyliodonium and downregulation of Nox5 suppresses ROS production, leading to decreased cell proliferation and increased apoptosis.10 Taken together, these lines of evidence indicate that upregulation of Nox ROS producers contributes to prostate cancer growth.

Upregulation of COX enzymes COXs include two isoforms, COX-1 and COX-2, which are rate-limiting enzymes in prostaglandin biosynthesis and produce ROS during their processes. Although COX-1 is constitutively expressed ubiquitously, COX-2 is only induced by stimulation. COX-2 expression was found to be increased in high-grade prostatic intraepithelial neoplasia (PIN) lesions compared with normal prostate tissue in a transgenic adenocarcinoma mouse model of prostate cancer (TRAMP).11 In addition, COX-2 expression is significantly higher in cancerous prostate tissues compared with benign prostate tissues, and higher in high-grade tumors.12 Based on these findings, nonsteroidal antiinflammatory drugs (NSAIDs), which inhibit COX activity, may be attractive potential chemopreventive agents. Several preclinical studies have demonstrated growthsuppressive and apoptosis-inducing effects of NSAIDs in prostate cancer.13

Hypoxia Tumor hypoxia is an inevitable characteristic of advanced prostate cancer, which increases intracellular ROS as an exogenous source.2 Hypoxia-induced ROS can activate numerous signaling components and stabilize hypoxia-inducible factor-1a (HIF-1a) through activation of proto-oncogenic signal-transduction pathways, including the phosphoinositide-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathway, which are inhibited by the ROS scavenger N-acetyl-cysteine.2 HIF-1a regulates its target genes that are pivotal in regulating cellular metabolism, cell cycle progression, antiapoptosis mechanisms, metastasis, and angiogenesis.2 Under hypoxic conditions, human prostate cancer cells show increased levels of ROS and accumulation of HIF-1a. Hypoxia thus acts as a regulator of oxidative stress as an exogenous source in prostate cancer, resulting in activation of proto-oncogenic pathways by HIF-1a.

Impaired antioxidant defenses Altered antioxidant enzymes Dismutases catalyzing the conversion of superoxides to water include intracellular copper-zinc SOD (SOD1) in the nucleus/cytoplasm, manganese SOD (SOD2) in mitochondria, and extracellular SOD. Catalase catalyzes the conversion of hydrogen peroxide to water and is located within peroxisomes and the cytoplasm. Several lines of evidence have shown a close relationship between antioxidant defense systems and prostate cancer. SOD1, SOD2, and catalase are suppressed in cancerous regions of human prostate cancer tissues compared with noncancer regions.14 Similarly, Bostwick et al. found lower expression of SOD1, SOD2, and catalase in human high-grade PIN and prostate cancer compared with benign prostate epithelium.15 However, Oberley et al. found lower levels of SOD2 in human primary prostate cancer tissues and higher levels in metastatic tissues compared with normal tissues.4 Taken together, these results indicate that SOD and catalase expression levels are decreased in tumor tissues compared with nontumor tissues although SOD2 is elevated in metastatic sites compared with primary regions, as depicted in Fig. 2. Glutathione S-transferase p (GSTP1) plays an important role in the detoxification of electrophilic compounds, such as carcinogens and cytotoxic drugs, by glutathione conjugation. Inactivation of GSTP1 can impair cellular antioxidant systems and augment intracellular oxidative stress. Hypermethylation of the promoter region in the GSTP1 gene is a common feature identified during prostate carcinogenesis, and GSTP1 gene methylation has been detected in 50%– 70% of PIN and 70%–95% of prostate cancers.16

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Noncancerous prostate SOD → Catalase → GSTπ →

Primary prostate cancer SOD ↓ Catalase ↓ GSTπ ↓

Oxidative stress Metastatic prostate cancer SOD2 ↑

Castration-resistant prostate cancer SOD2 ↓

FIG. 2 Changes in antioxidant enzymes during prostate carcinogenesis, prostate cancer progression, and castration resistance. Dysregulated antioxidant enzymes in prostate cancer reciprocally interact with oxidative stress.

Oxidative stress

Noncancerous prostate

Tissue injury Genomic alterations

Primary prostate cancer

Protein and lipid oxidations Signal-transduction pathway PI3K/Akt, MAPK AP-1

Metastatic prostate cancer

FIG. 3 Mechanisms of prostate carcinogenesis and prostate cancer progression mediated by oxidative stress. Tissue injury and signal transduction pathways induced by oxidative stress accelerate both prostate carcinogenesis and prostate cancer progression.

Thus, as shown in Fig. 3, a decrease in antioxidant enzymes, including SOD, catalase, and GSTP1, may lead to elevated levels of oxidative stress in cancers, whereas augmented oxidative stress may induce a compensating increase in antioxidant enzymes. However, SOD2 levels are reduced in castration-resistant prostate cancer,17 indicating that the alterations in antioxidant enzyme activities are complex.

Altered transcriptional factors related to the redox balance Transcription factors are also involved in the antioxidant system, including Nrf2, NF-kB, and AP-1, which are modulated by the redox status and inversely control the redox balance through regulation of their downstream genes.18 Nrf2 regulates the expression of phase II key protective enzymes through the antioxidant-response element.19 Recent studies have indicated that Nrf2 and several of its target genes are significantly downregulated in human prostate cancer, resulting in continuous exposure to increased oxidative stress.20 NF-kB and AP-1 are induced by oxidative stress and regulate the expression of genes involved in several processes including cell proliferation, antiapoptosis mechanisms,

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invasion, metastasis, and angiogenesis.21 Both NF-kB and AP-1 are thought to be constitutively activated and contribute to malignant transformation and progression as well as castration resistance in prostate cancer.21 Conversely, suppression of NF-kB and AP-1 reduces the invasive and metastatic properties of prostate cancer.22,23 These findings suggest that increased oxidative stress resulting from Nrf2 suppression may contribute to upregulation of proto-oncogenic transcription factors NF-kB and AP-1.

Role of oxidative stress in the pathogenesis of prostate cancer Although excessive physiological levels of oxidative stress are toxic, physiological levels of oxidative stress regulate many cellular functions, such as cell proliferation, cell cycle progression, invasion, metastasis, and angiogenesis, possibly resulting in prostate carcinogenesis and cancer progression through oxidative modifications of cellular components. Signaling induced by oxidation can activate signal transduction molecules, such as PI3K/Akt and MAPK, and elevate the transcriptional activity of Nrf2, NF-kB, and AP-1 via phosphorylation of Jun and dissociation of Nrf2 and NF-kB from inhibitory protein complexes.18 Low doses of hydrogen peroxide have been demonstrated to stimulate prostate cancer cell growth and migration.24 In contrast, oxidative stress has been shown to be involved in the inactivation of several important proteins including those involved in DNA repair, apoptosis, cell signaling, and essential enzymatic pathways.25 Thus, oxidative stress is thought to contribute to the pathogenesis of prostate cancer through oncogenic and antiapoptotic pathways.

Oxidative stress in carcinogenesis and cancer progression Increased oxidative damage has been shown to correlate with tumor progression in in vitro and in vivo models of prostate tumorigenesis.26,27 Among them, Kumar et al. clearly demonstrated higher production of ROS in prostate cancer cell lines compared with benign prostate cancer cell lines.10 Furthermore, in Nkx3.1/Pten knockout mice, DNA and proteins were increasingly damaged by oxidation during tumor progression.27 Similarly, Tam et al. demonstrated increased ROS-induced damage in the prostate gland of TRAMP mice during tumorigenesis.26 Intriguingly, it has been recently reported that periprostatic adipose tissue promotes prostate cancer cell invasion through intracellular ROS induced by Nox5 in prostate cancer cells.28 Thus, in addition to intrinsic stimuli, exogenous insults from the environment can drive prostate cancer progression through increased oxidative stress. In humans, numerous studies have shown a close relationship between oxidative stress in prostate carcinogenesis and prostate cancer development. Oxidative stress represented by urinary F2-isoprostane levels was increased in high-grade PIN and prostate cancer.29 Conversely, serum levels of the antioxidant a-tocopherol were decreased in prostate cancer compared with a control group. Intriguingly, most patients with metastatic prostate cancer received androgen-deprivation therapy, suggesting that such therapy may affect serum antioxidant and oxidative stress levels.30 Similarly, oxidative stress represented by the ratio of 8-hydroxydeoxyguanosine (8-OHdG) to creatinine in urine was increased in prostate cancer compared with age-matched healthy controls.31 Additionally, peroxide levels and the total equivalent antioxidant capacity in serum were increased and decreased in prostate cancer and nonprostate disease patients, respectively.32 Changes in the redox state have been demonstrated along with the progression of human prostate cancer. High levels of 8-OHdG were detected in primary prostate cancer compared with the normal prostate epithelium, whereas metastatic prostate cancer showed higher levels of ROS-induced damage products (4-hydroxy-2-nonenal-modified proteins and 8-OHdG) than either primary cancer or normal prostate epithelium.33 In addition, higher oxidative stress levels have been detected in aggressive prostate cancer cells compared with normal prostatic epithelium-derived cells and localized prostate cancer cells.34 Matrix metalloproteinases, which are known invasion/metastasis promoters, are activated by ROS, and attenuated by ROS production blockade through Nox inhibition and extracellular SOD overexpression in prostate cancer cells. These findings indicate that matrix metalloproteinases may mediate prostate cancer progression through increased oxidative stress.10 Oxidative stress also promotes angiogenesis in cancer cells.2 The ROS inducer Nox consistently induced angiogenesis in prostate cancer,35 whereas heme oxygenase 1, which counteracts oxidative stress, suppressed angiogenesis,36 indicating that oxidative stress regulates cancer progression through induction of angiogenesis. Recently, a systemic review of oxidative stress in prostate cancer patients summarized numerous data and reported the consensus and controversies regarding the findings.37 As summarized, consistently in most studies, oxidative biomarkers, including DNA damage (8-OHdG), lipid peroxidation (malondialdehyde, microsomal membrane, and isoprostanes), and protein peroxidation (NO2 /NO3 /iNOS/cGMP, carbonylation, and glycation), are increased, while antioxidant indicators, such as endogenous antioxidants (catalase and glutathione peroxidase) and exogenous antioxidants (vitamin A, vitamin C, and total antioxidants), are decreased.37 However, there are controversial results in terms of glutathione, glutathione reductase, glutathione S-transferase, SOD, and vitamin E.37

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Oxidative stress in castration resistance Despite the initial success of androgen-deprivation therapy to suppress advanced prostate cancer, castration-resistant cancer eventually develops with critical consequences. Oxidative stress is implicated in castration resistance of prostate cancer.38 It aberrantly activates androgen receptor (AR) signaling under low androgen levels through various pathways, including AR overexpression, AR cofactors, and signal transduction pathways, as indicated in Fig. 4,38 thereby promoting castration resistance. Hydrogen peroxide-resistant derivatives of androgen-dependent prostate cancer cells express high levels of AR and exhibit a castration-resistant phenotype.39 In addition, several lines of evidence have shown that oxidative stress is increased in castration-resistant cells, as indicated by higher intracellular ROS levels in castration-resistant cells compared with androgen-dependent cells40 as well as higher antioxidant protein levels and an increased ability to scavenge ROS in castration-resistant cells and tumors.38,41 Thus, AR activation by oxidative stress is thought to render prostate cancer cells resistant to castration. Intriguingly, a single-nucleotide polymorphism (SNP) in GSTM3 encoding glutathione S-transferase m is associated with sensitivity to androgen-deprivation therapy and prognosis, suggesting that antioxidants play a key role in castration resistance and progression of prostate cancer.42

Oxidative stress in resistance to other therapeutics In addition to androgen-deprivation therapy, several therapeutics, such as radiotherapy including novel radioisotope radium-223, taxane chemotherapy such as docetaxel and cabazitaxel, and novel AR axis-targeting agents such as abiraterone, apalutamide, and enzalutamide, have expanded their roles in prostate cancer treatment and emerged as novel agents for prostate cancer.43 Although most of these modalities are different from androgen-deprivation therapy in mode of action, oxidative stress is involved in the resistance to these therapies similarly to castration resistance. Irradiation and chemotherapeutic agents such as taxanes are well-known inducers of oxidative stress under various conditions, where induced oxidative stress, in part, exerts cytotoxic effects on prostate cancer cells and noncancer cells. Moreover, oxidative stress induced by cytotoxic modalities, in turn, induces a variety of antioxidative and antiapoptotic signal transductions, resulting in cellular resistance to cytotoxic therapies.44 In addition, the activity of AR itself is closely associated with cellular resistance to irradiation and taxanes through its prosurvival and antiapoptotic properties.45,46 Oxidative stress

Up regulation of AR expression Up regulation of AR cofactor Activated signal-transduction pathway

Augmented AR signaling

Prostate carcinogenesis Prostate cancer progression Castration resistance Resistance to other treatments

FIG. 4 Mechanism of acquired castration resistance by oxidative stress through augmented AR signaling. Oxidative stress leads to aberrant AR signaling, resulting in prostate carcinogenesis, prostate cancer progression, and oxidative stress.

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Intriguingly, novel AR axis-targeting agents have been suggested to induce oxidative stress in prostate cancer cells. Oxidative stress was increased after resistance to novel antiandrogen enzalutamide,42 suggesting a similar relationship to oxidative stress between androgen-deprivation therapy and AR axis-targeting agents. Taken together, these findings suggest that oxidative stress induced by therapeutic cytotoxicity and tumor-suppressive interventions for prostate cancer cells is a double-edged sword, where oxidative stress exerts antisurvival and proapoptotic effects, but augments antiapoptotic and aggressive phenotypes in prostate cancer cells, in contrast to therapeutic intent, indicating the importance of regulating oxidative stress during various cancer therapeutics.

Prostate cancer risk factors and their links to oxidative stress Several definitive prostate cancer risk factors have been identified, including age, race, and family history, as well as other possible risk factors including androgens, inflammation, diet, and lifestyle. These risk factors may result in increased oxidative stress, as shown in Fig. 5. To understand the relationship between oxidative stress and prostate cancer, this section reviews the possible links between risk factors affecting prostate cancer and oxidative stress.

Aging The incidence of prostate cancer increases with age. Specifically, the incidence of prostate cancer is low in men younger than 40, but it becomes common in men older than 80. Several studies have suggested that 42%–80% of men will develop prostate cancer after 80 years of age.47 Exposure to oxidative stress generated endogenously by the by-products of normal metabolic processes and exogenously by environmental exposure to toxic substances has been thought to increase with aging. Indeed, progressive accumulation of DNA strand breaks, DNA adducts, and oxidative modification of enzymes for DNA repair have been documented to increase, while antioxidant defenses including ROS detoxification enzyme activities decrease with age. Thus, increased oxidative stress associated with aging is thought to contribute to the pathogenesis of prostate cancer.

Genetic background (race and family history) African American and Caucasian men are at high risk for clinically relevant prostate cancer compared with Asian men. For example, Japanese men living or born in the United States have a lower risk of prostate cancer than African and Caucasian men in the United States,48 indicating a racial difference in prostate cancer development. Furthermore, the response to androgen-deprivation therapy is better in Japanese men compared with American men,49 suggesting that the racial difference affects not only prostate carcinogenesis but also castration resistance. A family history of prostate cancer is also a well-known risk factor.50 Definitive risk factors

Aging

Genetic background

Possible risk factors

Androgens

Inflammation

Diet

Lifestyle

Oxidative stress

Prostate cancer FIG. 5 Relationship among risk factors affecting prostate cancer and oxidative stress. Definitive and possible risk factors comorbid with prostate cancer are associated with oxidative stress.

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Genetic background also contributes to the differential incidence rates of prostate cancer among races and families, even after excluding the effects of exogenous factors. Several genetic variations have been reported to influence prostate cancer incidence. Associations between prostate cancer and the Ala16Val SNP in the SOD2 gene have been reported. A metaanalysis of 34 studies examining the association between the SOD2 Ala16Val polymorphism and prostate cancer found a significant association between Ala/Ala and prostate cancer compared with Val/Val.51 In addition, SNPs in the gene encoding the high-density lipoprotein-associated enzyme paraoxonase 1, which decreases oxidative stress, have been reportedly associated with an increased risk of aggressive prostate cancer. This result suggests that increased oxidative stress by altered paraoxonase 1 activity promotes the aggressiveness of prostate cancer.52 In addition, a SNP in the catalase gene has been correlated with an increased risk of prostate cancer diagnosed under the age of 65.53 Similarly, correlations between prostate cancer incidence and SNPs in xenobiotic metabolic enzyme genes, including glutamate-cysteine ligase, thioredoxin reductase 2, microsomal epoxide hydrolase 1, and myeloperoxidase, have been observed.54

Androgens The importance of androgens in prostate carcinogenesis has been suggested by classical observations of young castrated men and men deficient for 5a-reductase that converts testosterone to the more potent dihydrotestosterone. Recent preventive studies (PCPT and REDUCE trials using 5a-reductase inhibitors finasteride and dutasteride, respectively) also showed that 5a-reductase inhibitors reduce the total prostate cancer detection rate among men with high prostate-specific antigen levels.55,56 Furthermore, the growth and progression of most prostate cancers are dependent on androgen, as indicated by the initial remarkable therapeutic effect of androgen-deprivation therapy.57 Androgens have been implicated in oxidative stress. ROS levels are increased by androgens via the transcription factor JunD and the mitochondrial redox regulator p66Shc.58,59 However, castration clearly induces an oxidative state in the rat prostate, whereas testosterone replacement in castrated rats reduces oxidative stress levels by downregulating Nox expression and recovering antioxidants.9 Similarly, androgen deprivation induces oxidative stress in prostate cancer cells and human prostate cancer tissues.32,60 Intriguingly, oxidative stress levels are decreased at physiological androgen levels that stimulate cell growth, whereas oxidative stress levels dramatically increase at higher nonphysiological doses that inhibit cell growth.61 Taken together, these reports suggest that a nonphysiological androgen milieu involving either androgen deprivation or overload induces oxidative stress in the prostate, as shown in Fig. 6.

Inflammation ROS/RNS production by activated inflammatory cells and/or secreted inflammatory cytokines is thought to cause cellular injury and accumulation of genomic damage, leading to prostate carcinogenesis, prostate cancer progression, and castration resistance.25,62 This is supported by the fact that several inflammatory conditions in humans are accompanied by increased levels of oxidative DNA damage.63 Conversely, oxidative stress itself can induce inflammation, indicating a close mutual relationship between oxidative stress and inflammation.64 As a molecular mechanism, signal transducer and activator of transcription 3 (STAT3) and NF-kB, which are master regulators of inflammation, and their related molecules, such as interleukin-6 and interleukin-8, play critical roles in prostate cancer pathogenesis in relation to inflammation. Both STAT3 and NF-kB are well known to promote carcinogenesis and cancer progression in various cancers. Moreover, intriguingly, both are transcription factors that induce AR expression in prostate cancer cells, where castration resistance occurs.65

Oxidative stress

Androgens Starved level

Physiological level

Excessive level

FIG. 6 Relationship between the androgen level and oxidative stress level. Low and excessive levels of androgens are associated with increased oxidative stress levels.

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Chronic inflammation and proliferative inflammatory atrophy (PIA) are thought to be the first step in prostate carcinogenesis, as suggested by the pathological observations that chronic inflammation and PIA accompany high-grade PIN.62 Epidemiologically, a prior history of certain sexually transmitted infections or prostatitis increases the relative risk of prostate cancer,66 whereas the use of NSAIDs reduces prostate cancer risk, as described above. Thus, several lines of evidence suggest that inflammation may be closely implicated in prostate cancer by promoting oxidative damage.

Diet Japanese men who emigrated to Hawaii had a higher rate of prostate cancer than those living in Japan.48 This observation was supported by another report showing that Japanese men born in the United States had higher incidence rates of prostate cancer than Japanese immigrants born in Japan.67 In addition, epidemiological data regarding the ethnic and geographic variations in prostate cancer incidence and mortality suggest that dietary factors may influence prostate carcinogenesis and prostate cancer progression. The idea that high calorie and high-fat diets may affect prostate carcinogenesis is supported by the finding that these diets, which are thought to increase ROS/RNS production through metabolic processes, promote prostate cancer proliferation in mice. In contrast, vegetables rich in antioxidants are thought to protect against prostate cancer, and several antioxidants, including green tea, isoflavones, lycopene, vitamin E, and resveratrol, are all considered to help prevent prostate carcinogenesis.68

Lifestyle Similar to dietary factors, lifestyle may also affect the incidence of prostate cancer. Smoking is a well-known risk factor for lung, head and neck, and bladder cancers. Although the contribution of smoking to prostate cancer is obscure, the smoking rate was reportedly higher in men with advanced and critical prostate cancer,69 suggesting that it may contribute to prostate cancer progression. In addition, a metaanalysis of 24 cohort studies showed an increased incidence of prostate cancer among heavy smokers.70 Physiological activity and obesity, which can be linked to oxidative stress, have also been suggested to influence prostate cancer incidence.71

Conclusions Increased production of ROS by altered mitochondrial bioenergetics, upregulation of Nox, xanthine oxidase, and COX, hypoxia, and/or impairment of antioxidant defenses result in a redox imbalance and oxidative stress. Physiological levels of ROS promote a broad range of cellular processes, such as cell proliferation, invasion, metastasis, and angiogenesis, through the accumulation of genetic and epigenetic damage, as well as by activating proto-oncogenic signaling (PI3K/ Akt and MAPK) and proto-oncogenic transcription factors (NF-kB, AP-1, and AR). Some factors, including age, race, family history, androgens, inflammation, diet, and lifestyle, are associated with oxidative stress and involved in prostate cancer incidence and progression as well as castration resistance. Oxidative stress is thus implicated in prostate carcinogenesis, prostate cancer progression, and cellular resistance to various therapeutics including androgen-deprivation therapy. However, the current levels of knowledge are insufficient to ascertain the precise role of oxidative stress in the pathogenesis of prostate cancer. Therefore, further studies are needed to elucidate the implications of oxidative stress in prostate cancer.

Applications to other cancers or conditions In this chapter, I reviewed the effects of oxidative stress on prostate cancer pathogenesis. Here, the close association between oxidative stress and prostate cancer pathogenesis including carcinogenesis, cancer progression, and treatment resistance was described, where a dysregulated balance of prooxidant and antioxidant properties in prostate cancer were observed. Oxidative stress plays key roles in a variety of cancers including breast, lung, gastrointestinal, gynecological, and cutaneous cancers. Similar to prostate cancer, in various cancers, DNA damage in oncogenes and tumor-suppressor genes is a critical step to carcinogenesis, which is accelerated by epigenetic alterations induced by oxidative stress. Furthermore, in the progression processes, such as angiogenesis, invasion, and metastasis, oxidative stress plays crucial roles in various cancers as described in other chapters. Androgen-deprivation therapy for prostate cancer is a backbone therapy, where oxidative stress mediates castration resistance. Because hormone therapy is frequently employed for hormone receptorpositive breast cancer, this knowledge regarding prostate cancer may be applicable to breast cancer. Furthermore, classically, radiotherapy and chemotherapy in addition to surgical treatment have been the main treatments for various cancers

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for several decades. According to the data obtained from prostate cancer, oxidative stress is thought to play an important role in the effect and toxicity by such cytotoxic therapies as well as treatment resistance. Molecular targeted agents that inhibit angiogenesis and oncogenic signaling, and immune checkpoint inhibitors targeting PD-1/PD-L1 and CTLA-4 have been developed and applied as anticancer therapies to various cancers. However, the role of oxidative stress in the effects of these novel agents has not fully been explored yet, which should be investigated in the future.

Summary points l

l l

l

l

ROS production by altered mitochondrial bioenergetics, upregulation of NADPH oxidase, cyclooxygenase, and hypoxia is increased in prostate cancer. Antioxidant enzymes and their regulating transcription factors are deregulated in prostate cancer. Oxidative stress caused by redox imbalances between prooxidants and antioxidants contributes to prostate carcinogenesis and prostate cancer progression. Oxidative stress promotes castration resistance through aberrant AR activation and resistance to other therapeutics in prostate cancer. Definitive and possible risk factors affecting prostate cancer are linked to oxidative stress, implicating these factors in the pathogenesis of prostate cancer through oxidative stress.

References 1. Sauer H, Wartenberg M, Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 2001;11:173–86. 2. Galanis A, Pappa A, Giannakakis A, Lanitis E, Dangaj D, Sandaltzopoulos R. Reactive oxygen species and HIF-1 signalling in cancer. Cancer Lett 2008;266:12–20. 3. Koskenkorva-Frank TS, Weiss G, Koppenol WH, Burckhardt S. The complex interplay of iron metabolism, reactive oxygen species, and reactive nitrogen species: insights into the potential of various iron therapies to induce oxidative and nitrosative stress. Free Radic Biol Med 2013;65:1174–94. 4. Oberley TD. Oxidative damage and cancer. Am J Pathol 2002;160:403–8. 5. Petros JA, Baumann AK, Ruiz-Pesini E, Amin MB, Sun CQ, Hall J, et al. mtDNA mutations increase tumorigenicity in prostate cancer. Proc Natl Acad Sci U S A 2005;102:719–24. 6. Yu JJ, Yan T. Effect of mtDNA mutation on tumor malignant degree in patients with prostate cancer. Aging Male 2010;13:159–65. 7. Chowdhury SK, Raha S, Tarnopolsky MA, Singh G. Increased expression of mitochondrial glycerophosphate dehydrogenase and antioxidant enzymes in prostate cancer cell lines/cancer. Free Radic Res 2007;41:1116–24. 8. Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 2007;87:245–313. 9. Tam NN, Gao Y, Leung YK, Ho SM. Androgenic regulation of oxidative stress in the rat prostate: involvement of NAD(P)H oxidases and antioxidant defense machinery during prostatic involution and regrowth. Am J Pathol 2003;163:2513–22. 10. Kumar B, Koul S, Khandrika L, Meacham RB, Koul HK. Oxidative stress is inherent in prostate cancer cells and is required for aggressive phenotype. Cancer Res 2008;68:1777–85. 11. Shappell SB, Olson SJ, Hannah SE, Manning S, Roberts RL, Masumori N, et al. Elevated expression of 12/15-lipoxygenase and cyclooxygenase-2 in a transgenic mouse model of prostate carcinoma. Cancer Res 2003;63:2256–67. 12. Kirschenbaum A, Liu X, Yao S, Levine AC. The role of cyclooxygenase-2 in prostate cancer. Urology 2001;58:127–31. 13. Harris RE. Cyclooxygenase-2 (cox-2) blockade in the chemoprevention of cancers of the colon, breast, prostate, and lung. Inflammopharmacology 2009;17:55–67. 14. Baker AM, Oberley LW, Cohen MB. Expression of antioxidant enzymes in human prostatic adenocarcinoma. Prostate 1997;32:229–33. 15. Bostwick DG, Alexander EE, Singh R, Shan A, Qian J, Santella RM, et al. Antioxidant enzyme expression and reactive oxygen species damage in prostatic intraepithelial neoplasia and cancer. Cancer 2000;89:123–34. 16. Li LC. Epigenetics of prostate cancer. Front Biosci 2007;12:3377–97. 17. Sharifi N, Hurt EM, Thomas SB, Farrar WL. Effects of manganese superoxide dismutase silencing on androgen receptor function and gene regulation: implications for castration-resistant prostate cancer. Clin Cancer Res 2008;14:6073–80. 18. Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med 2010;49:1603–16. 19. Taguchi K, Motohashi H, Yamamoto M. Molecular mechanisms of the Keap1–Nrf2 pathway in stress response and cancer evolution. Genes Cells 2011;16:123–40. 20. Frohlich DA, McCabe MT, Arnold RS, Day ML. The role of Nrf2 in increased reactive oxygen species and DNA damage in prostate tumorigenesis. Oncogene 2008;27:4353–62. 21. Karin M, Takahashi T, Kapahi P, Delhase M, Chen Y, Makris C, et al. Oxidative stress and gene expression: the AP-1 and NF-kappaB connections. Biofactors 2001;15:87–9.

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22. Huang S, Pettaway CA, Uehara H, Bucana CD, Fidler IJ. Blockade of NF-kappaB activity in human prostate cancer cells is associated with suppression of angiogenesis, invasion, and metastasis. Oncogene 2001;20:4188–97. 23. Ozanne BW, McGarry L, Spence HJ, Johnston I, Winnie J, Meagher L, et al. Transcriptional regulation of cell invasion: AP-1 regulation of a multigenic invasion programme. Eur J Cancer 2000;36:1640–8. 24. Polytarchou C, Hatziapostolou M, Papadimitriou E. Hydrogen peroxide stimulates proliferation and migration of human prostate cancer cells through activation of activator protein-1 and up-regulation of the heparin affin regulatory peptide gene. J Biol Chem 2005;280:40428–35. 25. Palapattu GS, Sutcliffe S, Bastian PJ, Platz EA, De Marzo AM, Isaacs WB, et al. Prostate carcinogenesis and inflammation: emerging insights. Carcinogenesis 2005;26:1170–81. 26. Tam NN, Nyska A, Maronpot RR, Kissling G, Lomnitski L, Suttie A, et al. Differential attenuation of oxidative/nitrosative injuries in early prostatic neoplastic lesions in TRAMP mice by dietary antioxidants. Prostate 2006;66:57–69. 27. Ouyang X, DeWeese TL, Nelson WG, Abate-Shen C. Loss-of-function of Nkx3.1 promotes increased oxidative damage in prostate carcinogenesis. Cancer Res 2005;65:6773–9. 28. Laurent V, Toulet A, Attan
e C, Milhas D, Dauvillier S, Zaidi F, et al. Periprostatic adipose tissue favors prostate cancer cell invasion in an obesitydependent manner: role of oxidative stress. Mol Cancer Res 2019;17:821–35. 29. Barocas DA, Motley S, Cookson MS, Chang SS, Penson DF, Dai Q, et al. Oxidative stress measured by urine F2-isoprostane level is associated with prostate cancer. J Urol 2011;185:2102–7. 30. Yossepowitch O, Pinchuk I, Gur U, Neumann A, Lichtenberg D, Baniel J. Advanced but not localized prostate cancer is associated with increased oxidative stress. J Urol 2007;178:1238–43. 31. Miyake H, Hara I, Kamidono S, Eto H. Oxidative DNA damage in patients with prostate cancer and its response to treatment. J Urol 2004;171:1533–6. 32. Pace G, Di Massimo C, De Amicis D, Corbacelli C, Di Renzo L, Vicentini C, et al. Oxidative stress in benign prostatic hyperplasia and prostate cancer. Urol Int 2010;85:328–33. 33. Oberley TD, Zhong W, Szweda LI, Oberley LW. Localization of antioxidant enzymes and oxidative damage products in normal and malignant prostate epithelium. Prostate 2000;44:144–55. 34. Freitas M, Baldeiras I, Proenc¸a T, Alves V, Mota-Pinto A, Sarmento-Ribeiro A. Oxidative stress adaptation in aggressive prostate cancer may be counteracted by the reduction of glutathione reductase. FEBS Open Bio 2012;2:119–28. 35. Kim J, Koyanagi T, Mochly-Rosen D. PKCd activation mediates angiogenesis via NADPH oxidase activity in PC-3 prostate cancer cells. Prostate 2011;71:946–54. 36. Ferrando M, Gueron G, Elguero B, Giudice J, Salles A, Leskow FC, et al. Heme oxygenase 1 (HO-1) challenges the angiogenic switch in prostate cancer. Angiogenesis 2011;14:467–79. 37. Oh B, Figtree G, Costa D, Eade T, Hruby G, Lim S, et al. Oxidative stress in prostate cancer patients: a systematic review of case control studies. Prostate Int 2016;4:71–87. 38. Shiota M, Yokomizo A, Naito S. Oxidative stress and androgen receptor signaling in the development and progression of castration-resistant prostate cancer. Free Radic Biol Med 2011;51:1320–8. 39. Shiota M, Yokomizo A, Tada Y, Inokuchi J, Kashiwagi E, Masubuchi D, et al. Castration resistance of prostate cancer cells caused by castrationinduced oxidative stress through Twist1 and androgen receptor overexpression. Oncogene 2010;29:237–50. 40. Shigemura K, Sung SY, Kubo H, Arnold RS, Fujisawa M, Gotoh A, et al. Reactive oxygen species mediate androgen receptor- and serum starvationelicited downstream signaling of ADAM9 expression in human prostate cancer cells. Prostate 2007;67:722–31. 41. Kuruma H, Egawa S, Oh-Ishi M, Kodera Y, Satoh M, Chen W, et al. High molecular mass proteome of androgen-independent prostate cancer. Proteomics 2005;5:1097–112. 42. Shiota M, Fujimoto N, Itsumi M, Takeuchi A, Inokuchi J, Tatsugami K, et al. Gene polymorphisms in antioxidant enzymes correlate with the efficacy of androgen-deprivation therapy for prostate cancer with implications of oxidative stress. Ann Oncol 2017;28:569–75. 43. Fujimoto N. Novel agents for castration-resistant prostate cancer: early experience and beyond. Int J Urol 2016;23:114–21. 44. Shiota M, Yokomizo A, Naito S. Pro-survival and anti-apoptotic properties of androgen receptor signaling by oxidative stress promote treatment resistance in prostate cancer. Endocr Relat Cancer 2012;19:R243–53. 45. Chou FJ, Chen Y, Chen D, Niu Y, Li G, Keng P, et al. Preclinical study using androgen receptor (AR) degradation enhancer to increase radiotherapy efficacy via targeting radiation-increased AR to better suppress prostate cancer progression. EBioMedicine 2019;40:504–16. 46. Shiota M, Dejima T, Yamamoto Y, Takeuchi A, Imada K, Kashiwagi E, et al. Collateral resistance to taxanes in enzalutamide-resistant prostate cancer through aberrant androgen receptor and its variants. Cancer Sci 2018;109:3224–34. 47. Minelli A, Bellezza I, Conte C, Culig Z. Oxidative stress-related aging: a role for prostate cancer? Biochim Biophys Acta 2009;1795:83–91. 48. Hirayama T, editor. Comparative epidemiology of cancer in the U.S. and Japan—Mobidity. Japan Society for the Promotion of Science; 1978. p. 43–62. 49. Fukagai T, Namiki TS, Carlile RG, Yoshida H, Namiki M. Comparison of the clinical outcome after hormonal therapy for prostate cancer between Japanese and Caucasian men. BJU Int 2006;97:1190–3. 50. Madersbacher S, Alcaraz A, Emberton M, Hammerer P, Ponholzer A, Schr€oder FH, et al. The influence of family history on prostate cancer risk: implications for clinical management. BJU Int 2011;107:716–21. 51. Wang S, Wang F, Shi X, Dai J, Peng Y, Guo X, et al. Association between manganese superoxide dismutase (MnSOD) Val-9Ala polymorphism and cancer risk—a meta-analysis. Eur J Cancer 2009;45:2874–81. 52. Stevens VL, Rodriguez C, Talbot JT, Pavluck AL, Thun MJ, Calle EE. Paraoxonase 1 (PON1) polymorphisms and prostate cancer in the CPS-II nutrition cohort. Prostate 2008;68:1336–40.

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53. Choi JY, Neuhouser ML, Barnett M, Hudson M, Kristal AR, Thornquist M, et al. Polymorphisms in oxidative stress-related genes are not associated with prostate cancer risk in heavy smokers. Cancer Epidemiol Biomarkers Prev 2007;16:1115–20. 54. Koutros S, Andreotti G, Berndt SI, Hughes Barry K, Lubin JH, Hoppin JA, et al. Xenobiotic-metabolizing gene variants, pesticide use, and the risk of prostate cancer. Pharmacogenet Genomics 2011;21:615–23. 55. Thompson IM, Goodman PJ, Tangen CM, Lucia MS, Miller GJ, Ford LG, et al. The influence of finasteride on the development of prostate cancer. N Engl J Med 2003;349:215–24. 56. Andriole GL, Bostwick DG, Brawley OW, Gomella LG, Marberger M, Montorsi F, et al. Effect of dutasteride on the risk of prostate cancer. N Engl J Med 2010;362:1192–202. 57. Miyamoto H, Messing EM, Chang C. Androgen deprivation therapy for prostate cancer: current status and future prospects. Prostate 2004;61:332–53. 58. Mehraein-Ghomi F, Lee E, Church DR, Thompson TA, Basu HS, Wilding G. JunD mediates androgen-induced oxidative stress in androgen dependent LNCaP human prostate cancer cells. Prostate 2008;68:924–34. 59. Veeramani S, Yuan TC, Lin FF, Lin MF. Mitochondrial redox signaling by p66Shc is involved in regulating androgenic growth stimulation of human prostate cancer cells. Oncogene 2008;27:5057–68. 60. Shiota M, Song Y, Takeuchi A, Yokomizo A, Kashiwagi E, Kuroiwa K, et al. Antioxidant therapy alleviates oxidative stress by androgen deprivation and prevents conversion from androgen dependent to castration resistant prostate cancer. J Urol 2012;187:707–14. 61. Ripple MO, Henry WF, Rago RP, Wilding G. Prooxidant-antioxidant shift induced by androgen treatment of human prostate carcinoma cells. J Natl Cancer Inst 1997;89:40–8. 62. De Marzo AM, Platz EA, Sutcliffe S, Xu J, Gr€onberg H, Drake CG, et al. Inflammation in prostate carcinogenesis. Nat Rev Cancer 2007;7:256–69. 63. Shen Z, Wu W, Hazen SL. Activated leukocytes oxidatively damage DNA, RNA, and the nucleotide pool through halide-dependent formation of hydroxyl radical. Biochemistry 2000;39:5474–82. 64. Khurana N, Sikka SC. Targeting crosstalk between Nrf-2, NF-kB and androgen receptor signaling in prostate cancer. Cancer 2018;10:E352. 65. Shiota M, Yokomizo A, Naito S. Increased androgen receptor transcription: a cause of castration-resistant prostate cancer and a possible therapeutic target. J Mol Endocrinol 2011;47:R25–41. 66. Sutcliffe S. Sexually transmitted infections and risk of prostate cancer: review of historical and emerging hypotheses. Future Oncol 2010;6:1289–311. 67. Cook LS, Goldoft M, Schwartz SM, Weiss NS. Incidence of adenocarcinoma of the prostate in Asian immigrants to the United States and their descendants. J Urol 1999;161:152–5. 68. Hori S, Butler E, McLoughlin J. Prostate cancer and diet: food for thought? BJU Int 2011;107:1348–59. 69. Plaskon LA, Penson DF, Vaughan TL, Stanford JL. Cigarette smoking and risk of prostate cancer in middle-aged men. Cancer Epidemiol Biomarkers Prev 2003;12:604–9. 70. Huncharek M, Haddock KS, Reid R, Kupelnick B. Smoking as a risk factor for prostate cancer: a meta-analysis of 24 prospective cohort studies. Am J Public Health 2010;100:693–701. 71. Stein CJ, Colditz GA. Modifiable risk factors for cancer. Br J Cancer 2004;90:299–303.

Chapter 3

Oxidative stress in lung cancer Amir Mousapasandia,b, Wei Sheng Joshua Lokeb,c, Cristan A. Herbertd, and Paul S. Thomasb,c a

Prince of Wales’ Clinical School and Mechanisms of Disease and Translational Research, School of Medical Sciences, Faculty of Medicine, UNSW

Sydney, Sydney, NSW, Australia, b Department of Respiratory Medicine, Prince of Wales Hospital, Randwick, Sydney, NSW, Australia, c Prince of Wales’ Clinical School and Mechanisms of Disease and Translational Research, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia, d

Mechanisms of Disease and Translational Research, School of Medical Sciences, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia

List of abbreviations 8-OHdG ALK AP-1 COX-2 EGFR FOXO H2O2 HDL HIF1-a IARC IL-6 IL-8 iNOS KRAS LDCT miRNA mRNA NF-kB NNK NO NO2 NSCLC
O22 O3 OH2 ONOO2 PAH PD-L 1 PTEN
Q2
R RNS
RO
ROO ROS SCLC SO2 STAT3 TB

8-hydroxy-deoxyguanosine anaplastic lymphoma kinase activator protein 1 cyclooxygenase-2 epidermal growth factor receptor Forkhead box hydrogen peroxide high-density lipoprotein hypoxia-inducible factor-1a International Agency for Research on Cancer interleukin-6 interleukin-8 inducible nitric oxide synthase kirsten rat sarcoma low-dose computed tomography microRNA messenger RNA nuclear factor kappa B nicotine-derived nitrosamine ketone nitric oxide nitrogen dioxide non-small cell lung cancer superoxide ozone hydroxyl radicals peroxynitrite polycyclic aromatic hydrocarbons Programmed death-ligand 1 phosphatase and tensin homolog semiquinone carbon-centered radials reactive nitrogen species alkoxyl radicals peroxyl radicals reactive oxygen species small cell lung cancer sulfur dioxide transducer and activator of transcription 3 Tuberculosis

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00003-1 © 2021 Elsevier Inc. All rights reserved.

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TC TNF-a TP53

A Oxidative stress and cancer

total cholesterol tumor necrosis factor a tumor protein 53

Introduction The lung, mucous membranes, and the skin are subjected to both exogenous and endogenous oxidants and reactive nitrogen species. Reactive oxygen species (ROS) may be derived from exposure to exogenous sources, such as cigarette smoke, but can also result from normal cellular respiration. ROS are highly reactive molecules that can initiate cellular damage and are free radicals, meaning that they possess reactive unpaired electrons.1a An adult inhales 10,000 L of ambient air daily, which can be contaminated with cigarette smoke, ionizing radiation, particulate matter, sulfur dioxide (SO2), nitrogen dioxide (NO2), and pathogens. When inhaled, these harmful materials promote the formation of reactive oxygen and nitrogen species resulting in the initiation of oxidation and nitrosylation, which cause a cascade of signaling events and lung injury.1b In addition to exogenous sources, ROS are formed endogenously as part of the reduction of oxygen to water in the mitochondrial electron transport chain during the generation of ATP, by cellular enzymes (e.g., P450 oxidase and xanthine oxidase) and via the cyclooxygenase pathway. The mitochondrial electron transport chain is the major site of ROS production in most mammalian cells.1c This review outlines oxidative stress in lung cancer, the role of reactive oxygen and nitrogen species in pulmonary carcinogenesis, and the potential for exogenous antioxidants to counteract excessive ROS.

Lung cancer Lung cancer is the most common cause of death from cancer and the most commonly diagnosed cancer among males in the world. In 2018, there were 2.1 million diagnosed cases and 1.8 million deaths globally, accounting for almost 1 in 5 (18.4%) of all cancer deaths. Lung cancer is often diagnosed at a late stage and hence has a poor prognosis with a global 5-year relative survival varying greatly from 2% in Libya to 30% in Japan.1 Despite this significant burden of disease and poor prognosis, and apart from tobacco and radon control, there are no population-based programs to reduce the incidence of lung cancer or to screen for early disease. Recently, the National Lung Screening Trial in the United States led to the recommendation of an annual low-dose computed tomography (LDCT) screening for people aged 55–80 with a 30-pack-year smoking history.2 Despite this recommendation, screening has been ineffective, as many eligible subjects have a lower socioeconomic status. As a result, LDCT lung cancer screening has been accessed by only 4% of the 6.8 million people in the United States who are eligible.3 The two main subtypes of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC has an incidence of approximately 15%, while NSCLC has an incidence of approximately 85%. This classification is significant as histological subtypes differ in their invasiveness, response to treatment, and prognosis, therefore interventions should be done in a tailored manner.4 In addition, subtypes of NSCLC are now recognized according to genotypic expression. Subtypes of NSCLC include those with mutations in the genes for EGFR, KRAS, and ALK, together with PDL expression, which further subtype these neoplasms with the ability to modify the treatment approaches.

Etiology of lung cancer Cigarette smoking is the greatest risk factor for lung cancer, with 80%–90% of cases being associated with smoking, although lung cancer among nonsmokers is now the seventh leading cause of cancer mortality. Thus, other factors that contribute to lung cancer have been identified such as diet, radon exposure, air pollution, silica, asbestos, ionizing radiation, infective agents, and genetic predisposition.5

Tobacco smoking Tobacco combustion produces many known carcinogens. Lung cancer risk varies with the duration, type, and intensity of tobacco consumption, resulting a relative risk in smokers compared to nonsmokers of 6.99 in females and 7.33 males.6

Oxidative stress in lung cancer Chapter

3 29

Some of the most significant carcinogens are the constituents of tar, the polycyclic aromatic hydrocarbons (PAH) such as benzo(a)pyrenes, and the nicotine-derived nitrosamine ketone(NNK) such as N-nitrosamine 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone, inorganic compounds such as chromium, cadmium, arsenic, radioactive polonium (Po210), and organic compounds such as butadiene.7

Air pollution Passive tobacco smoking is also associated with an increased risk of lung cancer with nicotine and other tobacco byproducts being identified in the urine of nonsmokers passively exposed to cigarette smoke.8 Furthermore, nonsmoking spouses of smokers have a 20%–30% higher chance of developing lung cancer.9 Air pollutants, including biomass smoke, vehicle exhausts, etc., have been linked to the incidence and mortality of lung cancer and epidemiological studies have identified an association between elevated levels of NO2, PM2.5, and PM10 and lung cancer.10

Infection and inflammation The human papilloma virus (types 16 and 18) are well-known carcinogens and are commonly present in lung tumor tissue.11 In addition, the damage caused by inflammation and infection is a contributor to carcinogenesis. Patients with old tuberculous (TB) lesions are predisposed to having epidermal growth factor receptor (EGFR) mutations in these neoplasms; particularly exon 19 deletions.12 TB and pneumonia confer an increased odds ratio of 1.76 and 1.43 for the development of lung cancer, respectively. Moreover, other respiratory conditions which are coupled with recurrent chronic inflammation, such as idiopathic pulmonary fibrosis, and also COPD, confer an elevated relative risk of 1.46–2.44 for the development of lung cancer based on the degree of COPD severity.13

Radon 222

Radon is a radioactive element created from the decay of uranium with dose-related carcinogenicity. Radon and its daughters can be adsorbed to airborne particulates and enter the lung tissue. Radon undergoes decay and the elements (its progeny and a-rays) produced from the decay bombard lung cells resulting in DNA damage and hence contribute to lung cancer over time.14 There is also a well-established synergistic effect between smoking and exposure to radon as more than 85% of deaths from radon exposure were smokers. This is highlighted by the risk of radon-induced lung cancer with exposure to 4 pCi/L and 10 pCi/L of radon being 6 and 18 per 1000 in nonsmokers, and 62 and 150 per 1000 in smokers.15

Genetics A positive family history for lung cancer has been linked to a 1.7-fold increase in the chance of having lung cancer, and 8% of lung cancers are estimated to occur as a result of a genetic predisposition.16, 17 To date, 21 genes have been found for which there is robust evidence linking them with lung cancer with some of these genes being found on chromosomes 5p15, 15q25-26, and 6q21.18

Tobacco smoking and oxidants Tobacco smoke is a complex amalgamation of more than 4700 chemical compounds which are dispersed in the tar and gas phases. The radicals present in these phases differ.19 Tobacco smoke in the tar phase consists of extremely high concentrations of radicals (1017 per gram). These radicals are stable and are largely organic. Semiquinone (Q ), for example, is held in the tar matrix. It reacts with oxygen to form superoxide (O2) (Eq. 1) which consequently dismutates to form hydrogen peroxide (H2O2) (Eq. 2).





Q
 + O2 ! Q + O2


(1)

2O2
 + 2H + ! O2 + H2 O2

(2)

Furthermore, the tar contains metal ions (e.g., iron) which, through the Fenton reaction, generate highly oxidizing hydroxyl radicals (OH) from hydrogen peroxide (Eq. 3). H2 O2 + FeðIIÞ ! HO
+ HO + FeðIIIÞ

(3)

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Hydroxyl radicals can also be formed as a result of the decomposition of peroxynitrite (ONOO), which is a product of a reaction between nitric oxide and superoxide (Eq. 4).20, 21 O2
 + NO ! ONOO ! HO
+ NO2

(4)

15

Tobacco smoke in the gas phase contains more than 10 radicals per inhalation. Contrary to stable radicals found in the tar phase, the organic radicals in the gas phase are transient (i.e., lifetimes of less than 1 s) reactive oxygen- and carboncentered radicals. They are quickly quenched by the respiratory tract lining fluid. It is a paradox that, in spite of their short lifetimes, high concentrations of radicals can be maintained and even increased in the gas phase for more than 10 min. This is because gas phase radicals exist in a steady state where they are continuously made and destroyed.21 It has been postulated that this steady state involves the slow oxidation of nitric oxide (NO) to nitrogen dioxide (NO2) (Eq. 5). 2NO + O2 ! 2NO2

(5)


Nitrogen dioxide then reacts with isoprene present in tobacco smoke to form carbon-centered radials (R ) (Eq. 6). Carbon radicals then react with oxygen, forming peroxyl radicals (ROO ) (Eq. 7). They react with nitric oxide to form alkoxyl radicals (RO ) and more nitrogen dioxide (Eq. 8).20





ð6Þ R
+ O2 ! ROO


(7)

ROO + NO ! RO + NO2

(8)







The oxidant burden placed on the lung by the abovementioned exogenously derived oxidants is further intensified in smokers who have higher numbers of alveolar macrophages (two to fourfold) and leukocytes (up to 10-fold). Moreover, compared to nonsmokers, alveolar macrophages and leukocytes from tobacco smokers spontaneously release increased amounts of superoxide and hydrogen peroxide thereby exacerbating the oxidative burden in the lung (Fig. 3).22 In addition, hydrogen peroxide has been detected in increased amounts in the exhaled breath condensate in those with lung cancer.23

Silica and oxidants Silica can be inhaled during hard-rock mining, sand-blasting, or grinding. There are different forms of silica (vitreous, crystalline, synthetic/mineral, amorphous/natural, and biogenic). Exposure to silica results in severe alveolar inflammation sustained by oxidants present in the alveolar space. Upon inhalation, silica reaches the alveolar space where it is phagocytosed by alveolar macrophages. Depending on the surface characteristics, the silica particles are either cleared from the lungs by the macrophages or activate macrophages at the molecular and cellular levels. This results in the release of ROS and RNS. Eventually the macrophages undergo apoptosis and release the silica particles. Subsequent ingestion-re-ingestion cycles result in the release of more cell-derived ROS and RNS. Additionally, silica particles can react directly with the alveolar and bronchiolar epithelium to form particle-derived ROS and RNS (Fig. 1). They damage the alveolar and bronchiolar epithelium and may react with cell-derived ROS and RNS to yield peroxynitrite (ONOO) from superoxide (O2) and nitric oxide (NO).24 Oxidants can be either bound to the silica surface (surface radical) or formed when silica is placed in aqueous suspensions. The former develop when silica is fractured or ground. When this occurs, the silicon-oxygen bonds are cleaved. Molecular oxygen then reacts at the sites of cleavage and produces several “surface-bound ROS” including SiO3, SiO2, and Si+ O2.25 Oxidants can also be generated when silica is suspended in aqueous solution. Iron ions that are located in the redox and coordinative positions on the silica surface and silicon-based surface radicals serve as two active centers for oxidant production. The iron centers yield HO radicals via the Fenton reaction (Eq. 9) or the Haber-Weiss cycle (Eqs. 10–13) when a reductant and trace amounts of iron are present.

















Fe2 + ! H2 O2 ! Fe3 + + OH + HO


(9)

Fe3 + ! reductant ! Fe2 + + reductant

(10)

Fe2 + + O2 ! Fe3 + + O2 


(11)





+ H2 O ! HO2 + OH or O2 + 2H + e ! H2 O2

(12)

2HO2
! H2 O2 ! O2 O2 
+ H2 O2 ! HO
+ OH + O2

(13)

O2







+

Oxidative stress in lung cancer Chapter

3 31

Silica and oxidants in the alveolus Clearance

Silica

Alveolar macrophage

Ingestionre-ingestion cycles “frustrated phagocytosis”

Apoptosis

Cell-derived ROS and RNS Particular-derived ROS and RNS

Epithelium FIG. 1 Silica and oxidants in the alveolus. The inhaled silica reaches the alveolus and is ingested by alveolar macrophages. Alveolar macrophage ingested silica is either cleared, or persists in the macrophage, resulting in a process called “frustrated phagocytosis” which releases ROS and RNS (cell-derived). The alveolar macrophages then undergo apoptosis and release the silica, perpetuating the abovementioned process. Inhaled silica can also cause the alveolar epithelium to release ROS and RNS (particular-derived). The ROS and RNS produced via these two sources increases the oxidative burden on the alveolar epithelium.

Moreover, hydroxyl radicals can also be formed when surface radicals (SiO , SiO2, SiO3, Si+-O2) come into contact with water (Eq. 14) or hydrogen peroxide (Eqs. 15 and 16).26











SiO
+ H2 O ! SiOH + HO


(14)

SiOO
+ H2 O2 ! SiOH + HO
+ O2

(15)

Si O2 + H2 O2 ! SiOH + HO + O2

(16)

+







Asbestos and oxidants Asbestos fibers may be inhaled during the mining, extraction, processing, and use of this fiber. It is now most commonly a problem for those in the construction industry, but previously was used widely in shipbuilding, boiler making, plumbing, roofing as well as insulation for heat and electricity. It remains a common problem and continues to be used in countries such as Russia, India, and those in Southeast Asia. The inhalation of asbestos results in the accumulation of macrophages in the alveolar space.27 The mechanisms underlying ROS production following asbestosis inhalation are similar to that of silica (see above). Alveolar macrophages engulf the asbestos fibers and undergo a process of “frustrated phagocytosis”.28 Post inhalation, the asbestos fiber acquires redox-active iron on its surface which encourages the development of extremely reactive hydroxyl radicals from hydrogen peroxide through Fenton-catalyzed Haber-Weiss reactions (Eq. 17). The iron can also catalyze the production of alkoxyl radical from organic hydroperoxides (Eq. 18).28 O2  + H2 O2 ! HO + HO
+ O2

(17)

Fe2 + + ROOH ! Fe3 + + RO + HO

(18)

Alkoxyl and hydroxyl radical production leads to ROS-induced perturbation of DNA structure and function which leads to pulmonary carcinogenesis.29

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MicroRNAs and oxidative stress Neoplastic cells acquire biological capabilities that enable tumor growth and metastatic dissemination known as “the hallmarks of cancer.” Some of these hallmarks include sustained proliferative signaling, inducing angiogenesis, resisting cell death, and evading growth suppressors.30 MicroRNAs (miRNAs) are noncoding, single-stranded RNAs composed of 18–25 nucleotides, which are able to change the function of various signaling pathways by altering posttranscriptional gene expression via messenger RNA (mRNA) or by affecting protein translation. MicroRNA-induced alteration of gene expression has been associated with all the hallmarks of carcinogenesis.31 miRNAs contribute to carcinogenesis by interacting with tumor suppressor genes or oncogenes. This functional modulation results in deletion, overexpression, and epigenetic alterations of miRNAs resulting in altered levels of the target protein with the potential for oncogenicity.32 For instance, in lung cancer cells, at least 11 miRNAs are able to directly target EGFR which in turn cause its activation and promote excessive proliferation and cell cycle progression.33Another example is that of miR-494 inducing angiogenesis by targeting the phosphatase and tensin homolog (PTEN).34 Since both oxidative stress and miRNAs are dysregulated in cancer, it is a logical premise to attempt to understand the link between those two. It has been shown that exposure to hydrogen peroxide (H2O2) leads to changes in miRNA, thereby showing that changes in ROS can alter miRNAs. This is evident in how some microRNAs, so-called ROSmirs, are modulated by oxidative stress to regulate expressions of various pathways in response to ROS.35 ROS also regulate the miRNAs through transcription factors (p53, NF-kB, c-jun, and FOXO), biogenesis enzymes (regulating RNase-dependent steps), and epigenetic modifications (methylation and histone acetylation). In addition to the regulation of miRNAs by ROS, miRNAs could also play an important role in redox homeostasis by modulating the genes responsible for the production/inhibition of ROS.36 Therefore, through this interconnected regulatory mechanism, ROS and miRNAs may impact carcinogenesis synergistically or antagonistically.

Radon and oxidative stress Substantial evidence exists that exposure to radon is the second leading cause of lung cancer. 222Radon is a radioactive gas which produces alpha particles through its decay. Alpha particles are known to induce cellular damage through chromosome aberrations, and chromatid exchanges. Higher levels of intracellular superoxide anions (O2
) and hydrogen peroxide (H2O2) are recognized to occur after cellular exposure to radon gas.37 8-Hydroxy-deoxyguanosine (8-OHdG) adducts are reliable biomarkers for oxidative stress induced by radon exposure and elevated levels 8-OHdG in lung, liver, and blood are found in animal models of radon-induced carcinogenesis. Moreover, 8-OHdG levels have been used to determine the extent of oxidative DNA damage in lung cancer models and are also elevated in human subjects with a high background of radon.38, 39

Inflammation and oxidative stress During inflammation, granulocytes and leukocytes are recruited to the location of injury where they release large amount of ROS due to an increased uptake of oxygen in a phenomenon known as the respiratory burst. These inflammatory cells recruit additional cells to the damaged site via the release of chemokines and cytokines which further augments the oxidative stress in the area of inflammation. The expression of ROS by inflammatory mediators activate a cascade of signal transduction altering transcription factors such as nuclear factor kappa B (NF-kB), hypoxia-inducible factor-1a (HIF1-a), and signal transducer and activator of transcription 3 (STAT3) and induce enzymatic activation of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS). This causes an aberrant inflammatory response. Transcription factors NF-kB and activator protein 1 (AP-1) are responsible for the gene transcription of downstream inflammatory cytokines such as interleukin-8 (IL-8), tumor necrosis factor a (TNF-a), and interleukin-6 (IL-6) which attract more inflammatory cells such as alveolar macrophages, neutrophils, and eosinophils to generate an inflammatory cascade.40, 41 The recruited leukocytes in turn eliminate pathogens by producing oxidants as mentioned, including superoxide, nitric oxide, hydrogen peroxide, hydroxyl radical, peroxynitrite, and hypochlorous acid. The oxidants can inactivate pathogens via halogenation or protein or lipid peroxidation. Following the destruction and removal of these foreign pathogens, inflammation resolves.42 As such, in the attempt to eliminate toxins, neutrophils and macrophages produce oxidants which further augment the inflammatory response. The oxidative stress-induced inflammation creates a vicious cycle resulting in damage to epithelial and stromal cells, which consequently promotes carcinogenesis over time.41

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Oxidative stress leads to DNA mutations and lung cancer As outlined previously, RNS and ROS produced in chemical reactions and by inflammatory cells such as macrophages, and enzymes such as nitric oxide synthases combined with physiological production during mitochondrial respiration and peroxisomal metabolism lead to oxidative stress and lung damage.43 ROS causes DNA damage through transcriptional arrest, induction of transcription errors, or genomic instability, which are all linked to carcinogenesis. These alterations include single- or double-stranded DNA degradation, breakdown of purine/pyrimidine bases or sugar-complex changes, translocations, mutations, deletions, and cross-linking with proteins. Oxidants are highly reactive redox agents due the unbalanced electrons which readily attack DNA, protein, and lipid. Oxidants attack DNA bases to form DNA adducts, which are complexes of DNA covalently bound to a molecule. These adducts can invoke a miscoding during DNA replication resulting in a permanent mutation. Other alterations by oxidants include base alteration, base insertion or deletion, chromosomal translocation, single- or double-strand breaks, microsatellite instability, and oncogene activation, which are directly associated with lung cancer.44 Once these injuries from oxidative stress have accumulated, they may result in carcinogenesis. This is particularly the case when the critical regions coding for oncogenic or tumor suppressor activity are modified and result in uncontrolled proliferation and loss of apoptosis. The most common mutation that occurs in the tumor suppressor gene is on TP53 in lung cancer and is caused as the result of guanine➔ thymine transversions followed by guanine➔ adenine transitions often occurring at codons 157, 158, 245, 248, 249, and 273. DNA mechanisms to proofread and correct any miscoding may repair or remove the damaged sections via double-strand break repair, cross-link repair, direct repair, base excision, or nucleotide excision. However, if the damage is beyond repair, a permanent mutation occurs in DNA that may result in oncogenesis45 (Fig. 2). Over 40% of patients with lung cancer have a mutation in their mitochondrial DNA, highlighting the significance of mtDNA (mitochondrial DNA) in lung cancer. mtDNA mutations can also be detected in the exhaled breath condensate of patients with lung cancer.46 A mtDNA mutation causes disruption in electron transport chain function and metabolism.47 These disruptions could result in the acceleration of aerobic glycolysis during the high demands of metastasis and eventually lead to mtDNA genome depletion. Therefore, preserving the integrity of mtDNA could be a potential therapeutic approach.48 Balanced levels of ROS have an important role in promoting cell death and hence preventing the development of cancer. For instance, ROS can defend the host against neoplastic cells by attacking their DNA and inducing apoptosis, thus preventing cancer development by interrupting the tumor cell life cycle and apoptotic behavior.49 This was identified in Calu-6 and A549 lung cancer cell lines as hydrogen peroxide (H2O2) inhibited growth of these neoplastic cells through caspasedependent apoptosis, necrosis, and G1-phase arrest of the cell cycle and this cytotoxic effect could be further studied as a potential therapeutic approach.50

DNA adducts and mutations e.g., in Carcinogens oncogenes and oxidants and tumour suppressor genes Tobacco smoke, Silica, asbestos, domestic cooking etc.

Loss of growth control and apoptosis

Proliferative growth

Lung cancer

Repair

Normal DNA FIG. 2 Stepwise progression toward lung carcinogenesis. Tobacco smoke and other sources of oxidants cause DNA adduct formation and mutation. These lead to mutations and loss of cellular growth controls, resulting in unencumbered proliferative growth and, finally, lung cancer. This process can be mitigated or halted when DNA is repaired.

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Lipid and protein peroxidation Oxidants can also attack lipid molecules and result in lipid peroxidation products. Specifically, ROS attack polyunsaturated fatty acids (PUFAs) invoking membrane structural and functional alterations. Consequently, lipid peroxidation results in the formation of highly reactive aldehydes which potently form new covalent bonds with cellular targets. Common lipid peroxidation products include acrolein, malondialdehyde (MDA), and 4-hydroxynonenal (HNE). This lipid peroxidation is capable of covalently binding to protein targets such as histidine, cysteine, or lysine residues resulting in functional and membranous changes, it can attack DNA structure and induce cytotoxicity through other signaling pathways and therefore possibly cause carcinogenesis.51 Perhaps this is why plasma aldehyde levels have been found to be significantly higher in patients with lung cancer and other forms of cancer.52 Oxidative stress in lung cancer patients is strongly associated with lipid metabolic disturbances. An elevated oxidative stress state was associated with the increase in TC:HDL (total cholesterol:high-density lipoprotein ratio) and a decrease in HDL concentrations.53 The decrease in HDL, “good” cholesterol, may be explained by the antioxidant properties of HDL in a high oxidative state, which function in slowing down the oxidation of LDL and mediating the enzyme esterase-paraoxonase-1 (PON-1) which in turn inhibit/delay oxidation of lipid peroxides.54 Additionally, oxidants also react with protein side-chain constituents such as arginine, proline, and threonine leading to the formation of reactive carbonylation derivatives. Oxidative stress and protein carbonylation have been identified in

1. 2. 3.

4. 5.

Exogenous sources Tobacco smoke Infections Exposure to environmental smoke Occupational exposure Domestic exposure

ROS/RNS

Increased oxidant burden

Endogenous sources 1. Respiratory chain 2. Cellular enzymes (xanthine oxidase and P450 oxidase) 3. Cyclooxygenase pathway

Depletion of oxidant defense Oxidative stress

Protein peroxidation

Lipid peroxidation

Neutrophils and macrophages sequestration

Further ↑ Oxidants Impaired DNA reparation proteins

Transcription of proinflammatory cytokines interleukins and TNF-α

Activation of transcription factors NF-kB and AP-1

Chronic inflammation DNA damage and mutation and loss of repair Inhibition of apoptosis Initiation, promotion, cell proliferation, cell transformation Lung cancer

FIG. 3 Oxidative stress and lung carcinogenesis. Endogenous and exogenous sources of oxidants result in an oxidant/antioxidant disequilibrium which causes cell damage. Prolonged oxidative injury can lead to lung cancer.

Oxidative stress in lung cancer Chapter

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several pathological processes perhaps due to irreversible protein modification and loss of their functional capacity. Oxidative stress-induced protein carbonylation therefore induces protein dysfunction and may contribute to carcinogenesis.55

Antioxidants and lung cancer prevention As the effect of ROS in carcinogenesis has been elucidated, the concept that antioxidants protect against cancer has been the foundation of the antioxidant food supplement industry. However, a growing body of evidence indicates that the beneficial effects of antioxidants may not be what was initially assumed.56 In the case of lung cancer, a recent meta-analysis investigating 1,233,096 person-years of follow-up found no association between antioxidant intake and lung cancer, and even suggested that a higher intake of retinol may be linked with an increased risk.57

Conclusion In conclusion, oxidants are an important part of normal physiology; however, high concentrations of oxidants from exogenous and endogenous sources can result in oxidative stress and injury. Oxidative stress causes cellular damage such as DNA mutation, protein carbonylation, and lipid peroxidation and contributes to the development of lung cancer. In lung cancer, the two main risk factors, cigarette smoking and radon exposure, are well recognized and the mechanism by which they exacerbate the production of ROS/NOS and cause neoplastic mutations and carcinogenesis have been identified. Moreover, other factors such miRNA alteration and chronic inflammation increase the frequency of lung cancer through oxidative stress. Finally, the role of antioxidants in lung cancer remains unclear and more evidence is required to understand their exact role, if any, in the prevention or development of this disease.

Summary points l

l

l

l

l

There are endogenous and exogenous sources of oxidative stress. Some important exogenous sources of oxidants include radon exposure and cigarette smoking which are the leading causes of lung cancer. Inflammatory mediators contribute to oxidative stress and is evident in how chronic inflammation pathologies such as COPD confers an increased relative risk of lung cancer. Oxidative stress leads to lung carcinogenesis by causing cellular damages such as protein carbonylation, lipid peroxidation, dysfunction of antioxidants and metabolism, and genome changes in an accumulative manner. Despite the significant role of oxidative stress in the development of lung cancer, antioxidants use has not been beneficial as a preventative measure for lung cancer. A summary of oxidative stress pathways leading to lung carcinogenesis is depicted in Fig. 3.

References 1. Cheng T-YD, et al. The International Epidemiology of Lung Cancer: latest trends, disparities, and tumor characteristics. J Thorac Oncol 2016;11 (10):1653–1671. 1a. Phaniendra A, Jestadi DB, Periyasamy L. Free radicals: properties, sources, targets, and their implication in various diseases. Indian J Clin Biochem 2015;30(1):11–26. 10.1007/s12291-014-0446-0. 1b. Kurt OK, Zhang J, Pinkerton KE. Pulmonary health effects of air pollution. Curr Opin Pulm Med 2016;22(2):138–43. 10.1097/MCP. 0000000000000248. 1c. Li X, Fang P, Mai J, et al. Targeting mitochondrial reactive oxygen species as novel therapy for inflammatory diseases and cancers. J Hematol Oncol 2013;6:19. https://doi.org/10.1186/1756-8722-6-19. 2. Hoffman RM, Sanchez R. Lung cancer screening. Med Clin North Am 2017;101(4):769–85. 3. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69(1):7–34. 4. Pola
nski J, et al. Histological subtype of lung cancer affects acceptance of illness, severity of pain, and quality of life. J Pain Res 2018;11:727–33. 5. McCarthy WJ, et al. Chapter 6: Lung cancer in never smokers: epidemiology and risk prediction models. Risk Anal 2012;32(Suppl 1):S69–84. 6. O’Keeffe LM, et al. Smoking as a risk factor for lung cancer in women and men: a systematic review and meta-analysis. BMJ Open 2018;8(10) e021611. 7. Sun S, Schiller JH, Gazdar AF. Lung cancer in never smokers—a different disease. Nat Rev Cancer 2007;7(10):778–90. € 8. Oberg M, et al. Worldwide burden of disease from exposure to second-hand smoke: a retrospective analysis of data from 192 countries. Lancet 2011;377(9760):139–46. 9. de Groot PM, et al. The epidemiology of lung cancer. Transl Lung Cancer Res 2018;7(3):220–33. 10. Eckel SP, et al. Air pollution affects lung cancer survival. Thorax 2016;71(10):891–8. 11. Koshiol J, et al. Assessment of human papillomavirus in lung tumor tissue. J Natl Cancer Inst 2011;103(6):501–7.

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12. Shiels MS, et al. Increased risk of lung cancer in men with tuberculosis in the alpha-tocopherol, beta-carotene cancer prevention study. Cancer Epidemiol Biomarkers Prev 2011;20(4):672–8. 13. Zhang X, et al. Chronic obstructive pulmonary disease and risk of lung cancer: a meta-analysis of prospective cohort studies. Oncotarget 2017;8 (44):78044–56. 14. Kim SH, et al. Indoor radon and lung cancer: estimation of attributable risk, disease burden, and effects of mitigation. Yonsei Med J 2018;59 (9):1123–30. 15. Lantz PM, Mendez D, Philbert MA. Radon, smoking, and lung cancer: the need to refocus radon control policy. Am J Public Health 2013;103 (3):443–7. 16. Kanwal M, Ding X-J, Cao Y. Familial risk for lung cancer. Oncol Lett 2017;13(2):535–42. 17. Lissowska J, et al. Family history and lung cancer risk: International Multicentre Case–Control Study in Eastern and Central Europe and Meta-Analyses. Cancer Causes Control 2010;21(7):1091–104. 18. Wang J, et al. Genetic predisposition to lung cancer: comprehensive literature integration, meta-analysis, and multiple evidence assessment of candidate-gene association studies. Sci Rep 2017;7(1):8371. 19. Rahman I, Biswas SK, Kode A. Oxidant and antioxidant balance in the airways and airway diseases. Eur J Pharmacol 2006;533(1):222–39. 20. Pryor WA, Stone K. Oxidants in cigarette smoke radicals, hydrogen peroxide, peroxynitrate, and peroxynitritea. Ann N Y Acad Sci 1993;686(1):12–27. 21. Valavanidis A, Vlachogianni T, Fiotakis K. Tobacco smoke: involvement of reactive oxygen species and stable free radicals in mechanisms of oxidative damage, carcinogenesis and synergistic effects with other respirable particles. Int J Environ Res Public Health 2009;6(2):445–62. 22. Sarir H, et al. IL-8 production by macrophages is synergistically enhanced when cigarette smoke is combined with TNF-a. Biochem Pharmacol 2010;79(5):698–705. 23. Chan HP, et al. Elevated levels of oxidative stress markers in exhaled breath condensate. J Thorac Oncol 2009;4(2):172–8. 24. Fubini B, Hubbard A. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis. Free Radic Biol Med 2003;34(12):1507–16. 25. Fubini B, et al. Chemical functionalities at the silica surface determining its reactivity when inhaled. Formation and reactivity of surface radicals. Toxicol Ind Health 1990;6(6):571–98. 26. Fubini B, et al. Variability of biological responses to silicas: effect of origin, crystallinity, and state of surface on generation of reactive oxygen species and morphological transformation of mammalian cells. J Environ Pathol Toxicol Oncol 2001;20(Suppl. 1):95–108. 27. Guidotti TL, et al. Diagnosis and initial management of nonmalignant diseases related to asbestos. Am J Respir Crit Care Med 2004;170(6):691–715. 28. Kamp DW, et al. The role of free radicals in asbestos-induced diseases. Free Radic Biol Med 1992;12(4):293–315. 29. Liu G, et al. Molecular mechanisms of asbestos-induced lung epithelial cell apoptosis. Chem Biol Interact 2010;188(2):309–18. 30. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144(5):646–74. 31. Wu K-L, et al. The roles of MicroRNA in lung cancer. Int J Mol Sci 2019;20(7):1611. 32. Ventura A, Jacks T. MicroRNAs and cancer: short RNAs go a long way. Cell 2009;136(4):586–91. 33. Lawrence WCC, Feng FW, William CSC. Genomic sequence analysis of EGFR regulation by MicroRNAs in lung cancer. Curr Top Med Chem 2012;12(8):920–6. 34. Mao G, et al. Tumor-derived microRNA-494 promotes angiogenesis in non-small cell lung cancer. Angiogenesis 2015;18(3):373–82. 35. He J, Jiang B-H. Interplay between reactive oxygen species and MicroRNAs in cancer. Curr Pharmacol Rep 2016;2(2):82–90. 36. Gong Y-Y, et al. MicroRNAs regulating reactive oxygen species in cardiovascular diseases. Antioxid Redox Signal 2017;29(11):1092–107. 37. Robertson A, et al. The cellular and molecular carcinogenic effects of radon exposure: a review. Int J Mol Sci 2013;14(7):14024–63. 38. Nie J-H, et al. Oxidative damage in various tissues of rats exposed to radon. J Toxicol Environ Health A 2012;75(12):694–9. 39. Yanxiao G, et al. Changes of 8-OHdG and TrxR in the residents who bathe in radon Hot Springs. Dose-Response 2019;17(1)1559325818820974. 40. Drost EM, et al. Oxidative stress and airway inflammation in severe exacerbations of COPD. Thorax 2005;60(4):293–300. 41. Federico A, et al. Chronic inflammation and oxidative stress in human carcinogenesis. Int J Cancer 2007;121(11):2381–6. 42. Azad N, Rojanasakul Y, Vallyathan V. Inflammation and lung cancer: roles of reactive oxygen/nitrogen species. J Toxicol Environ Health B 2008;11 (1):1–15. 43. Boukhenouna S, et al. Reactive oxygen species in chronic obstructive pulmonary disease. Oxid Med Cell Longev 2018;2018:5730395. 44. Liguori I, et al. Oxidative stress, aging, and diseases. Clin Interv Aging 2018;13:757–72. 45. Srinivas US, et al. ROS and the DNA damage response in cancer. Redox Biol 2018;25:101084. https://doi.org/10.1016/j.redox.2018.101084. 46. Yang Ai SS, et al. Mitochondrial DNA mutations in exhaled breath condensate of patients with lung cancer. Respir Med 2013;107(6):911–8. 47. Liu X, Chen Z. The pathophysiological role of mitochondrial oxidative stress in lung diseases. J Transl Med 2017;15(1):207. 48. Ishikawa K, et al. ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis. Science 2008;320(5876):661. 49. Valko M, et al. Role of oxygen radicals in DNA damage and cancer incidence. Mol Cell Biochem 2004;266(1):37–56. 50. Park W. Hydrogen peroxide inhibits the growth of lung cancer cells via the induction of cell death and G1-phase arrest. Oncol Rep 2018;40:1787–94. 51. Yoshida Y, Umeno A, Shichiri M. Lipid peroxidation biomarkers for evaluating oxidative stress and assessing antioxidant capacity in vivo. J Clin Biochem Nutr 2013;52(1):9–16. 52. Erejuwa OO, Sulaiman SA, Ab Wahab MS. Evidence in support of potential applications of lipid peroxidation products in cancer treatment. Oxid Med Cell Longev 2013;2013:931251. 53. Zabłocka-Słowi
nska K, et al. Oxidative stress in lung cancer patients is associated with altered serum markers of lipid metabolism. PLoS One 2019; 14(4): e0215246.

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Jurek A, et al. LDL susceptibility to oxidation and HDL antioxidant capacity in patients with renal failure. Clin Biochem 2006;39(1):19–27. Mateu-Jim
enez M, et al. Redox imbalance in lung cancer of patients with underlying chronic respiratory conditions. Mol Med 2016;22:85–98. Birben E, et al. Oxidative stress and antioxidant defense. World Allergy Organ J 2012;5(1):9–19. Narita S, et al. Dietary consumption of antioxidant vitamins and subsequent lung cancer risk: the Japan Public Health Center-based prospective study. Int J Cancer 2018;142(12):2441–60.

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Chapter 4

Endogenous antioxidants in the prognosis and treatment of lung cancer Laurie Freire Boullosa∗, Jinthe Van Loenhout∗, and Christophe Deben∗ Center for Oncological Research, University of Antwerp, Wilrijk, Belgium

List of abbreviations Akt ARE ASK1 ATP BSO EGFR FDA GCL GPx GSH GSR GSS GSSH H2O2 HIF-1alpha HO-1 JAK1 Keap1 LUAD mRNA mTOR NADPH NQO1 Nrf2 NSCLC OS PI3K PRDX Prx PTEN ROS siRNA STAT3 TCGA Trx TrxR TXN

protein kinase B antioxidant responsive element apoptosis signal regulating kinase 1 adenosine triphosphate buthionine sulfoximine epidermal growth factor receptor Food and Drug Administration glutamate cysteine ligase glutathione peroxidases glutathione glutathione reductase glutathione synthetase glutathione disulfide hydrogen peroxide hypoxia-inducible transcription factor-1 alpha eme oxygenase-1 janus kinase 1 kelch-like ECH-associated protein 1 lung adenocarcinoma messenger ribonucleic acid mammalian target of rapamycin nicotinamide adenine dinucleotide phosphate NAD(P)H quinone oxidoreductase 1 nuclear factor erythroid 2-related factor 2 non-small cell lung cancer overall survival phosphoinositide 3-kinase peroxiredoxin peroxiredoxins protein phosphatase and tensin homolog reactive oxygen species small interfering ribonucleic acid signal transducer and activator of transcription 3 The Cancer Genome Atlas thioredoxin thioredoxin reductase thioredoxin

∗All authors contributed equally to the manuscript. Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00004-3 © 2021 Elsevier Inc. All rights reserved.

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Txnip TXNRD1 ZnPP

A Oxidative stress and cancer

thioredoxin interacting protein thioredoxin reductase 1 zinc protoporphyrin

Double-edged sword of antioxidants in cancer Oxidative stress, induced by endogenous and exogenous sources of oxidants, is related to the carcinogenesis of lung cancer and elevated reactive oxygen species (ROS) levels sustain cancer growth. However, it is important to note that the role of ROS in cancer is rather complex and paradoxical. In general, cancer is characterized by higher cellular ROS levels compared to their normal counterparts.1 These persistent high levels of ROS can be explained by the imbalance between oxidants and antioxidants in cancer cells, resulting in oxidative stress.2 This imbalance is due to oncogenic transformations including alteration in the tumor genetics, metabolism, and microenvironment.3 For instance, hypoxia is a characteristic feature of cancer resulting from an imbalance between oxygen supply and consumption due to uncontrollable cell proliferation, altered metabolism, and abnormal tumor blood vessels. This results in a reduced transport of oxygen and nutrients.4 It is essential for cancer cells to adapt to these hypoxic conditions by altering their metabolism. Cancer cells maintain their high energy levels through a high rate of glycolysis followed by lactic acid fermentation even in the presence of abundant oxygen, which is called aerobic glycolysis or the Warburg-effect. This process is followed by oxidation in mitochondria, resulting in an increased ROS generation.5 In addition, cancer cells evolved mechanisms to protect themselves from this intrinsic oxidative stress and developed an adaptation mechanism by upregulation of pro-survival molecules and their antioxidant defense system to maintain the redox balance.2 Increase of intracellular ROS levels may result in the activation of oncogenes, which are involved in cell proliferation and inactivation of tumor suppressor genes, angiogenesis, and mitochondrial dysfunction.5 A low-to-moderate increase of ROS in cancer cells serves as signaling molecule in cancer survival. For instance, hydrogen peroxide (H2O2) reversibly oxidizes cysteine thiol groups of phosphatases such as phosphatase and tensin homolog (PTEN) which cause loss of their activity and promote activation of the PI3K/Akt/mTOR survival pathway.6 Conversely, when the levels of ROS are further elevated, it can overcome the defensive antioxidant system of the cancer cells and it can cause cell death through various mechanisms.7 Consequently, there are two different approaches based on the redox balance to counteract cancer cells (Fig. 1).8 In the first approach, oxidative stress can be decreased by scavenging intracellular ROS. For example, by increasing the intake of dietary antioxidants, as mostly described in this book, oxidative stress can be depleted, subsequently causing growth inhibition, and increased susceptibility to cell death in cancer cells. The second approach is by increasing ROS levels in cancer cells. This can be done either by direct production of ROS by exogenous agents (e.g., ionizing radiation) or indirectly by increasing intracellular ROS concentrations via targeted inhibition of antioxidant systems in cancer cells. In this chapter,

ROS homeastasis

Tumor promoting - Increased proliferation - Increased metastasis - Increased angiogenesis

Tumor inhibition - Oxidative damage to proteins - Oxidative damage to DNA - Increased lipid peroxidation

Cell death threshold

FIG. 1 Reactive oxygen species (ROS) can both promote and inhibit cancer, depending on the endogenous ROS levels. In normal cells antioxidants are responsible for the elimination of ROS, which are produced during the metabolism, to maintain redox homeostasis. Cancer cells have higher steady-state levels of ROS, which are counterbalanced by an increased antioxidant capacity. This moderate increase of ROS can have tumorpromoting effects, such as induction of metastasis, angiogenesis, and tumor proliferation. Further elevation of ROS levels can cause oxidative damage of proteins and DNA and an increase in lipid peroxidation. Consequently, cancer cells will cross the threshold of cell death. Therapeutic antioxidants scavenge these toxic ROS levels and decrease the tumor-promoting effects. Conversely, therapeutic inhibition of endogenous antioxidants, chemotherapy, and radiotherapy increases ROS levels and induce cancer cell death.

ROS

Therapeutic antioxidants

Therapeutic inhibition of endogenous antioxidants Chemotherapy Radiotherapy

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we focus on the second approach and discuss alterations in the major antioxidant systems in non-small cell lung cancer (NSCLC), their prognostic value, and the use of antioxidant inhibitors for the treatment of NSCLC.

Nrf2: Transcriptional regulator of the redox balance Role of Nrf2 The Nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor is the first line of defense against oxidative stress by acting as a master transcription regulator of >200 oxidative stress-related genes.9 Its expression is tightly regulated by kelch-like ECH-associated protein 1 (Keap1) which targets Nrf2 for polyubiquitination and degradation under normal unstressed physiological conditions (Fig. 2).10 In cancer cells, Nrf2 is often constitutively activated and supports cancer cell proliferation and protection against chemo- and radiotherapy.10

Nrf2 in NSCLC

Keap1

Keap1

Several studies examined the prognostic value of Nrf2 expression in NSCLC patients. Yang et al. report that patients (n ¼ 60) with high Nrf2 expression (56.7%) have a significantly worse overall and progression-free survival and respond poorly to platinum-based chemotherapy.11 Inoue et al. studied Nrf2 expression in 109 NSCLC cases and conclude that positive staining for Nrf2 (34%) is an independent factor predicting worse lung cancer-specific survival.12 Solis et al. detected Nrf2 expression in 26% of NSCLC patients (n ¼ 304) and multivariate analysis shows that Nrf2 expression was associated with worse overall survival. In addition, they report that nuclear Nrf2 expression was associated with worse progression-free survival in squamous cell carcinoma patients who received adjuvant platinum-based therapy.13 Romero et al. performed an in-depth analysis of the LUAD (lung adenocarcinoma) data set (n ¼ 458) available in The Cancer Genome Atlas (TCGA). They report that core Nrf2 target genes were significantly upregulated in advanced stage tumors and that tumors associated with this Nrf2 core signature had significantly worse survival.14 One of the main mechanisms leading to Nrf2 activation is the presence of loss-of-function mutations in KEAP1 resulting in reduced affinity of Keap1 to Nrf2 (Fig. 2). KEAP1 is the third most frequently mutated gene in the LUAD cohort, further supporting the relevance of the Keap1/Nrf2 pathway in NSCLC.14

Ubiquitination Nrf2 Degradation Oxidative stress response genes Trx system members GSH system members

Mu Ke t ap 1

Ke

ap

1

Luteolin

Nrf2

ML385 Chemoresistance Drug-metabolizing enzymes Drug-efflux transporters Cytoprotective genes

Nrf2 Activation

ARE Poor NSCLC prognosis Increased NSCLC proliferation

FIG. 2 Nrf2 transcription factor. Nrf2 is a master transcriptional regulator of the oxidative stress response. Its expression is tightly regulated by Keap1, which targets Nrf2 for proteasomal degradation. One of the main mechanisms leading to Nrf2 activation is the presence of loss-of-function mutations in KEAP1 which frequently occurs in NSCLC. Mutations result in reduced affinity of Keap1 to Nrf2. Increased activation of Nrf2 leads to transcription of genes related to chemoresistance and the Trx and GSH system leading to increased cancer cell proliferation and poor prognosis. Luteolin and ML385 are experimental therapies used for the inhibition of Nrf2 in NSCLC. ARE, antioxidant response element; NSCLC, nonsmall cell lung cancer.

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Nrf2-mediated chemoresistance and therapeutic strategies The studies discussed above clearly show that Nrf2 expression negatively affects the prognosis of NSCLC patients which is potentially mediated by chemoresistance. Several chemotherapeutics exert their therapeutic effect through increased cellular ROS levels. Consequently, a constitutively activated Nrf2 antioxidant response can limit this chemotherapy-induced ROS accumulation through the expression of cytoprotective genes. In addition, genes encoding for drug-metabolizing enzymes (e.g., NAD(P)H quinone oxidoreductase 1, NQO1) and efflux transporters (e.g., multidrug resistance proteins) are also under the transcriptional control of Nrf2 via binding to antioxidant responsive elements (ARE) and contribute to an accelerated metabolism and efflux of chemotherapeutic agents.15 This makes Nrf2 an interesting therapeutic target either as monotherapy or in combination with conventional chemotherapeutics for the treatment of lung cancer. Singh et al. identified ML385 as a molecule that binds Nrf2 and inhibits its transcriptional activity in KEAP1-deficient NSCLC cancer cells. Furthermore, they show that ML385 enhanced cytotoxicity of paclitaxel and carboplatin in vitro and they report superior efficacy of ML385 + carboplatin therapy compared to single-agent treatment in A549 and H460 mouse xenografts.16 Tang et al. reported that the flavonoid luteolin (30 ,40 ,5,7tetrahydroxyflavone) to be a potent and selective Nrf2 inhibitor. Luteolin is found in celery, green pepper, parsley, perilla leaf, and chamomile tea. Moreover, they demonstrate that luteolin significantly enhanced the response to oxaliplatin, bleomycin, and doxorubicin in A549 NSCLC cancer cells.17 Inhibition of Nrf2-driven heme oxygenase-1 (HO-1) in A549 cells by siRNA or ZnPP, a specific HO-1 inhibitor, augments cisplatin-induced cytotoxicity by markedly increasing cisplatininduced ROS.18 These studies show that inhibition of Nrf2 or its downstream transcriptional targets can enhance chemosensitivity in Nrf2 overexpressing NSCLC cancer cells.

Glutathione and thioredoxin antioxidant systems Members of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems are under the transcriptional regulation of Nrf2 and their expression supports tumor formation and proliferative signaling pathways in cancer cells while avoiding high damaging levels of ROS that can induce cancer cell death.19 Therefore, inhibition of both the GSH and Trx system is proposed as an anticancer strategy. Using gene expression data from 500 LUAD patients available in TCGA we determined which members are highly expressed in NSCLC patients and how this expression affects their prognosis. In addition, we highlight promising therapeutic strategies that act on these antioxidants in NSCLC.

GSH system The GSH system catalyzes H2O2 into H2O in the cytoplasm and mitochondria of cells. GSH is a reduced tripeptide consisting of three amino acids glutamine, cysteine, and glycine. De novo synthesis of GSH is a two-step process (Fig. 3). The first step involves sequential ATP-dependent formation of amide bonds between cysteine and glutamate to produce the dipeptide g-glutamylcysteine. This reaction is catalyzed by the enzyme glutamate cysteine ligase (GCL). The cellular import of cystine by the cystine/glutamate antiporter system xC-, which is under transcriptional control of Nrf2, is an important rate-limiting step in GSH synthesis. In the second step, GSH is formed by glutathione synthetase (GSS) which adds glycine to the dipeptide.20 This tripeptide is the most abundant nonenzymatic antioxidant in the cell with different functions as (i) predominant scavenger of ROS in cells, (ii) detoxification enzyme, (iii) regenerator/reducer of vitamins C and E, and (iv) cofactor of several oxidative stress detoxifying enzymes.19 For the latter when H2O2 is increased, two GSH molecules are oxidized to glutathione disulfide (GSSG) by glutathione peroxidases (GPx). The GPx protein family consists of eight members (GPx 1–8) with different substrate specificity. To recycle GSH, glutathione reductase (GSR) oxidizes GSSG back to GSH by NADPH-dependent mechanism (Fig. 3).21

GSH system in NSCLC The GSH system is often dysregulated in lung cancer, where it plays a dual role in its progression. It is necessary for the removal and detoxification of carcinogens, but increased GSH levels in lung cancer cells have a protective function by leading to chemotherapeutic drug resistance.22 Data extracted from TCGA indicate that several members of the GSH system are highly expressed in LUAD patients (Fig. 4A) which have opposing effects on prognosis. Fig. 4B shows that high mRNA expression of both GSR and GPX3 is associated with better prognosis of lung adenocarcinoma patients. On the contrary, high expression of GPX2 and GPX8 is linked to low patient survival. Liu et al. came to

Endogenous antioxidants in lung cancer Chapter

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mutp53

ARE

SLC7A11

System xC-

Erastin Sulfasalazine

SLC3A2

Nrf2

4

Cystine Ferroptosis

Cys

Glu GCL γ-Glu-Cys

BSO

GSS +

NADP

Gly

LO•

Fe2+ LOOH

GSH GR

GPx4

RSL3 Withaferin A

LOH GSSG NADPH FIG. 3 Glutathione system. Glutathione (GSH) synthesis is dependent on the cellular import of cystine by the cystine/glutamate antiporter system xC-. Transcription of one of its components SLC7A11 is regulated by Nrf2 and (mutant) p53. The enzyme glutamate cysteine ligase (GCL) catalyzes the binding of cysteine (Cys) and glutamate to produce g-glutamylcysteine (g-Glu-Cys). Finally, GSH is formed by glutathione synthetase (GSS) which adds glycine to the dipeptide. Two GSH molecules are oxidized to glutathione disulfide (GSSG) by glutathione peroxidases (GPx). Finally, GSSG is recycled to GSH by glutathione reductase (GSR). Glutathione peroxidase 4 (GPx4) reduces lipid hydroperoxides (LOOH) to harmless lipid alcohols (LOH) to prevent iron-mediated lipid peroxidation (LO), thereby protecting cells against ferroptosis. Inhibition of the GSH system using compounds like erastin, sulfasalazine, buthionine sulfoximine (BSO), RSL3, and withaferin A can induce ferroptosis in cancer cells.

(A)

(B)

P < .01

P < .0165

P < .01

P < .001

FIG. 4 Glutathione system and prognosis in NSCLC adenocarcinoma. (A) Gene expression of the glutathione system members in 500 lung adenocarcinoma patients. FPKM (fragment per kilobase million) values were extracted from the lung adenocarcinoma (LUAD) cohort of the cancer genome atlas (TCGA) RNA-seq dataset using the Human Protein Atlas available from www. proteinatlas.org.23 (B) KaplanMeier survival curves were plotted based on the best expression cutoff, i.e., the FPKM value that yields maximal difference with regard to survival using the Human Protein Atlas. Only GSH system members that significantly affected patient survival are presented. A cutoff of a log-rank P-value 2 cm but ≤4 cm, and ≤ 10 mm DOI

T3

Tumor >4 cm or any tumor >10 mm DOI

T4

Moderately advanced or very advanced local disease

T4a

Moderately advanced local disease: (lip) tumor invades through cortical bone or involves the inferior alveolar nerve, floor of mouth, or skin of face (i.e., chin or nose); (oral cavity) tumor invades adjacent structures only (e.g., through cortical bone of the mandible or maxilla, or involves the maxillary sinus or skin of the face); note that superficial erosion of bone/tooth socket (alone) by a gingival primary is not sufficient to classify a tumor as T4

T4b

Very advanced local disease; tumor invades masticator space, pterygoid plates, or skull base and/or encases the internal carotid artery

Regional nodal metastases NX

Regional lymph nodes cannot be assessed

N0

No regional lymph node metastasis

N1

Metastasis in a single ipsilateral lymph node, 3 cm or less in greatest dimension and ENE-negative

N2

Metastasis in a single ipsilateral lymph node, 3 cm or less in greatest dimension and ENE-positive; or more than 3 cm but not more than 6 cm in greatest dimension and ENE-negative; or metastases in multiple ipsilateral lymph nodes, none more than 6 cm in greatest dimension and ENE-negative; or metastasis in bilateral or contralateral lymph nodes, none more than 6 cm in greatest dimension, ENE-negative

N2a

Metastasis in a single ipsilateral or contralateral lymph node 3 cm or less in greatest dimension and ENE-positive; or metastasis in a single ipsilateral lymph node more than 3 cm but not more than 6 cm in greatest dimension and ENE-negative

N2b

Metastasis in multiple ipsilateral lymph nodes, none more than 6 cm in greatest dimension and ENE-negative

N2c

Metastasis in bilateral or contralateral lymph nodes, none more than 6 cm in greatest dimension and ENE-negative

N3

Metastasis in a lymph node more than 6 cm in greatest dimension and ENE negative; or metastasis in a single ipsilateral lymph node more than 3 cm in greatest dimension and ENE-positive; or metastasis in multiple ipsilateral, contralateral, or bilateral lymph nodes, with any ENE-positive

N3a

Metastasis in a lymph node more than 6 cm in greatest dimension and ENE-negative

N3b

Metastasis in a single ipsilateral node more than 3 cm in greatest dimension and ENE-positive; or metastasis in multiple ipsilateral, contralateral, or bilateral lymph nodes, with any ENE-positive

Distant metastases MX

Unable to assess for distant metastases

M0

No distant metastases

M1

Distant metastases

TNM staging Stage 0

Tis N0 M0

Stage I

T1 N0 M0

Stage II

T2 N0 M0

Stage III

T3 N0 M0 T1 to T3 N1 M0

Stage IVa

T4a N0 M0 T4a N1 M0 T1 to T4a N2 M0 Continued

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TABLE 1 American Joint Committee on Cancer Staging (AJCC) For Oral Cavity Cancer 2017, 8th edition Staging System— cont’d Stage IVb

Any T N3 M0 T4b Any N M0

Stage 4c

Any T Any N M1

DOI: depth of invasion, ENE: Extranodal extension. American Joint Committee on Cancer Staging (AJCC) 2017, 8th Edition Staging System is seen in the table. AJCC Staging System is the most commonly accepted and used staging system for head and neck cancer. Staging of a cancer guide the treatment and follow-up of the cancer patient.

TABLE 2 Common reactive oxygen and nitrogen species in oral cancer. Reactive oxygen species

Reactive nitrogen species

Hidroxyl radical (.OH)

Nitric oxide (NO)

Superoxide anion (oxygen with an unpaired electron) (O2 %)

Nitrite (NO2 )

Hydrogen peroxide (H2O2)

Nitrate (NO–) 3

Hypochlorous acid (HOCl)

Peroxynitrite (ONOO )

Common reactive oxygen and nitrogen species and their abbreviations in Oral Cancer are seen in the table.

Free radicals may react with other molecules due to their unpaired electrons, and generate more free radicals through chain reactions. This accumulation of free radicals may impair all cellular components such as DNA, RNA, proteins, lipids, etc. on the occasion of OS.11 ROS may also change the cellular signaling pathways while acting as cellular messengers in redox signaling.12 OS is blamed in the development of many diseases such as Alzheimer’s and Parkinson’s, atherosclerosis, myocardial infarction, and cancer.11 In the aerodigestive tract, malignancies distinct from other cancer types, such as the exogenous factors, mainly tobacco smoke and alcohol consumption, are declared to be the main factors in the etiology. This issue is important for the aerodigestive malignancies as oral cancer may be seen as a preventable disease.

The causes of oxidative stress in the oral cavity The causes of OS in the oral cavity can be classified as endogenous and exogenous causes:

Endogenous causes The endogenous causes of OS are the natural by-products of the metabolism. Cytochrome P450 metabolism, inflammatory cells, mitochondria, and peroxisomes are the main endogenous sources.13 These causes may be seen in various types of diseases such as oral cancer. Due to the incomplete reduction of molecular oxygen in the mitochondrial oxygen metabolism, O2 , H2O2, and HO are produced.14 In inflammation, ROS and RNS are mainly produced by NADPH oxidase in activated leukocytes.10 Detoxification systems have antioxidant (AO) mechanisms. Vitamins A, C, and E and Glutathione (GSH) are the nonenzymatic metabolites. Various peroxidases, catalase, superoxide dismutase (SOD) are the enzymes that participate in the AO mechanisms. The low levels of nonenzymatic AO metabolites or the diseases that cause the insufficiency of them or inhibition of the AO enzymes also cause OS.10

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Exogenous causes Exogenous causes are the distinctive properties of aerodigestive tract malignancies. As the orifice of this tract, the oral cavity is the first region that is exposed to the cancerous materials. The main exogenous causes of OS in oral cancers can be sorted as tobacco smoke, food (especially alcohol consumption), loss of oral hygiene, chronic inflammation, and dental materials.10

Tobacco smoke and chewing Cigarette and other tobacco products have numerous chemical components that take part in the pathologic processes of many diseases.15 Additionally, heat and pH disturbance of the body fluids (during tobacco chewing) also contribute to the pathologic process with free radical production.16 Tobacco smoke is the foremost proven cancerous material not only for lung cancer but also for most of the aerodigestive tract malignancies. Betel (areca) nut chewing is also a widespread use of tobacco and the cause of oral cancer in South and Southeastern Asia.17 Tobacco smoke and chewing increase ROS such as O2 , H2O2, and OH. NO, which is the most prominent RNS of tobacco smoke, may react with O2 and form nitrite (NO2 ). The reactivity of RNS increases with this reaction, and NO2 forms a considerable part of OS in the oral cavity of tobacco smokers.18 The unsaturated, chemically active aldehydes such as crotonaldehyde, acrolein, etc. react with thiol ( SH) groups of proteins like arginine, histidine, lysine, and cysteine residues.19 This reaction, which is called Michael addition, induces modifications in the structure of proteins. Additionally, this reaction may not only affect molecules like DNA, RNA, enzymes, etc. but also may inhibit antioxidant metabolites such as GSH.19

Food As the orifice of the digestive tract, the oral cavity is subjected to various kinds of food and beverage. Before digestion and entering into the systemic circulation, these substances show a local effect on the tissues of the oral cavity. Considering the local effect, not only the ingredients but also the temperature, solidity, acidity, etc. become important. High temperature and heat stress induce the nuclear translocation of Nuclear factor-erythroid 2 related factor 2 (Nrf2) and increase the target genes of this factor in human dental pulp cells. The target enzyme genes like the heme oxygenase-1 (HO-1) gene increase the level of ROS.20 The cooking style is another factor affecting OS. Heated cooking oil was shown to emit acrolein which is a chemically active aldehyde.21 As mentioned in the part of tobacco smoke and chewing, acrolein disrupts the structure of proteins, DNA, RNA, AO enzymes, etc. and causes OS.19 Alcohol consumption is a well-known cancerous factor as tobacco smoke for oral cancer. Alcohol shows its effect on the oral cavity locally and systemically. The decrease in AO mechanisms and accumulation of free radicals result as OS.22 Vitamins C and E deficiencies are seen in alcoholics, as a result of being used in nonenzymatic AO mechanisms.23 Alcohol abuse results as increased levels of cytochrome P450 2E1 (CYP2E1), which is the oxidizing enzyme of ethanol. CYP2E1 generates ROS and increases OS.24 Alcohol also increases the cancerous effect of tobacco smoke synergistically.2

Loss of oral hygiene and chronic inflammation As a result of oral hygiene loss, the accumulation of oral bacteria occurs. Chronic inflammation of the gingiva (gingivitis) and, in advanced cases, loss of tooth-supporting bone and teeth (periodontitis) may result in insufficient leukocyte functions.25 Fungal and viral infections, especially in immune system disorders and autoimmune disorders like Lichen Planus and Pemphigus Vulgaris, also create inflammatory processes.10 In phagocytes (monocytes and neutrophils), O2 by NADPH oxidase is the primary ROS to destroy pathogens. H2O2, HOCl, and NO are the other main ROS and RNS of this enzymatic mechanism. SOD, myeloperoxidase, NO synthase are additional enzymes.14 Nonenzymatic reactions of these molecules increase reactivity and form OH and NO2 . These mechanisms may also damage surrounding tissue to destroy intracellular pathogens. This hyperresponsiveness is also the mechanism of autoimmune disease.26 Studies suggest that OS, chronic inflammation, and cancer are closely linked.27

Dental materials In dental medicine, various kinds of materials are used to restore teeth function. It was detected that OS in the oral cavity may result from the metallic and incomplete curing of resins and acrylic restorations.7

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Hydroxyethyl methacrylate (HEMA) is a resin monomer that induces a Michael addition reaction of GSH, prevents the oxidation to GSH, and increased H2O2 levels resulting in the inhibition of SOD. OS occurs according to the inhibition of AO mechanisms and enzymes.28 Dental biomaterials are known to release ions of Ag, Co, Cu, Hg, etc. These ions also alter intracellular GSH levels. The blue light irradiation used in dental medicine is shown to increase H2O2 and OH levels, lipid peroxidation, and apoptosis.10

Oxidative stress in oral precancer and cancer The development of cancer is the result of the cumulative effect of multiple processes. The stages can be sorted by (1) initiation, (2) promotion, (3) progression, and OS takes part in all three stages.22 In the structural base of the cell, proteins are the major cell targets of ROS.29 The main mechanisms of ROS in the initiation stage are oxidative nuclear and mitochondrial DNA damage.30 In the promotion stage, clonal expansion of the tumor cells can be induced by ROS.31 OS may conduce to the modification of the enzyme genes. In the progression stage, ROS may inhibit enzymes like antiproteinases, activate enzymes like matrix metalloproteinases, and trigger the angiogenesis response which is crucial in invasion and metastasis.32–34 In the study by Beevi et al., nitric oxide products like NO2 , nitrate (NO3 ), and total nitrite (TNO2 ), and lipid peroxidation products like lipid hydroperoxide (LHP) and malondialdehyde (MDA) were determined to be significantly elevated, whereas antioxidants were determined to be significantly lowered in late-stage OSCC, compared with healthy individuals.35 Additionally, no difference was observed in terms of NO levels and NO synthase activity between the tumor and surrounding tumor-free tissues of the same patients with oral cancer in our recent study.36 The deficiency of AO mechanisms and the presence of OS are evident in carcinogenesis. However, OS is not likely to be limited to the tumor tissue. Tobacco and alcohol consumption are the major risk factors of oral cancer. Human oral cancer cells were observed to produce free radicals when incubated with tobacco extract in an in vitro study.37 Oxidative DNA damage, lipid peroxidation, damage of the cell structures, inhibition of AO systems are the main effects of OS induced by tobacco smoke and chewing that may take part in carcinogenesis.30 In the cancer initiation stage, the modification of DNA bases such as 8-hydroxy-deoxyguanosine (8-ox-odG) increases by 35%–50% in tobacco smokers.38 The levels of 8-ox-odG and 8-nitroguanosine were also found to increase in oral tissues of the patients with precancerous lesion leukoplakia and OSCC.39, 40 In the study by Kawanishi et al., it was found that 8-oxodG and 8-nitroguanosine may contribute to the development of OSCC from precancerous lesions like leukoplakia and lichen planus. In the same study, as a contribution to the tumor promotion stage, p53 accumulation was also demonstrated in OSCC, leukoplakia, and lichen planus.41 Lu et al. observed a significant increase in ROS in a betel nut treated OSCC cell in their study; they stated that the betel nut may take part in oral carcinogenesis with ROS generation. In the same study, upregulation of mitogen activated protein kinase-1. (MKP-1) resulting in increased levels of ROS, and increased autophagy and subsequent protection from apoptosis were observed. These findings were commented on as the pathogenesis-promoting effects of OS of the betel nut.42 The modification of AO enzymes also gives insight into OS mechanisms in oral carcinogenesis. The polymorphism of the AO mitochondrial SOD enzyme, SOD2 rs4880, has been determined to be significantly increased in smokers with OSCC.43 Chronic alcohol consumption increases the oxidative enzyme of ethanol, CYP2E1 activity. Increased ROS generation may lead to lipid peroxidation. The product of lipid peroxidation 4-hydroxynonenal (4HNE) binds and mutates DNA.24 In chronic alcoholics, the deficiency of Vitamins A, C, and E as nonenzymatic AO systems may initiate oral carcinogenesis.22 In head and neck cancers, various viruses such as Epstein–Barr virus in nasopharyngeal cancer and human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma are detected to take part in carcinogenesis. HPV 16 and 18 are known to be high-risk oncogenic virus subtypes. The incidence is 1.0% and associated with the sexual partner number and smoking habit.44 E2 proteins of the mitochondrial membranes of HPV 16 and 18 were observed to increase mitochondrial ROS generation, compared to E2 proteins of low-risk virus subtype HPV 6.45, 46 Even more in the study by Williams et al., E6 protein of high-risk oncogenic virus subtypes were also demonstrated to increase ROS with decreasing SOD2 and glutathione peroxidase (GPX) expressions in both HPV-positive and HPV-negative cells.47 Other HPV oncoproteins E6 and E7 also inhibit tumor suppressor genes such as TP53 and RB1 and thus may induce malignant transformation. The carcinogenic and prognostic effects of HPV are more evident in oropharyngeal cancer, compared with oral cancer.1

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Applications to other cancers or conditions In this chapter, oral cancer and its association with oxidative stress were reviewed. As the presence of certain carcinogenic factors is 90%–95% in oral cancers, certain risk factors in oral cancer and their association with oxidative stress were focused on. The leading risk factors such as tobacco smoke and alcohol consumption also constitute the risk for malignancy in the aerodigestive system. The oral cavity is the entrance of the aerodigestive tract. These exogenous carcinogenic substances show their first effects on the oral cavity. For example, for tobacco smoke, no filtration systems protect against the carcinogenic particles in the tobacco products to the alveolar system of the lung. In the oral cavity, there are few enzymes to metabolize the carcinogenic substances in the saliva. Thus, the consecutive anatomic regions such as the oropharynx, hypopharynx, larynx, and esophagus are also exposed to these substances as unmetabolized. The oxidative stress mechanisms in carcinogenesis and its association with the cancer of the head and neck anatomic regions may be similar. Therefore, the associated mechanisms, especially for tobacco smoke and alcohol consumption, are considered to be applicable for cancer of the anatomic regions such as the oropharynx, hypopharynx, larynx, and esophagus in the head and neck region. The studies also showed the associations with these regions.

Summary points l l l

l l

l l

l l

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l

This chapter focuses on oral cancer and its association with oxidative stress. Oral cancer is the most common cancer of the head and neck region. Certain risk factors such as tobacco smoke and chewing, alcohol consumption, etc. explain 90%–95% of the cases, especially with oral squamous cell carcinoma. As a preventable and early detectable disease, to understand the carcinogenesis process of oral cancer is crucial. Lip cancer is the most common of oral cancers. Solar ultraviolet light exposure is also associated with the development of lip cancer. The causes of oxidative stress in the oral cavity can be classified as endogenous and exogenous causes. Exogenous causes of oxidative stress in the oral cavity can be sorted as: tobacco smoke and chewing, food (especially alcohol consumption), loss of oral hygiene, chronic inflammation, and dental materials. Tobacco smoke and alcohol consumption synergistically increase the risk of oral cancer development. Tobacco smoke and chewing increase ROS such as O2 , H2O2, and OH. NO, which is the most prominent RNS of tobacco smoke, may react with O2 and form NO2 . The reactivity of RNS increases with this reaction, and NO2 forms a considerable part of oxidative stress in the oral cavity of tobacco smokers. Oxidative DNA damage, lipid peroxidation, damage of cell structures, inhibition of antioxidant systems are the main effects of oxidative stress induced by tobacco smoke and chewing that may take part in carcinogenesis. The decrease in antioxidant mechanisms and accumulation of free radicals result as oxidative stress. Vitamin C and E deficiencies are seen in alcoholics, as a result of being used in nonenzymatic antioxidant mechanisms. In chronic alcoholics, the deficiency of Vitamins A, C, and E as nonenzymatic AO systems may initiate oral carcinogenesis.

References 1. Wein RO, Weber RS. Malignant neoplasms of the oral cavity. In: Flint PW, Haughey BH, Lund V, Niparko JK, Robbins KT, Thomas JR, Lesperance MM, editors. Cummings otolaryngology: head and neck surgery. Philadelphia, PA: Elsevier Mosby; 2005. p. 1591–607. 2. Blot WJ, McLaughlin JK, Winn DM, Austin DF, Greenberg RS, Preston-Martin S, et al. Smoking and drinking in relation to oral and pharyngeal cancer. Cancer Res 1988;48(11):3282–7. 3. Koch WM, Lango M, Sewell D, Zahurak M, Sidransky D. Head and neck cancer in CNonsmokers: a distinct clinical and molecular entity. Laryngoscope 1999;109(10):1544–51. 4. Winn DM, Blot WJ, Shy CM, Pickle LW, Toledo A, Fraumeni Jr JF. Snuff dipping and oral cancer among women in the southern United States. N Engl J Med 1981;304(13):745–9. 5. Baden E. Prevention of cancer of the oral cavity and pharynx. CA Cancer J Clin 1987;37(1):49–62. 6. Sharan RN, Mehrotra R, Choudhury Y, Asotra K. Association of betel nut with carcinogenesis: revisit with a clinical perspective. PLoS One 2012;7(8) e42759. 7. Lohbauer U, Belli R, Ferracane JL. Factors involved in mechanical fatigue degradation of dental resin composites. J Dent Res 2013;92(7):584–91.

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8. Funk GF, Karnell LH, Robinson RA, Zhen WK, Trask DK, Hoffman HT. Presentation, treatment, and outcome of oral cavity cancer: a National Cancer Data Base report. Head Neck 2002;24(2):165–80. 9. Lydiatt WM, Patel SG, O’Sullivan B, Brandwein MS, Ridge JA, Migliacci JC, et al. Head and neck cancers—major changes in the American Joint Committee on cancer eighth edition cancer staging manual. CA Cancer J Clin 2017;67(2):122–37. 10. Avezov K, Reznick AZ, Aizenbud D. Oxidative stress in the oral cavity: sources and pathological outcomes. Respir Physiol Neurobiol 2015;209:91–4. 11. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39(1):44–84. 12. Droge W. Free radicals in the physiological control of cell function. Physiol Rev 2002;82(1):47–95. 13. Inoue M, Sato EF, Nishikawa M, Park AM, Kira Y, Imada I, et al. Mitochondrial generation of reactive oxygen species and its role in aerobic life. Curr Med Chem 2003;10(23):2495–505. 14. Lavie L. Obstructive sleep apnoea syndrome–an oxidative stress disorder. Sleep Med Rev 2003;7(1):35–51. 15. Kuper H, Adami HO, Boffetta P. Tobacco use, cancer causation and public health impact. J Intern Med 2002;251(6):455–66. 16. Stich HF, Anders F. The involvement of reactive oxygen species in oral cancers of betel quid/tobacco chewers. Mutat Res 1989;214(1):47–61. 17. Ko YC, Huang YL, Lee CH, Chen MJ, Lin LM, Tsai CC. Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J Oral Pathol Med 1995;24(10):450–3. 18. Hasnis E, Bar-Shai M, Burbea Z, Reznick AZ. Mechanisms underlying cigarette smoke-induced NF-kappa B activation in human lymphocytes: the role of reactive nitrogen species. J Physiol Pharmacol 2007;58(5):275–87. 19. Kehrer JP, Biswal SS. The molecular effects of acrolein. Toxicol Sci 2000;57(1):6–15. 20. Chang SW, Lee SI, Bae WJ, Min KS, Shin ES, Oh GS, et al. Heat stress activates interleukin-8 and the antioxidant system via Nrf2 pathways in human dental pulp cells. J Endod 2009;35(9):1222–8. 21. Indart A, Viana M, Clapes S, Izquierdo L, Bonet B. Clastogenic and cytotoxic effects of lipid peroxidation products generated in culinary oils submitted to thermal stress. Food Chem Toxicol 2007;45(10):1963–7. 22. Choudhari SK, Chaudhary M, Gadbail AR, Sharma A, Tekade S. Oxidative and antioxidative mechanisms in oral cancer and precancer: a review. Oral Oncol 2014;50(1):10–8. 23. Nordmann R. Alcohol and antioxidant systems. Alcohol Alcohol 1994;29(5):513–22. 24. Seitz HK, Stickel F. Molecular mechanisms of alcohol-mediated carcinogenesis. Nat Rev Cancer 2007;7(8):599. 25. Scott DA, Singer DL. Suppression of overt gingival inflammation in tobacco smokers—clinical and mechanistic considerations. Int J Dent Hyg 2004; 2(3):104–10. 26. Giannopoulou C, Krause KH, Muller F. The NADPH oxidase NOX2 plays a role in periodontal pathologies. Semin Immunopathol 2008;30:273–8. https://doi.org/10.1007/s00281-008-0128-1. 27. Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med 2010; 49(11):1603–16. 28. Krifka S, Spagnuolo G, Schmalz G, Schweikl H. A review of adaptive mechanisms in cell responses towards oxidative stress caused by dental resin monomers. Biomaterials 2013;34(19):4555–63. 29. Gieseg S, Duggan S, Gebicki JM. Peroxidation of proteins before lipids in U937 cells exposed to peroxyl radicals. Biochem J 2000;350(1):215–8. 30. Wiseman H, Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J 1996;313(Pt 1):17. 31. Trueba GP, Sa´nchez GM, Giuliani A. Oxygen free radical and antioxidant defense mechanism in cancer. Front Biosci 2004;9:2029–44. 32. Mori K, Shibanuma M, Nose K. Invasive potential induced under long-term oxidative stress in mammary epithelial cells. Cancer Res 2004; 64(20):7464–72. 33. Shinohara M, Adachi Y, Mitsushita J, Kuwabara M, Nagasawa A, Harada S, et al. Reactive oxygen generated by NADPH oxidase 1 (Nox1) contributes to cell invasion by regulating matrix metalloprotease-9 production and cell migration. J Biol Chem 2010;285(7):4481–8. 34. Maulik N. Redox signaling of angiogenesis. Antioxid Redox Signal 2002;4(5):805–15. 35. Beevi SSS, Rasheed AMH, Geetha A. Evaluation of oxidative stress and nitric oxide levels in patients with oral cavity cancer. Jpn J Clin Oncol 2004;34(7):379–85. 36. Ant A, Inal E, Avci A, Genc M, Tuncel U, Sencan Z, et al. Oxidative stress in relation to adenosine deaminase, nitric oxide, nitric oxide synthase and xanthine oxidase in oral cavity cancer. Eur Respir J 2018;4(1):38–43. 37. Bagchi M, Bagchi D, Patterson EB, Tang L, Stohs SJ. Age-related changes in lipid peroxidation and antioxidant defense in Fischer 344 rats a. Ann N Y Acad Sci 1996;793(1):449–52. 38. Sies H. Oxidative stress: oxidants and antioxidants. Exp Physiol 1997;82(2):291–5. 39. Ma N, Murata M, Thanan R, Ohnishi S, Kawanishi S, Hiraku Y. 8-nitroguanine, a potential biomarker to evaluate the risk of inflammation-related carcinogenesis. INTECH Open Access Publisher; 2012. 40. Ma N, Tagawa T, Hiraku Y, Murata M, Ding X, Kawanishi S. 8-Nitroguanine formation in oral leukoplakia, a premalignant lesion. Nitric Oxide 2006;14(2):137–43. 41. Kawanishi S, Hiraku Y, Pinlaor S, Ma N. Oxidative and nitrative DNA damage in animals and patients with inflammatory diseases in relation to inflammation-related carcinogenesis. Biol Chem 2006;387(4):365–72. 42. Lu HH, Kao SY, Liu TY, Liu ST, Huang WP, Chang KW, et al. Areca nut extract induced oxidative stress and upregulated hypoxia inducing factor leading to autophagy in oral cancer cells. Autophagy 2010;6(6):725–37.

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43. Liu G, Zhou W, Wang LI, Park S, Miller DP, Xu LL, et al. MPO and SOD2 polymorphisms, gender, and the risk of non-small cell lung carcinoma. Cancer Lett 2004;214(1):69–79. 44. Kesarwala AH, Krishna MC, Mitchell JB. Oxidative stress in oral diseases. Oral Dis 2016;22(1):9–18. 45. Blachon S, Bellanger S, Demeret C, Thierry F. Nucleo-cytoplasmic shuttling of high risk human Papillomavirus E2 proteins induces apoptosis. J Biol Chem 2005;280(43):36088–98. 46. Lai D, Tan CL, Gunaratne J, Quek LS, Nei W, Thierry F, et al. Localization of HPV-18 E2 at mitochondrial membranes induces ROS release and modulates host cell metabolism. PLoS One 2013;8(9):e75625. 47. Williams VM, Filippova M, Filippov V, Payne KJ, Duerksen-Hughes P. Human papillomavirus type 16 E6* induces oxidative stress and DNA damage. J Virol 2014;88(12):6751–61.

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Chapter 7

Oxidative stress, epigenetics, and bladder cancer Chanchai Boonla Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

List of abbreviations BCG HPLC IHC LINE-1 RNS ROS SCC TCC TCGA TURBT

bacillus Calmette-Gu
erin high-pressure liquid chromatography immunohistochemistry long interspersed nuclear element-1 reactive nitrogen species reactive oxygen species squamous cell carcinoma transitional cell carcinoma The Cancer Genome Atlas transurethral resection of bladder tumor

Introduction Cancer is a leading cause of death worldwide. Bladder cancer is a malignancy of the urinary bladder. Statistically, bladder cancer is the 12th most common cancer reported in the GLOBOCAN 2018,1 and its incidence is progressively increasing. Bladder cancer is about three to four times more prevalent in men than women, occurring in later life around the sixth and seventh decades for men and women, respectively.2 Typically, bladder cancer diagnosed in women is more advanced and has a higher mortality rate than in men. According to the cellular origin, bladder cancer (90%) mostly arises from urothelial or transitional cells, the so-called urothelial carcinoma or previously known as transitional cell carcinoma (TCC). The rest (10%) are originated from nonurothelial cells including squamous cell carcinoma (SCC), adenocarcinoma, small-cell carcinoma, and sarcoma (very rare).2 The main important risk factors of bladder cancer remain cigarette smoking (RR: 3.5, 95% CI: 3.1–3.9 for current vs never smokers) and exposure to industrial carcinogens, e.g., aromatic amines and chemicals in paints and hair dyes.3 Chronic parasitic infection (urinary schistosomiasis) is a well-known bladder cancer risk (mostly for SCC) in the Middle East and Africa, where Schistosoma haematobium is endemic.4 Dietary components, including low intake of fluid, citrus fruits, and vegetables as well as high intake of processed meat and animal protein, increase the risk for bladder cancer.3 Additionally, genetic predisposition of bladder cancer development is demonstrated to some extent, for instances, polymorphisms in detoxification (NAT2, GSTM1) and urea transporter (SLC14A1) genes.3 Painless hematuria is a classic clinical presentation of bladder cancer. An infographic of the clinical overview of bladder cancer is shown in Fig. 1. In Thailand, bladder cancer is truly one of the life-threatening malignancies with an estimated incidence rate of 4.2 per 100,000 population in males and 1.3 per 100,000 population in women.5 The major risk factor found in Thai bladder cancer patients is cigarette smoking, but in some cases it is associated with pesticide/chemical exposure.6 In order to reduce the incidence of bladder cancer, a mechanistic insight into the urinary bladder carcinogenesis needs to be uncovered. Carcinogenesis is a process of transformation of normal cells to malignant cells that is primarily driven by the progressive accumulation of genetic mutations and epigenetic alterations. Bladder cancer is a highly and heterogeneously mutated malignancy with several mutations in signaling pathways and epigenetic regulatory genes.7 Several factors are known to initiate and promote genetic mutations and epigenetic changes in cancer, of which oxidative stress is one of the critical elements.8 Oxidative stress is caused by the overwhelming production of reactive oxygen species (ROS)

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00007-9 © 2021 Elsevier Inc. All rights reserved.

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FIG. 1 Infographic of overview of clinical features of bladder cancer. About 90% of bladder cancers arise from urothelium, called urothelial carcinoma. Urothelial carcinoma is classified into nonmuscle invasive (80%) and muscle invasive (20%) tumors. The nonmuscle invasive tumor recurs frequently, about 60%–70%. BCG, bacillus Calmette-Gu
erin; TCC, transitional cell carcinoma; TURBT, transurethral resection of bladder tumor.

together with depletion of defensive antioxidants that leads to oxidative cell injury and eventually cell death.9 This chapter focuses on how ROS and oxidative stress regulate changes in epigenetics in bladder cancer, specifically urothelial carcinoma.

Urothelial carcinoma Urothelial carcinoma is classified into two types based on spreading into the bladder wall, nonmuscle invasive or superficial tumors (80%) and muscle invasive tumors (20%) (Fig. 1). Muscle invasive tumors are more lethally aggressive, while nonmuscle invasive tumors are more frequently recurrent (about 60%–70%).2 Histological grading of nonmuscle invasive tumors categorizes the tumors into the least aggressive (low-grade, 93%) and the most aggressive (high-grade, 7%) tumors.2 The high-grade tumors usually progress toward the invasive stage. To justify the optimal treatment of bladder cancer, accurate tumor staging is crucial. The widely accepted T-stage classifies urothelial tumors based on the depth of penetration through the bladder layers. Nonmuscle invasive tumors are defined in stage 0 (consisting of Ta: papillary tumor and Tis: carcinoma in situ) and stage 1 (penetrated to laminar propia). Muscle invasive tumors are divided into three stages, stage 2 (muscle layer invasion), stage 3 (invaded to fat layer and perivesical tissues), and stage 4 (invaded to neighboring tissues and organs).10 Transurethral resection of bladder tumor (TURBT) is generally the treatment option for noninvasive tumors (stages 0 and 1). Adjuvant therapy, either intravesical chemotherapy or BCG immunotherapy or the combination following TURBT, is recommended in all nonmuscle invasive patients to prevent recurrence and progression toward muscle invasive tumors.11 The treatment approaches for muscle invasive tumors (stages 2–4) include cystectomy

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with neobladder reconstruction and the combination of three therapeutic modalities (deep TURBT, chemotherapy, and radiation).2 Five-year survival rates of the patients greatly depend on the tumor stages. It is drastically reduced from 98% in stage 0 to 15% in stage 4 (Fig. 1).2 Carcinogenesis of urothelial carcinoma is heterogeneous. Evidences demonstrate that nonmuscle invasive and muscle invasive bladder cancers have distinct molecular pathogenic pathways, called the dual-track development.10 Papillary Ta low-grade tumors arise from a simple hyperplasia of normal urothelial cells with oncogenic mutation of FGFR3 (accounted for 60%–70%). Invasive carcinoma is directly derived from the flat dysplasia and carcinoma in situ (Tis) with underlying TP53 mutation and RB1 loss.10 Additionally, activation of the MAPK signaling pathway is preferentially found in the noninvasive papillary tumors. According to the TCGA (The Cancer Genome Atlas) cohort, molecular subtypes of urothelial carcinoma are grouped into luminal and basal subtypes.12 In urothelial multilayers, the upper layer contains urothelial cells with the most differentiated phenotypes (called luminal cells), but the lower/basal layer (near the basement membrane) contains uroprogenitor cells (basal cells) with the least differentiation and self-renewal capacity. All low-grade papillary noninvasive (Ta) tumors are virtually originated from urothelial cells at the upper layer and designated as a luminal molecular subtype. In contrast, about half of the muscle invasive (T2) tumors are originated from the basal cells (progressively transformed from normal basal cells to Tis to T1, and then T2), the so-called basal molecular subtype. The other half of the muscle invasive tumors, however, are of luminal subtype deriving from the preexisting noninvasive papillary tumors (from normal luminal cells to Ta to T1, and then T2).12 Notably, luminal and basal subtypes have clearly distinct clinical behaviors and response different from chemotherapy. Therefore, the molecular subtype is important for implementing the appropriate therapeutic means.

Oxidative stress in bladder cancer Oxidative stress is a condition in which the defensive antioxidant system cannot cope with the abnormally produced ROS, resulting in oxidative damage. Relative to normal cells, ROS in cancer cells is persistently elevated both intracellularly and extracellularly, which is a consequence of gene mutation and oncogenic signaling, increased aerobic glycolysis (Warburg effect), and mitochondrial energetics, hypoxia, and chronic inflammation.13 ROS directly causes DNA lesions, and the high rate of oxidized lesion formation together with failure of DNA repair introduces a bunch of mutations in cells undergoing oxidative stress. Both direct and indirect oncogenic roles of ROS and oxidative stress in carcinogenesis and cancer progression are well established.13 The question is how cancer cells adapt and survive under the highly oxidative microenvironment. The main adaptive mechanisms of cancer cells employed to overcome oxidative stress are reprograming metabolism and empowering the antioxidative response through the Nrf2/Keap1/ARE axis. Oxidative stress vitally contributes to the development of bladder cancer.14 Several human studies reported an increased oxidative stress in patients with bladder cancer.15,16 ROS directly damages the cellular DNA and promotes tumor development not only through genetic mutations, but also through epigenetic alterations.17 Our study in patients with bladder cancer shows a significantly increased urinary level of 8-hydroxydeoxyguanosine (8-OHdG) and malondialdehyde (MDA) relative to healthy controls.6 An increased expression of 8-OHdG and 4-hydroxynonenol (4-HNE) in bladder cancer tissues is also demonstrated.16,18,19 These emphasize an increase in the oxidative stress extent in bladder cancer patients.

Epigenetics in cancer Epigenetics combines epi- and -genetics. The prefix epi, derived from Greek, means on, above, over, in addition to, after or near. In this sense, epigenetics refers to any modifications that occurred near genetic sequences or DNA, and those modifications must have a property of genetic inheritance. A widespread definition of epigenetics is the heritable chemical modifications such as DNA methylation and histone modification that alter gene expression without directly affecting or changing the DNA sequences. This raises an argument that some of the epigenetic modifications, for instance, modification of histones in response to DNA damage, are too transient to be regarded as heritable epigenetic marks. The redefined definition of epigenetic events is proposed to be “the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states.”20 It is now well accepted that the epigenetic pattern is altered in diseased states relative to normal physiological states, and the epigenetic alteration is one of the significant hallmarks of cancers. Two main epigenetic mechanisms that have been intensively investigated in cancers are DNA methylation and histone modification. DNA methylation reaction requires S-adenosylmethionine (SAM) methyl donor and DNA methyltransferase (DNMT) enzyme. The preference site for DNA methylation is cytosine (C) residue at the CpG dinucleotides to yield the 5-methylated cytosine (5mC) product. A large number of CpG dinucleotides are found in the promoter regions of genes, the so-called CpG islands. These CpG islands are usually unmethylated in actively expressing genes. Methylation of CpG islands

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interferes with binding of transcription factors and triggers heterochromatin formation, hence silencing the gene exp1ression. The common alterations of DNA methylation in human cancers are regional (site-specific CpG island promoter) hypermethylation of tumor suppressor genes and global DNA hypomethylation (genome-wide hypomethylation). Promoter hypermethylation of several tumor suppressor genes has been reported in several human cancers, exerting the oncogenic role to drive genetic instability, uncontrollable proliferation, apoptotic resistance, and abnormal differentiation.21 The pioneer studies of global DNA hypomethylation in human cancers appeared in 1983.22,23 Feinberg and Vogelstein used methylation-sensitive restriction enzymes to demonstrate a substantial proportion of methylated CpG dinucleotides in normal tissues and unmethylated CpGs in cancer cells.22 Gama-Sosa et al. measured 5mC content in DNA by HPLC and showed that metastatic tumors had significantly lower genomic 5mC content than benign tumors and normal tissues, respectively.23 The human genome contains a huge amount of the repetitive elements, and therefore hypomethylation of the cancer DNA usually occurs on these repetitive sequences. The hypomethylation of repetitive elements reactivates retrotransposons that further promote genomic instability and alter gene expression favoring oncogenesis.24 The most abundant and best characterized retrotransposon in the mammalian genome is the long interspersed nuclear element-1 (LINE-1). The methylation level of LINE-1 elements is widely used as a surrogate marker of global DNA hypomethylation. Hypomethylation of LINE-1 elements is demonstrated in many human cancers including bladder cancer.25 Regulation of gene expression through histone modification requires a remodeling of chromatin structures between euchromatin and heterochromatin states. Euchromatin is an open or active chromatin state that allows genes to be transcribed. Heterochromatin is a closed or compacted state of chromatin that blocks the accessibility of gene promoters to transcription factors, hence repressing the gene expression. Chemical modifications create epigenetic marks on histone proteins, the so-called histone marks or histone codes, and the lysine (K) residues located in the histone tails are the positions for such chemical modification. The H3K9me3 mark denotes trimethylation (me3) of the ninth lysine (K9) in the tail of histone subunit 3 (H3). The enzyme that generates the mark is called the epigenetic writer (such as histone methyltransferases, HMT), the protein that binds to the mark is called the epigenetic reader (such as heterochromatin protein 1, HP1), and the enzyme that removes the mark is called the epigenetic eraser (such as histone deacetylases, HDAC).26 Histone acetylation, catalyzed by histone acetylases (HAT), is a general signal for euchromatin formation and gene expression. In contrast, histone deacetylation, catalyzed by HDAC, is associated with heterochromatin formation and gene repression. In summary, histone modification exhibits roles for activating and silencing gene transcription depending upon the chromatin marks. The well-known marks for euchromatin formation include H3K4me3 and acetylated histone marks. The best characterized marks for heterochromatin are H3K9me3, H3K27me3, and H4K20me3.

Epigenetic alterations in urothelial carcinoma In urothelial carcinoma, global DNA hypomethylation and promoter hypermethylation are the key epigenetic alterations involved in carcinogenesis and progression.27 The promoters of many tumor suppressor genes (for instance, ARF, GSTP1, CDH1, and CDKN2A) are hypermethylated in bladder cancer.28,29 The Illumina GoldenGate methylation assay showed that nonmuscle invasive and muscle invasive tumors had different epigenetic regulated pathways.30 Invasive tumors had more hypermethylated loci (mostly located in the CpG islands) than nonmuscle invasive tumors. In contrast, hypomethylated loci (located outside CpG islands) are more prevalent in the nonmuscle invasive tumors. The expression of 5mC in urinary exfoliated cells from bladder cancer patients is lower than that in the exfoliative urothelial cells from healthy volunteers,31 suggesting that the global DNA hypomethylation in urothelial carcinoma could be detected by urine cytology. An immunohistochemical (IHC) study demonstrates that 5mC expression in urothelial carcinoma tissues is lower than in normal urothelial tissues, and the decreased 5mC expression is associated with an advanced tumor stage and high degree of inflammation.32 Our IHC data confirm that 5mC expression in bladder cancer tissues is diminished relative to the noncancerous tissues.18 In addition, the level of 5mC in the leukocyte DNA of bladder cancer patients is significantly lower than that in the matched controls.33 These human studies verify the occurrence of global DNA hypomethylation in urothelial carcinoma. The hypomethylation of LINE-1 elements in urothelial carcinoma is firstly demonstrated in urothelial cell lines and tumor tissues by Schulz’s group using methylation-sensitive restriction enzymes (HpaII/MspI) and Southern blotting.34 An increased LINE-1 hypomethylation in bladder tumor tissues is later confirmed by bisulfite-PCR pyrosequencing.35 Hypomethylation of LINE-1 is associated with increased LINE-1 transcript expression.24 Hypomethylation of LINE-1 in peripheral blood cells is associated with an increased risk for bladder cancer.36 In addition to LINE-1 elements, DNA hypomethylation of other retroelements such as HERV-K and AluY elements are also demonstrated in human bladder cancer tissues and bladder cancer cell lines.37 Additionally, the degree of DNA hypomethylation in the muscle invasive cell lines is higher than in the papillary-origin cell lines and normal urothelial cell lines.

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We firstly report the hypomethylation of LINE-1 in urinary exfoliated cells of bladder cancer patients, and we show that the combination of two measurements, level of LINE-1 hypomethylation loci in urinary exfoliated cells and level of protein carbonyl in plasma (as oxidative stress marker), provides the best accuracy for bladder cancer diagnosis.38 Later, we demonstrate that 5mC expression is decreased, but 8-OHdG and 4-HNE expression are increased in human bladder cancer tissues relative to the noncancerous bladder tissues.18,19 The findings underline the fact that urothelial carcinoma has global DNA hypomethylation that coincidentally occurs with increased oxidative stress. Normal chromatin remodeling is dependent on intact functions of chromatin modifying enzymes (e.g., histone methyltransferases, demethylases, and acetylases) and members of the large chromatin remodeling protein complex (e.g., polycomb complex and SWI/SNF complex). Thus, mutations in these genes are investigated to see if they have any oncogenic contribution to bladder cancer development. A molecular analysis of the TCGA cohort of 412 muscle invasive bladder cancers showed that about 3% (10/39) of significantly mutated genes were genes related to chromatin remodeling (such as KDM6A, CREBBP, and ARID1A).39 Furthermore, a truncating ARID1A mutation (Q403*) is found to associate with poor prognosis in patients with urothelial carcinoma, and the loss of ARID1A protein expression is observed in the more aggressive tumors.40 Therefore, mutations in this group of genes drive and/or promote the development of urothelial carcinoma. Alteration of histone modification in urothelial cancer has not yet been intensively investigated. Seven chromosomal regions (containing tumor suppressor genes such as PLCD1, DLEC1, and HOXA5) silenced by an epigenetic mechanism are identified in bladder cancer and called the multiple regional epigenetic silencing (MRES) phenotype.41 This silencing phenotype is associated with H3K9 and H3K27 methylation and H3K9 hypoacetylation. Additionally, the MRES phenotype is associated with the gene expression signature of the Tis tumor rather than of the Ta papillary tumor, suggesting that this silencing phenotype contributes to the development and progression of carcinoma in situ of bladder cancer. The tissue microarray study showed that the global level of heterochromatin marks (H3K9me3 and H3K27me3) in bladder cancer tissues was lower than that in the normal urothelium.42 Later, this group reported a significantly decreased level of H3ac in bladder cancer tissues compared to normal urothelial samples, but a significant change in H3K18ac and H4ac levels between the two groups was not observed.43 Global reduction of H4K20me3 expression was observed in bladder cancer tissues relative to normal urothelial tissues, and it was associated with poor prognosis in patients with muscle invasive tumors.44 Our study showed that H3K9me3 and its reader HP1a were overexpressed in bladder cancer tissues relative to the noncancerous counterparts, and they were positively associated with 8-OHdG expression.18 These evidences indicate that histone modification is altered in urothelial cancer, and this histone modification alteration coexisted with increased oxidative stress. Taken together, the alteration of epigenetics (both DNA methylation and histone modification) in cancer frequently coincided with increased oxidative stress, suggesting that they might have a cause-and-effect relationship.

ROS alters DNA methylation in urothelial carcinoma Many evidences suggest that epigenetic alteration is a consequence of ROS attack and a response to oxidative stress.45,46 ROS-induced oxidative stress is associated with promoter hypermethylation of tumor suppressor genes and hypomethylation of repetitive elements,46 suggesting that the mechanisms of ROS-induced aberrant DNA methylation between gene and nongene sequences are different. ROS-induced promoter hypermethylation has been proposed to mediate via at least three mechanisms.46 First, ROS acts as a catalyst of DNA methylation reaction to accelerate the addition of the methyl group to C residue.47 Second, a large silencing complex containing DNMTs (e.g., polycomb repressive complex 4) is formed and recruited to promoters of genes under oxidative stress in response to ROS-induced DNA damage, and DNMTs in this polycomb complex facilitate DNA methylation at the promoter regions causing hypermethylation and transcriptional silencing.48 Third, ROS directly upregulates DNMT expression.49 ROS induces DNA hypomethylation via at least four possible mechanisms.46,50 First, ROS directly causes oxidized lesions, notably 8-OHdG. The 8-OHdG lesion at CpG sites strongly inhibits the catalytic function of DNMTs leading to DNA hypomethylation. Second, 8-OHdG is a premutagenic lesion that must be fixed. A failure of 8-OHdG repair at the CpG site introduces a G-to-T transversion resulting in loss of the CpG site. Third, ROS actively triggers the DNA demethylation process. ROS directly attacks the 5mC and converts it into 5-hydroxymethylcytosine (5-hmC). The 5hmC is also produced from the oxidation of 5mC by the ten-eleven translocation (TET) enzymes. These 5hmC lesions are passively changed to C residues through the normal DNA replication process. Alternatively, these 5-hmC lesions are actively reverted to C residues through the iterative oxidation and thymine DNA glycosylase (TDG)-mediated base excision repair.51 Fourth, oxidative stress indirectly induces DNA hypomethylation through SAM depletion. SAM is an important cellular methyl donor produced from methionine in the one-carbon metabolism pathway. This one-carbon metabolism pathway is connected to glutathione (GSH) biosynthesis via the transsulfuration pathway, a pathway to generate

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cysteine (Cys) from homocysteine (Hcy).52 The common intermediate of these two pathways is Hcy. During the antioxidative response to ROS, GSH progressively and actively produced, and Hcy is fluxed to transsulfuration for synthesizing Cys, a GSH precursor. This leads to less production of methionine and subsequently causes inadequacy of SAM synthesis. SAM depletion directly affects DNA methylation reaction and results in DNA hypomethylation. We reported that exposure of the bladder cancer cell line to H2O2 caused hypomethylation of LINE-1 elements, and the treatment with tocopheryl acetate (antioxidant) prevented the LINE-1 hypomethylation.53 Later, we demonstrated that ROS was able to induce LINE-1 hypomethylation in both normal kidney cells (HK-2) and bladder cancer cell lines (UM-UC-3 and TCCSUP), and this ROS-induced LINE-1 hypomethylation, at least in part, mediates through oxidative stress-induced SAM depletion.52 However, it should be noted that the oxidative stress-induced DNA methylation change might appear to be a temporal effect rather than a permanent change.49

ROS causes alteration of histone modification in bladder cancer Oxidative stress-induced chromatin alterations mediate several cellular processes involved in adaptively responding to stress and driving the disease pathogenesis.45 ROS and reactive nitrogen species (RNS) can directly modify histone proteins, causing changes in protein folding and stability. In vitro, H1 and H3 histones exposed to peroxynitrite causes tyrosine nitration, and these nitrated histones have increased thermostability. Reactive aldehydes readily react with histone proteins forming carbonyl adducts that further lead to histone degradation and nucleosome loss. Glutathionylation, an addition of GSH to Cys residue in protein, is a frequent chemical modification of proteins (including histones) in cells undergoing oxidative stress. Increased glutathionylated proteins are associated with increased GSH level and extent of drug resistance. Glutathionylation of histones is shown to decrease the stability of nucleosomes.45 In urothelial carcinoma, we show that ROS induces tumor progressivity,19 and causes profound changes in histone modifications (both methylation and acetylation) in bladder cancer cell lines.54 In some bladder cancer cell lines, ROS induced LINE-1 protein expression, and this ROS-induced LINE-1 expression was accompanied by increased formation of active chromatin on full-length LINE-1 elements. Our finding suggests that ROS induces a change in histone modification that favors active chromatin formation on full-length LINE-1 elements and allows LINE-1 protein expression, hence enhancing tumor progression. In summary, ROS behaves as a double-edged sword with both physiological and pathological functions depending on the level of production and state of balance. Increased ROS production is the main cause of oxidative stress that is critically involved in the initiation and progression of bladder cancer. Oxidative stress globally and locally influences epigenetic alteration that further drives the disease progression. The more the understanding of how oxidative stress induces changes in DNA methylation and chromatin structure, the more the chances of finding the target for drug development. Inhibiting both oxidative stress and epigenetic change could be an effective therapeutic approach for oxidative stress-mediated pathologies, including urothelial carcinoma.

Oxidative stress and epigenetic change in other diseased conditions Oxidative stress is implicated not only in the pathogenesis of cancer, but also in several other chronic diseases. Amyloid plaque formation is induced by ROS in Alzheimer’s disease. ROS oxidizes LDL and contributes to atherosclerotic plaque formation. Hyperglycemia induces endothelial ROS formation and vesicular dysfunction in diabetes. Calcium oxalate crystals induce ROS production and inflammatory reaction in renal tubular cells that mediate the urinary stone pathogenesis.9 Epigenetic alterations are parallelly found in these ROS-mediated pathologies. ROS is proposed to be a cause of dysregulated epigenetics. A study in bronchial epithelial cells shows that H2O2 causes increased H3K4me3, but decreased H3K9ac and H4K8ac expression, and vitamin C attenuates these changes.55 As Fe(II) is a cofactor of the histone demethylase enzyme, it is proposed that ROS oxidizes Fe(II) to form Fe(III), causing a reduction of histone demethylase activity. Additionally, this study shows that the altered histone modification can be reversed after removal of H2O2. Therefore, it is concluded that oxidative stress transiently alters the epigenetic pattern via modulation of the histone modifying enzyme activity.55 We demonstrate an increased extent of oxidative stress in hepatocellular carcinoma (HCC). Plasma protein carbonyl content is higher, but plasma total antioxidant capacity (TAC) is lower in HCC patients than in healthy controls.56 Nrf2 and 8-OHdG expression are increased in cancerous liver tissues of HCC patients.57 Furthermore, increased 8-OHdG expression in HCC tissues is associated with shorter survival. Experimentally, H2O2 induces Nrf2 and 8-OHdG expression and enhances migration and invasion in the HCC cell line.57 This suggests the oncogenic role of ROS in HCC progression. Change in Nrf2 promoter methylation was not observed in the H2O2-treated HCC cells, suggesting that ROS-induced

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Nrf2 expression was not likely to be regulated through the promoter DNA methylation. In case of the tumor suppressor gene, we demonstrate that RUNX3 promoter methylation in peripheral blood mononuclear cells of HCC patients is higher than that of healthy subjects, and H2O2 experimentally increases RUNX3 promoter methylation in the HCC cell line. For histone modification, our IHC data show that histone marks for heterochromatin formation (H4K20me3, H3K9me3, and H3K27me3) are upregulated in human HCC tissues relative to the noncancerous liver tissues. In vitro experiment in HCC cell lines demonstrate that H2O2 causes upregulation of these three heterochromatic marks. Our findings support the hypothesis that ROS and oxidative stress promote cancer genesis and progression through epigenetic dysregulation.

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Oxidative stress is increased in bladder cancer patients. Global DNA hypomethylation, promoter hypermethylation of tumor suppressor genes, and altered histone modification are frequently found in urothelial carcinoma, and these epigenetic alterations play critical roles in tumorigenesis and progression. Increased oxidative stress is associated with LINE-1 hypomethylation in patients with bladder cancer. LINE-1 protein expression is increased in bladder cancer tissues, and its elevation is associated with increased oxidative stress and advanced tumor stage. ROS and oxidative stress cause alterations of DNA methylation and histone modification in urothelial carcinoma. ROS induces tumor aggressiveness in bladder cancer cell lines. ROS induces hypomethylation of LINE-1, at least in part, through depletion of methyl donor SAM. ROS enhances reactivation of full-length LINE-1 elements in some bladder cancer cell lines by the increased formation of active chromatin at full-length LINE-1 promoters. Attenuation of oxidative stress together with reversal of epigenetic abnormality could be the new therapeutic approach for preventing the development and decelerating the progression of bladder cancer.

References 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68(6):394–424. 2. Berdik C. Unlocking bladder cancer. Nature 2017;551(7679):S34–5. 3. Cumberbatch MGK, Jubber I, Black PC, Esperto F, Figueroa JD, Kamat AM, et al. Epidemiology of bladder cancer: a systematic review and contemporary update of risk factors in 2018. Eur Urol 2018;74(6):784–95. 4. Sarant L. Egypt: the flatworm’s revenge. Nature 2017;551(7679):S46–7. 5. Sriplung H. Urinary bladder. In: Khuhaprema T, Srivatanakul P, Sriplung H, Wiangnon S, Sumitsawan Y, Attasara P, editors. Cancer in Thailand. Bangkok: National Cancer Institute Thailand; 2007. 6. Opanuraks J, Boonla C, Saelim C, Kittikowit W, Sumpatanukul P, Honglertsakul C, et al. Elevated urinary total sialic acid and increased oxidative stress in patients with bladder cancer. Asian Biomed 2010;4(5):703–10. 7. Audenet F, Attalla K, Sfakianos JP. The evolution of bladder cancer genomics: what have we learned and how can we use it? Urol Oncol 2018; 36(7):313–20. 8. Moloney JN, Cotter TG. ROS signalling in the biology of cancer. Semin Cell Dev Biol 2018;80:50–64. 9. Boonla C. In:Filip C, Albu E, editors. Oxidative stress in urolithiasis, reactive oxygen species (ROS) in living cells. IntechOpen; 2018. https://doi.org/ 10.5772/intechopen.75366 (May 23rd 2018). Available from: https://www.intechopen.com/books/reactive-oxygen-species-ros-in-living-cells/oxi dative-stress-in-urolithiasis. 10. Knowles MA, Hurst CD. Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity. Nat Rev Cancer 2015; 15(1):25–41. 11. Babjuk M, Burger M, Comperat EM, Gontero P, Mostafid AH, Palou J, et al. European Association of Urology guidelines on non-muscle-invasive bladder cancer (TaT1 and carcinoma in situ)—2019 update. Eur Urol 2019;76(5):639–57. 12. Czerniak B, Dinney C, McConkey D. Origins of bladder cancer. Annu Rev Pathol 2016;11:149–74. 13. Liao Z, Chua D, Tan NS. Reactive oxygen species: a volatile driver of field cancerization and metastasis. Mol Cancer 2019;18(1):65. 14. Michaud DS. Chronic inflammation and bladder cancer. Urol Oncol 2007;25(3):260–8. 15. Akcay T, Saygili I, Andican G, Yalcin V. Increased formation of 8-hydroxy-20 -deoxyguanosine in peripheral blood leukocytes in bladder cancer. Urol Int 2003;71(3):271–4. 16. Soini Y, Haapasaari KM, Vaarala MH, Turpeenniemi-Hujanen T, Karja V, Karihtala P. 8-hydroxydeguanosine and nitrotyrosine are prognostic factors in urinary bladder carcinoma. Int J Clin Exp Pathol 2011;4(3):267–75. 17. Wachsman JT. DNA methylation and the association between genetic and epigenetic changes: relation to carcinogenesis. Mutat Res 1997;375(1):1–8. 18. Whongsiri P, Pimratana C, Wijitsettakul U, Sanpavat A, Jindatip D, Hoffmann MJ, et al. Oxidative stress and LINE-1 reactivation in bladder cancer are epigenetically linked through active chromatin formation. Free Radic Biol Med 2019;134:419–28.

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19. Whongsiri P, Pimratana C, Wijitsettakul U, Jindatip D, Sanpavat A, Schulz WA, et al. LINE-1 ORF1 protein is up-regulated by reactive oxygen species and associated with bladder urothelial carcinoma progression. Cancer Genomics Proteomics 2018;15(2):143–51. 20. Bird A. Perceptions of epigenetics. Nature 2007;447:396. 21. Pfeifer GP. Defining driver DNA methylation changes in human cancer. Int J Mol Sci 2018;19(4):1166. 22. Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 1983;301(5895): 89–92. 23. Gama-Sosa MA, Slagel VA, Trewyn RW, Oxenhandler R, Kuo KC, Gehrke CW, et al. The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Res 1983;11(19):6883–94. 24. Aporntewan C, Phokaew C, Piriyapongsa J, Ngamphiw C, Ittiwut C, Tongsima S, et al. Hypomethylation of intragenic LINE-1 represses transcription in cancer cells through AGO2. PLoS One 2011;6(3):e17934. 25. Chalitchagorn K, Shuangshoti S, Hourpai N, Kongruttanachok N, Tangkijvanich P, Thong-ngam D, et al. Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene 2004;23(54):8841–6. 26. Falkenberg KJ, Johnstone RW. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat Rev Drug Discov 2014;13(9):673–91. 27. Schulz WA, Goering W. DNA methylation in urothelial carcinoma. Epigenomics 2016;8(10):1415–28. 28. Porten SP. Epigenetic alterations in bladder cancer. Curr Urol Rep 2018;19(12):102. 29. Besaratinia A, Cockburn M, Tommasi S. Alterations of DNA methylome in human bladder cancer. Epigenetics 2013;8(10):1013–22. 30. Wolff EM, Chihara Y, Pan F, Weisenberger DJ, Siegmund KD, Sugano K, et al. Unique DNA methylation patterns distinguish noninvasive and invasive urothelial cancers and establish an epigenetic field defect in premalignant tissue. Cancer Res 2010;70(20):8169–78. 31. Seifert HH, Schmiemann V, Mueller M, Kazimirek M, Onofre F, Neuhausen A, et al. In situ detection of global DNA hypomethylation in exfoliative urine cytology of patients with suspected bladder cancer. Exp Mol Pathol 2007;82(3):292–7. 32. Chung CJ, Chang CH, Chuu CP, Yang CR, Chang YH, Huang CP, et al. Reduced 5-methylcytosine level as a potential progression predictor in patients with T1 or non-invasive urothelial carcinoma. Int J Mol Sci 2014;16(1):677–90. 33. Moore LE, Pfeiffer RM, Poscablo C, Real FX, Kogevinas M, Silverman D, et al. Genomic DNA hypomethylation as a biomarker for bladder cancer susceptibility in the Spanish Bladder Cancer Study: a case-control study. Lancet Oncol 2008;9(4):359–66. 34. Jurgens B, Schmitz-Drager BJ, Schulz WA. Hypomethylation of L1 LINE sequences prevailing in human urothelial carcinoma. Cancer Res 1996; 56(24):5698–703. 35. Choi SH, Worswick S, Byun HM, Shear T, Soussa JC, Wolff EM, et al. Changes in DNA methylation of tandem DNA repeats are different from interspersed repeats in cancer. Int J Cancer 2009;125(3):723–9. 36. Wilhelm CS, Kelsey KT, Butler R, Plaza S, Gagne L, Zens MS, et al. Implications of LINE1 methylation for bladder cancer risk in women. Clin Cancer Res 2010;16(5):1682–9. 37. Kreimer U, Schulz WA, Koch A, Niegisch G, Goering W. HERV-K and LINE-1 DNA methylation and reexpression in urothelial carcinoma. Front Oncol 2013;3:255. 38. Patchsung M, Boonla C, Amnattrakul P, Dissayabutra T, Mutirangura A, Tosukhowong P. Long interspersed nuclear element-1 hypomethylation and oxidative stress: correlation and bladder cancer diagnostic potential. PLoS One 2012;7(5):e37009. 39. Robertson AG, Kim J, Al-Ahmadie H, Bellmunt J, Guo G, Cherniack AD, et al. Comprehensive molecular characterization of muscle-invasive bladder cancer. Cell 2017;171(3):540–56. e525. 40. Balbas-Martinez C, Rodriguez-Pinilla M, Casanova A, Dominguez O, Pisano DG, Gomez G, et al. ARID1A alterations are associated with FGFR3wild type, poor-prognosis, urothelial bladder tumors. PLoS One 2013;8(5):e62483. 41. Vallot C, Stransky N, Bernard-Pierrot I, Herault A, Zucman-Rossi J, Chapeaublanc E, et al. A novel epigenetic phenotype associated with the most aggressive pathway of bladder tumor progression. J Natl Cancer Inst 2011;103(1):47–60. 42. Ellinger J, Bachmann A, G€oke F, Behbahani TE, Baumann C, Heukamp LC, et al. Alterations of global histone H3K9 and H3K27 methylation levels in bladder cancer. Urol Int 2014;93(1):113–8. 43. Ellinger J, Schneider AC, Bachmann A, Kristiansen G, Muller SC, Rogenhofer S. Evaluation of global histone acetylation levels in bladder cancer patients. Anticancer Res 2016;36(8):3961–4. 44. Schneider AC, Heukamp LC, Rogenhofer S, Fechner G, Bastian PJ, von Ruecker A, et al. Global histone H4K20 trimethylation predicts cancerspecific survival in patients with muscle-invasive bladder cancer. BJU Int 2011;108(8 Pt 2):E290–6. 45. Kreuz S, Fischle W. Oxidative stress signaling to chromatin in health and disease. Epigenomics 2016;8(6):843–62. 46. Wu Q, Ni X. ROS-mediated DNA methylation pattern alterations in carcinogenesis. Curr Drug Targets 2015;16(1):13–9. 47. Afanas’ev I. New nucleophilic mechanisms of ros-dependent epigenetic modifications: comparison of aging and cancer. Aging Dis 2014;5(1):52–62. 48. O’Hagan HM, Wang W, Sen S, Destefano SC, Lee SS, Zhang YW, et al. Oxidative damage targets complexes containing DNA methyltransferases, SIRT1, and polycomb members to promoter CpG Islands. Cancer Cell 2011;20(5):606–19. 49. Campos AC, Molognoni F, Melo FH, Galdieri LC, Carneiro CR, D’Almeida V, et al. Oxidative stress modulates DNA methylation during melanocyte anchorage blockade associated with malignant transformation. Neoplasia 2007;9(12):1111–21. 50. Franco R, Schoneveld O, Georgakilas AG, Panayiotidis MI. Oxidative stress, DNA methylation and carcinogenesis. Cancer Lett 2008;266(1):6–11. 51. Kohli RM, Zhang Y. TET enzymes, TDG and the dynamics of DNA demethylation. Nature 2013;502(7472):472–9. 52. Kloypan C, Srisa-Art M, Mutirangura A, Boonla C. LINE-1 hypomethylation induced by reactive oxygen species is mediated via depletion of S-adenosylmethionine. Cell Biochem Funct 2015;33:375–85.

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53. Wongpaiboonwattana W, Tosukhowong P, Dissayabutra T, Mutirangura A, Boonla C. Oxidative stress induces hypomethylation of LINE-1 and hypermethylation of the RUNX3 promoter in a bladder cancer cell line. Asian Pac J Cancer Prev 2013;14(6):3773–8. 54. Whongsiri P, Phoyen S, Boonla C. Oxidative stress in urothelial carcinogenesis: measurements of protein carbonylation and intracellular production of reactive oxygen species. Methods Mol Biol 2018;1655:109–17. 55. Niu Y, DesMarais TL, Tong Z, Yao Y, Costa M. Oxidative stress alters global histone modification and DNA methylation. Free Radic Biol Med 2015;82:22–8. 56. Poungpairoj P, Whongsiri P, Suwannasin S, Khlaiphuengsin A, Tangkijvanich P, Boonla C. Increased oxidative stress and RUNX3 hypermethylation in patients with hepatitis B virus-associated hepatocellular carcinoma (HCC) and induction of RUNX3 hypermethylation by reactive oxygen species in HCC cells. Asian Pac J Cancer Prev 2015;16(13):5343–8. 57. Ma-On C, Sanpavat A, Whongsiri P, Suwannasin S, Hirankarn N, Tangkijvanich P, et al. Oxidative stress indicated by elevated expression of Nrf2 and 8-OHdG promotes hepatocellular carcinoma progression. Med Oncol 2017;34(4):57.

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Chapter 8

Linking oxidative stress and ovarian cancers Tsukuru Amanoa and Tokuhiro Chanob a

Department of Obstetrics & Gynecology, Shiga University of Medical Science, Ostu, Japan, b Department of Clinical Laboratory Medicine and Medical

Genetics, Shiga University of Medical Science, Ostu, Japan

Lists of abbreviations LGSC HGCS MC EC OCCC HNF1B ROS SOD2 RTK MSI-H PI3K/AKT/mTOR PIK3CA MMR SWI/SNF PTEN HER2 ARL4C TERT EZH2 BET

low-grade serous carcinoma high-grade serous carcinoma mucinous carcinoma endometrioid carcinoma ovarian clear cell carcinoma hepatocyte nuclear factor 1 homeobox B reactive oxygen species mitochondrial superoxide dismutase receptor tyrosine kinase high-level microsatellite instability phosphoinositide 3-kinase/AKT/mammalian target of rapamycin EGF/Ras/MAPK epidermal growth factor/Ras/mitogen-activated protein kinase phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha mismatch repair switch/sucrose nonfermentable phosphatase and tensin homolog human epidermal growth factor receptor 2 ADP-ribosylation factor-like 4C telomerase reverse transcriptase enhancer of zeste homolog 2 bromodomain and extra-terminal domain

Introduction Ovarian cancer is the eighth most common cancer affecting women in the world and the estimated number of new cases in 2018 is 295,000.1 Ovarian cancer has a high mortality rate; in 2018, approximately 14,000 and 4800 deaths due to ovarian cancer were reported in the United States and Japan, respectively.2, 3 Ovarian cancers can be classified into the following five pathological types: Low-grade serous carcinomas (LGSCs), high-grade serous carcinomas (HGSCs), mucinous carcinomas (MCs), endometrioid carcinomas (ECs), and clear cell carcinomas. LGSCs harbor somatic mutations such as KRAS, BRAF, PTEN, PIK3CA, CTNNB1, ARID1A, and PPP2R1A; however, neither TP53 mutation nor BRCA mutations have been detected in LSGCs.4 Contrastingly, in HGSC cases, TP53 mutations are detected in a majority of the cases, and homologous recombination deficiency (HRD) including BRCA inactivation is also reported in approximately 50% of the cases.5, 6 Recent studies advocate that most of the HGSCs and LGSCs arise from epithelial lesions on the fimbriated end of the fallopian tube.7–9 LGSCs develop through a serous borderline tumor and some HGSCs evolve from serous tubal intraepithelial carcinoma. Ovarian MCs are often associated with KRAS mutations. Lately, a majority of them are considered as metastatic tumors derived from the gastrointestinal tract, while only 3% of MCs are regarded as truly originating from the ovary.10 Although ovarian ECs and ovarian clear cell carcinomas (OCCCs) are both known as endometriosis associated

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00008-0 © 2021 Elsevier Inc. All rights reserved.

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cancers, molecular biological features and gene expression profiling between ovarian ECs and OCCCs are different. Ovarian ECs display PTEN loss and abnormalities not only in CTNNB1, but also in positive estrogen and progesterone receptors.11 This suggests that ovarian ECs may be hormone-induced cancers. In contrast, OCCCs rarely show positive estrogen and progesterone receptors, and are often associated with ARID1A and PIC3CA mutations and overexpression of hepatocyte nuclear factor 1 homeobox B (HNF1B)12–14 (Table 1). Among these five pathological types of ovarian cancers, OCCCs are most strongly linked to oxidative stress. Oxidative stress causes cancer by directly damaging DNA. Recently, it has been elucidated that molecular abnormalities involved in oxidative stress generation are deeply involved not only in carcinogenesis, but also in cancer progression, such as cancer invasion and metastasis. The involvement of oxidative stress in OCCC can be divided into the following two categories: (a) carcinogenesis from endometriosis to OCCC; (b) factors related to treatment after carcinogenesis. In this review, the process and prevention of carcinogenesis, nature of tumors, and treatment after carcinogenesis, particularly with respect to OCCCs will be discussed.

Oxidative stress promotes carcinogenesis from endometriosis to OCCCs A pooled analysis of case-control studies indicated that endometriosis is associated with a significantly increased risk of OCCC.15 The chocolate (endometriotic) cysts contain old blood with excess iron. Iron and its metabolites contribute to the generation of reactive oxygen species (ROS) through the Fenton reaction, acting as inducers of DNA damage and subsequent carcinogenesis.16 In fact, the carcinogenicity of iron compounds has been clearly demonstrated in past animal experiments. Interestingly, several studies have shown that renal clear cell carcinomas, which have molecular features similar to OCCCs, were produced by intraperitoneal injection of iron chelates.17, 18 Oxidative stress induced by excess heme production and iron accumulation was also revealed to be an important trigger in the malignant transformation of endometriosis to OCCC.19

Attempts to prevent development of OCCCs from endometriosis In OCCCs, abnormalities are often found in genes corresponding to oxidative stress and metabolism of ROS.20 Elimination of persistent inflammation and ROS are important to prevent carcinogenesis from endometriosis to OCCC. Surgical treatment is one of the beneficial procedures to prevent the cancellation from endometriosis. A nested case-control study in Sweden revealed that compared to controls, one-sided oophorectomy or radical extirpation of all visible endometriosis reduced the risk of later development of ovarian cancer to 19% and 30%, respectively.21 Hormonal therapy using low dose estrogen-progestin or dienogest (progestin medication) suppresses the progression of endometriosis to OCCC. After 5 years of oral contraception use, a 21.3% reduction in risk of OCCC has been reported.22 However, several cases of OCCCs arising TABLE 1 Genetic and biological features of ovarian carcinomas. Pathological type

Precursor lesion

LGSC

Genetic abnormality

Other features

SBT

KRAS, BRAF, PTEN, PIK3CA, CTNNB1, ARID1A, PPP2R1A

Moderate sensitivity to conventional chemotherapy

HGSC

STIC

TP53, BRCA

High sensitivity to conventional chemotherapy

MC

MBT

KRAS

Low sensitivity to chemotherapy, mostly metastasis from gastrointestinal tract

EC

Endometriosis

CTNNB1, PTEN

positive estrogen and progesterone receptor, occasionally sensitive to hormonal therapy

OCCC

Endometriosis

ARID1A, PIC3CA

Low sensitivity to chemotherapy, overexpression of antioxidant molecules (HNF-1B, SOD2)

LGSC, low-grade serous carcinom; SBT, serous borderline tumor; HGSC, high-grade serous carcinoma; STIC, serous intraepithelial carcinoma; MC, mucinous carcinoma; MBT, mucinous borderline tumor; EC, endometrioid carcinoma; OCCC, ovarian clear cell carcinoma; HNF-1B, hepatocyte nuclear factor 1 homeobox B; SOD2, mitochondrial superoxide dismutase. Although ECs and OCCCs arise from endometriosis, they differ in the genetic abnormalities and biological features. Among five pathological types of ovarian cancers, OCCCs are considered to be strongly linked to oxidative stress.

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from endometrioma during hormonal treatments have also been reported.23 In particular, it is unclear whether dienogest prevents the growth of OCCC from endometrioma. As oxidative stress is involved in the formation of OCCC, antioxidants intake may be effective in preventing its development. Vitamin C, vitamin E, carotenes (vitamin A), flavonoids, and isothiocyanate are known antioxidant supplements.24–27 Diets rich in vegetables and fruits, which are good sources of antioxidants, have been found to be healthy. Antioxidants may prevent or delay various steps associated with carcinogenesis.28–30 Carotenoid astaxanthin reduces oxidative stress and inflammation,31, 32 and exerts highly protective antioxidant33 and anticancer properties.34–36 Flavonoids inhibit multiple enzymes involved in cancer cell growth and arrest the cell cycle and tumor regression by activating the mitochondrial pathway of apoptosis.37, 38 Isothiocyanate inhibits the growth of ovarian cancer cells by inducing apoptosis in in vitro experiments.39 Animal experiments have demonstrated that isothiocyanate exerts inhibitory effects on the carcinogenesis of both forestomach and lung cancers induced by the carcinogen benzopyrene.40 Based on the results of studies, the effects of daily intake of antioxidant supplements on cancer were investigated in large-scale clinical trials (Table 2).41–49 The Physicians’ Health Study II randomized controlled trial revealed that neither vitamin C nor vitamin E supplements prevents prostate cancer,41 and the Women’s Antioxidant Cardiovascular Study concluded that vitamin C, vitamin E, or beta carotene supplementation is not beneficial in the primary prevention of total cancer incidence or cancer mortality.43 A large-scale population-based prospective study in Japan suggested that the intake of cruciferous vegetables containing abundant isothiocyanate might be associated with reduction in lung cancer risk among men who are currently nonsmokers.47 However, it is unclear whether this effect was entirely dependent on isothiocyanate consumption. Flavonoids have been reported to prevent carcinogenesis in some meta-analyses. Although there are limited evidences, dietary flavonoids have been reported to be associated with decreased risk of lung, stomach, and ovarian cancers.48, 49 In these studies, the subjects comprised members of the general population who did not necessarily have increased oxidative stress. In addition, to date, it has not been considered that some of the antioxidants may become prooxidants, nor has it been concluded that antioxidants cannot prevent cancers. Therefore, antioxidants intake may help to prevent this disease in people with increased oxidative stress, in the same way as extirpation of endometriosis reduced the risk of later development of ovarian cancer. Again, because OCCC carcinogenesis is particularly affected by oxidative stress, antioxidants may be effective for its prevention. At present, surgical treatment and hormonal therapy are suitable for preventing carcinogenesis from chocolate cysts. However, therapies consisting of strong antioxidants, flavonoids, and isothiocyanate require further investigation.

TABLE 2 Antioxidant supplements or food for cancer prevention. Clinical research

Antioxidant

Result

Reference

PHS II

Vitamin C, vitamin E

No reduction in the risk of prostate or general cancer

41

SU.VI.MAX

Vitamin C, vitamin E, Beta carotene

Lowered cancer incidence and all-cause mortality in men

42

WACS

Vitamin C, vitamin E, Beta carotene

No prevention of cancer incidence or cancer mortality

43

SELECT

Vitamin E

increased the risk of prostate cancer

44

CARET

Carotenoid (vitamin A)

Adverse effect on the incidence of lung cancer

45

ATBC

Vitamin E, Beta carotene

Adverse effect on the incidence of lung cancer

46

JPHC

Isothiocyanate

Reduction in lung cancer risk among men

47

Meta-analysis

Flanovoid

Decreased risk of lung and stomach cancers

48

Meta-analysis

Flanovoid

reduce the risk of ovarian cancer

49

PHS II, Physicians’ Health Study II; SU.VI.MAX, Supplementation en Vitamines et Mineraux Antioxydants study; WACS, Women’s Antioxidant Cardiovascular Study; SELECT, Selenium and Vitamin E Cancer Prevention Trial; CARET, Carotene and Retinol Efficacy Trial; ATBC, Alpha-Tocopherol, Beta-Carotene Cancer Prevention study; JPHC, Japan Public Health Center study. Large-scale clinical trials and meta-analysis to study the effect of daily intake of antioxidant supplements or food on cancer were performed. Despite the beneficial effects of antioxidants in basic research, it has not been considered that some of antioxidants may become prooxidants, nor been concluded that antioxidants cannot prevent cancer.

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Antioxidative pathway in OCCC In OCCC, abnormalities are often detected in genes that are associated with oxidative stress response and ROS metabolism.20 Several antioxidant molecules are involved in OCCC carcinogenesis. Among them, the overexpression of hepatocyte nuclear factor 1 homeobox B (HNF1B), a major homeobox-containing protein also known as transcription factor-2, holds high importance. Under hypoxia and acidosis, HNF1B can modify and adapt cancer cells to survive using a process facilitated by increased glucose consumption and glycolysis, commonly known as the Warburg effect.50 Tsuchiya et al.14 first reported HNF1B overexpression in OCCC, and showed that reduced HNF1B expression considerably increased the apoptosis rate in two OCCC cell lines. The overexpression of HNF1B was observed in endometrial tissues adjacent to OCCC tumors, suggesting that HNF-1B overexpression is an early event in OCCC carcinogenesis. HNF1B overexpression in OCCC is caused by epigenetic changes rather than by mutations. Kato et al.51 revealed that the hypomethylation of the CpG island of HNF1B induces its overexpression in OCCC. Moreover, recent research has revealed that HNF1B promotes dedifferentiation to cancer stem cells via activation of the Notch pathway, and enhances invasive potential and epithelialmesenchymal transition in cancer cells.52 Mitochondrial superoxide dismutase (SOD2), an antioxidant enzyme that metabolizes superoxide in mitochondria and plays an important role in maintaining mitochondrial function through oxidative stress tolerance, was found to be highly expressed in the ectopic endometrium compared to normal endometrium, and it promoted cell proliferation and migration in ovarian endometriosis.53 SOD2 is also highly expressed in OCCC, and its oxidative stress tolerance seems to contribute to carcinogenesis.54, 55 Antioxidative pathways are deeply involved not only in carcinogenesis, but also in treatment resistance in OCCC. As oxidative stress tolerance represents therapeutic resistance, OCCC usually exhibits poor and fatal prognosis, even during gradual progression. OCCC has low sensitivity to platinum and taxane-based chemotherapy. Therefore, the prognosis of OCCC is extremely poor, particularly in the advanced stages.56, 57 Previous studies have revealed the roles of HNF1B in driving the expression of several characteristic genes associated with OCCC,58 stimulating metabolic changes to promote gluconeogenesis, glycogen accumulation, and aerobic glycolysis,59 inducing chemotherapeutic resistance through the suppression of sulfatase-1 (Sulf-1), an extracellular sulfatase catalyzing the 6-O desulfation of heparan sulfate glycosaminoglycans,60 and reducing the activity of immunological checkpoints against tumors. Thus, HNF1B plays an important role in therapeutic resistance via oxidative stress tolerance in OCCC. The overexpression of SOD2 also advances tumor growth and metastasis in OCCC. Hemachandra et al.54 revealed that SOD2 is more highly expressed in OCCC than in any other epithelial ovarian cancer subtypes, and its overexpression contributes to tumor growth and metastasis in a chorioallantoic membrane model. Their study also indicated that SOD2 expression is associated with increased cell proliferation, migration, outgrowth on collagen, spheroid attachment, and Akt phosphorylation in ES-2 OCCC cells. Therefore, SOD2 is regarded as a pro-tumorigenic or metastatic factor in OCCC. Clinical studies recently demonstrated that high SOD2 expression is observed in 76% (33 out of 41) of OCCC cases, and SOD2 overexpression is correlated with a poor prognosis for OCCC.55 Accordingly, SOD2 is also considered to be involved in therapeutic refraction through oxidative stress resistance in OCCC.

Therapeutic targets for OCCC As mentioned above, conventional standard treatment is less effective for OCCC due to its strong oxidative stress tolerance. To overcome the therapeutic difficulties for ovarian cancers, especially for OCCC, the development of novel therapeutics for recurrent or refractory cases is urgently required. Recently, several new molecular targets have been proposed for OCCC, which are categorized into the following groups: downstream pathways of receptor tyrosine kinases (RTKs), antioxidative stress molecules, AT-rich interactive domain 1A (ARID1A)-related chromatin remodeling factors, and genomic instability including high-level microsatellite instability (MSI-H) related to the programmed death ligand 1/programmed cell death 1 (PD-L1/PD-1) pathways. Here, these categorized targets have been reviewed and several novel proposals have been made (Fig. 1). RTKs are receptors located on the surface of cells. They play an important role in regulating cell proliferation, differentiation, survival, metabolism, and migration. Both phosphoinositide 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) and epidermal growth factor/Ras/mitogen-activated protein kinase (EGF/Ras/MAPK) pathways are downstream pathways of RTKs. Mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), loss of phosphatase and tensin homolog (PTEN), amplification of human epidermal growth factor receptor 2 (HER2), and overexpression of MET (also known as hepatocyte growth factor receptor; HGFR) and ADP-ribosylation factor-like 4C (ARL4C) have been shown to activate these pathways in OCCC.12, 61–63 In several studies, inhibition of these molecules was shown to suppress OCCC. MET inhibitors significantly decreased the proliferation and increased the

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FIG. 1 Activating pathways and targeting proposals in ovarian clear cell carcinomas. In ovarian clear cell carcinomas, activating pathways are categorized into the following groups—downstream pathways of receptor tyrosine kinases (RTKs), antioxidative stress molecules like HNF1B and SOD2, AT-rich interactive domain 1A (ARID1A)-related chromatin remodeling factors, and genomic instability including MSI-H. These pathways can be targeted in future therapeutic strategies, and other targets such as RECQL1, WRN, and TERT can be used to induce cancer-specific synthetic lethality.

apoptosis of OCCC cells in vitro, and also suppressed in vivo tumor growth in xenograft models of OCCC.62 Despite its effectiveness in vitro, no clinical advantages have been observed for inhibitors for downstream pathways of RTKs in treating OCCC. MET inhibitor cabozantinib was clinically ineffective in treating thirteen patients with recurrent OCCC.64 The combination of temsirolimus with carboplatin/paclitaxel was also investigated in patients with advanced OCCC. However, compared to conventional treatments, this regimen did not significantly increase the rate of progression-free survival.65 Further investigations are required to determine the drugs that are more effective in combination with PI3K/ AKT/mTOR inhibitors, and the mutations associated with OCCC that can be targeted by PI3K/AKT/mTOR inhibitors. As mentioned above in the section on “antioxidative pathway in OCCC,” molecules related to oxidative stress resistance such as HNF1B and SOD2 are deeply involved not only in their carcinogenesis, but also in therapeutic refraction. HNF1B is a major potential target for the treatment of OCCC; however, its molecular inhibitors have not been identified and difficulties pertaining to the development of chemical agents inhibiting HNF1B are discussed presently. In the treatment of SOD2-abundant OCCC, drugs that suppress mitochondrial function may be effective. Replacement therapy or drug repositioning using biguanides, agents for treating diabetes mellitus, may target tumor cell mitochondria and thereby improve the therapeutic effect on OCCC. The follow-up program postsurgical resection of OCCC can consider drug repositioning using biguanides, and extensive clinical cohort studies in this direction are required in the future. ARID1A chromatin remodeling abnormalities are also useful candidates for therapeutic targets of OCCC.13 ARID1A gene encodes BAF250a, a subunit of the switch/sucrose nonfermentable (SWI/SNF) chromatin-remodeling complex which modifies chromatin structure by histone octamer ejection, octamer sliding, or local chromatin unwrapping in order to allow binding of other transcription factors.13 Additionally, mutations in ARID1A contribute to AKT phosphorylation and induce activation of the PI3K pathway.66 In ARID1A-mutated OCCC, inhibition of enhancer of zeste homolog 2 (EZH2) histone methyltransferase activity-induced synthetic lethality, including PI3K/AKT signaling suppression.67 Additionally, Berns et al.68 have recently revealed that small-molecule inhibitors of the bromodomain and extra-terminal domain (BET) family of proteins inhibit the proliferation of ARID1A-mutated OCCC cells by reducing the expression of multiple SWI/SNF members in vitro and in vivo. Furthermore, recent research demonstrated that ARID1A-deficient cancer cells have low levels of glutathione due to decreased expression of SLC7A11 and are specifically vulnerable to inhibition of antioxidant glutathione and glutamate-cysteine ligase synthetase catalytic subunit, a rate-limiting enzyme for glutathione synthesis.69 APR-246, one of glutathione inhibitors, has the potential to act as an effective agent inducing synthetic lethality in ARID1A-deficient cancer cells.69 Thus, EZH2 or BET inhibitors, and APR-246 represent promising new drugs to treat ARID1A-mutated OCCC in the future. Recently, immune checkpoint blockade by anti-PD-1/PD-L1 antibodies for treating solid tumors resisting conventional therapy has garnered attention. Especially, mismatch repair (MMR) deficient tumors are found to be sensitive to this immunotherapy.70, 71 Recent research demonstrated that about 7% (4 out of 57) of OCCC cases had MSI-H cancers without any

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MMR mutations.72 For MSI-H OCCC, immunotherapy by anti-PD-1/PD-L1 antibodies has high potential to be used as an effective treatment strategy. Moreover, in a clinical phase II trial, 2 out of 20 platinum-resistant recurrent OCCC cases showed complete remission upon treatment with an anti-PD-1 antibody, and these two patients also showed microsatellite stability (MSS).73 Taken together, not only MMR status, but also various genetic and epigenetic modifications contribute to sensitivity to the immune checkpoint blockade, whose efficiency can be evaluated in further studies. To maintain unlimited replicating potential, most cancer cells evolve mechanisms to maintain telomere length by increasing the activity of telomerase reverse transcriptase (TERT), the catalytic subunit of the telomerase that adds sixnucleotide telomere sequences to the end of chromosomes.74, 75 TERT promoter mutations are most common in OCCC among gynecological malignancies.76 Thus, TERT is regarded as a potential therapeutic target in OCCC. OBP-301, a genetically modified adenovirus that has strong antitumor activity and lyses cancer cells (especially with high telomerase activity) by viral proliferation, has the potential to develop as a theoretically suitable therapeutic agent for OCCC.77 Recently, applications of poly-ADP-ribose polymerase (PERP) inhibitors such as olaparib are able to induce synthetic lethality and achieve better prognosis for ovarian cancers.78, 79 These inhibitors can be extended to OCCC, especially under follow-up program after tumor mass reduction by primary surgery. Targeting DNA helicases is another strategy to induce synthetic lethality and treat OCCC, as indicated by the reports on RECQL1,80 and WRN targeting.81 In order to improve therapeutic achievements in ovarian cancers, especially in OCCC, more options for treatments are required to be explored. As it has been difficult to develop small-molecular inhibitors against HNF1B, different therapeutic options need to be prepared for individual tumor characteristics in the era of precision medicine. It is advisable to implement nucleic acid-based drugs such as siRNA to treat OCCC effectively and to adapt it to the transcriptional profile of individual tumor. Also, we should develop approaches to inhibit RECQL180, 82 and WRN helicases.81

Conclusion Here, we have briefly summarized recent discoveries in the process of carcinogenesis, prevention of carcinogenesis, characteristic nature of tumors and treatment post refractory OCCC, which is highly linked to oxidative stress. Removal of oxidative stress suppresses development from endometriosis to OCCC. Strong antioxidants, flavonoid, or isothiocyanate, may be useful for preventing carcinogenesis of OCCC. However, the stress tolerance properties of OCCC induce therapeutic resistance and make the treatment difficult. The genetic and biological characteristics of OCCC are gradually evaluated, and the therapeutic effects of various anticancer drugs, molecular targeting drugs, drug repositioning strategies, and immunotherapies are being verified. Not only further investigations to identify novel molecular targets are required, but also studies on precision medicine that combines multiple treatments based on the genetic and molecular characteristics of individual tumors are desired. As it is challenging to develop small molecular inhibitors for some undruggable molecules, we believe that it is absolutely essential to improve therapeutic approaches against OCCC and implement nucleic acidbased drug and multi-combinatorial treatment corresponding to transcriptional profile of each tumor.

Applications to other cancers or conditions Antioxidants and antioxidative pathways are deeply involved not only in the prevention of carcinogenesis, but also in therapeutics against OCCC. Surgical treatment and hormonal therapy are suitable for preventing carcinogenesis from endometriosis, and strong antioxidants such as flavonoid and isothiocyanate are possibly effective in the prevention of OCCC. Under the severe oxidative stress, OCCC abundantly expresses HNF1B and SOD2, and acquires the resistance. By targeting HNF1B or SOD2, antioxidative pathway can become an advisable therapeutic candidate for OCCC. Regarding to cancers other than ovarian cancer, intake of isoflavone was associated with reduced risk of prostate, colorectal, breast and lung cancers, while the precise mechanisms have not yet revealed.83–86 Isothiocyanate could also reduce the risk of prostate, gastric, colorectal, and lung cancers, especially in person with common deletion polymorphisms of glutathione-S-transferases M1 and T1.87–90 Furthermore, isothiocyanate could enhance radiosensitivity of HeLa cancer cells in vitro and in vivo, and the combination therapy with radiation is expected.91 Renal clear cell carcinoma (RCCC) holds similarly pathological and molecular phenotype to OCCC, and can benefit from a lot of aspects of therapeutics mentioned in this review. In fact, SOD2 abundance has correlated with a clinically worse outcome in metastatic RCCC,92 and has become an expectantly candidate to therapeutics.93 Whereas the MET inhibitor cabozantinib showed therapeutic excellence for advanced RCCC,94 the clinical effectiveness was poor in recurrent OCCC.64 The differences in the treatment outcomes between both may be caused by the prevalence of von Hippel-Lindau mutations in RCCC, or due to the increased incidence of active abnormalities of the PI3K/mTOR/AKT pathway and of ARID1A in OCCC64: paying also attention to precise differences in RCCC and OCCC.

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Summary points l l l l

l l

Oxidative stress promotes carcinogenesis from endometriosis to ovarian clear cell carcinomas (OCCC). Antioxidant intake is possibly effective for its prevention, together with surgical treatment and hormonal therapy. Accompanied by HNF1B and SOD2 overexpression, OCCC acquires resistance to oxidative stress and therapy. To treat OCCC effectively, several therapeutic targets are studied; RTKs, antioxidative stress molecules, ARID1Arelated factors, and genomic instability, etc. Targeting DNA helicases like RECQL1 and WRN is another strategy to induce synthetic lethality in OCCC. It is advisable to implement nucleic acid-based drugs and multi-combinatorial treatment corresponding to transcriptional profile of each OCCC.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

http://gco.iarc.fr/today/home. https://www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2018.html. https://ganjoho.jp/reg_stat/statistics/stat/short_pred.html. Shih IM, Kurman RJ. Ovarian tumorigenesis: a proposed model based on morphological and molecular genetic analysis. Am J Pathol 2004; 164(5):1511–8. K€ obel M, Reuss A, du Bois A, Kommoss S, Kommoss F, Gao D, et al. The biological and clinical value of p53 expression in pelvic high-grade serous carcinomas. J Pathol 2010;222(2):191–8. Network CGAR. Integrated genomic analyses of ovarian carcinoma. Nature 2011;474(7353):609–15. Kurman RJ, Vang R, Junge J, Hannibal CG, Kjaer SK, Shih IM. Papillary tubal hyperplasia: the putative precursor of ovarian atypical proliferative (borderline) serous tumors, noninvasive implants, and endosalpingiosis. Am J Surg Pathol 2011;35(11):1605–14. Labidi-Galy SI, Papp E, Hallberg D, Niknafs N, Adleff V, Noe M, et al. High grade serous ovarian carcinomas originate in the fallopian tube. Nat Commun 2017;8(1):1093. Vang R, Shih IM, Kurman RJ. Fallopian tube precursors of ovarian low- and high-grade serous neoplasms. Histopathology 2013;62(1):44–58. Seidman JD, Kurman RJ, Ronnett BM. Primary and metastatic mucinous adenocarcinomas in the ovaries: incidence in routine practice with a new approach to improve intraoperative diagnosis. Am J Surg Pathol 2003;27(7):985–93. K€ obel M, Kalloger SE, Boyd N, McKinney S, Mehl E, Palmer C, et al. Ovarian carcinoma subtypes are different diseases: implications for biomarker studies. PLoS Med 2008;5(12). e232. Kuo KT, Mao TL, Jones S, Veras E, Ayhan A, Wang TL, et al. Frequent activating mutations of PIK3CA in ovarian clear cell carcinoma. Am J Pathol 2009;174(5):1597–601. Jones S, Wang TL, Shih IM, Mao TL, Nakayama K, Roden R, et al. Frequent mutations of chromatin remodeling gene ARID1A in ovarian clear cell carcinoma. Science 2010;330(6001):228–31. Tsuchiya A, Sakamoto M, Yasuda J, Chuma M, Ohta T, Ohki M, et al. Expression profiling in ovarian clear cell carcinoma: identification of hepatocyte nuclear factor-1 beta as a molecular marker and a possible molecular target for therapy of ovarian clear cell carcinoma. Am J Pathol 2003; 163(6):2503–12. Pearce CL, Templeman C, Rossing MA, Lee A, Near AM, Webb PM, et al. Association between endometriosis and risk of histological subtypes of ovarian cancer: a pooled analysis of case-control studies. Lancet Oncol 2012;13(4):385–94. Toyokuni S. Role of iron in carcinogenesis: cancer as a ferrotoxic disease. Cancer Sci 2009;100(1):9–16. Li JL, Okada S, Hamazaki S, Ebina Y, Midorikawa O. Subacute nephrotoxicity and induction of renal cell carcinoma in mice treated with ferric nitrilotriacetate. Cancer Res 1987;47(7):1867–9. Liu M, Okada S. Induction of free radicals and tumors in the kidneys of Wistar rats by ferric ethylenediamine-N, N’-diacetate. Carcinogenesis 1994; 15(12):2817–21. Munksgaard PS, Blaakaer J. The association between endometriosis and ovarian cancer: a review of histological, genetic and molecular alterations. Gynecol Oncol 2012;124(1):164–9. Yamaguchi K, Mandai M, Oura T, Matsumura N, Hamanishi J, Baba T, et al. Identification of an ovarian clear cell carcinoma gene signature that reflects inherent disease biology and the carcinogenic processes. Oncogene 2010;29(12):1741–52. Melin AS, Lundholm C, Malki N, Swahn ML, Spare`n P, Bergqvist A. Hormonal and surgical treatments for endometriosis and risk of epithelial ovarian cancer. Acta Obstet Gynecol Scand 2013;92(5):546–54. Beral V, Doll R, Hermon C, Peto R, Reeves G. Cancer CGoESoO. Ovarian cancer and oral contraceptives: collaborative reanalysis of data from 45 epidemiological studies including 23,257 women with ovarian cancer and 87,303 controls. Lancet 2008;371(9609):303–14. Yoshino O, Minamisaka T, Ono Y, Tsuda S, Samejima A, Shima T, et al. Three cases of clear-cell adenocarcinoma arising from endometrioma during hormonal treatments. J Obstet Gynaecol Res 2018;44(9):1850–8. Nishida Y, Yamashita E, Miki W. Quenching activities of common hydrophilic and lipophilic antioxidants against singlet oxygen using chemiluminescence detection system. Carotenoid Sci 2007;11:16–20. Martin HD, Ruck C, Schmidt M, Sell S, Beutner IS, Mayer B, et al. Chemistry of carotenoid oxidation and free radical reactions. Pure Appl Chem 1999;71(12):2253–3363.

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26. Kuroki T, Ikeda S, Okada T, Maoka T, Kitamura A, Sugimoto M, et al. Astaxanthin ameliorates heat stress-induced impairment of blastocyst development in vitro: astaxanthin colocalization with and action on mitochondria. J Assist Reprod Genet 2013;30(5):623–31. 27. Park JS, Mathison BD, Hayek MG, Zhang J, Reinhart GA, Chew BP. Astaxanthin modulates age-associated mitochondrial dysfunction in healthy dogs. J Anim Sci 2013;91(1):268–75. 28. Ames BN. Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative diseases. Science 1983;221(4617):1256–64. 29. Dusinska´ M, Kazimı´rova´ A, Barancokova´ M, Beno M, Smolkova´ B, Horska´ A, et al. Nutritional supplementation with antioxidants decreases chromosomal damage in humans. Mutagenesis 2003;18(4):371–6. 30. Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C. Chronic inflammation and oxidative stress in human carcinogenesis. Int J Cancer 2007;121(11):2381–6. 31. Choi HD, Youn YK, Shin WG. Positive effects of astaxanthin on lipid profiles and oxidative stress in overweight subjects. Plant Foods Hum Nutr 2011;66(4):363–9. 32. Wolf AM, Asoh S, Hiranuma H, Ohsawa I, Iio K, Satou A, et al. Astaxanthin protects mitochondrial redox state and functional integrity against oxidative stress. J Nutr Biochem 2010;21(5):381–9. 33. Aoi W, Naito Y, Takanami Y, Ishii T, Kawai Y, Akagiri S, et al. Astaxanthin improves muscle lipid metabolism in exercise via inhibitory effect of oxidative CPT I modification. Biochem Biophys Res Commun 2008;366(4):892–7. 34. Kavitha K, Kowshik J, Kishore TK, Baba AB, Nagini S. Astaxanthin inhibits NF-kB and Wnt/b-catenin signaling pathways via inactivation of Erk/ MAPK and PI3K/Akt to induce intrinsic apoptosis in a hamster model of oral cancer. Biochim Biophys Acta 2013;1830(10):4433–44. 35. Kowshik J, Baba AB, Giri H, Deepak Reddy G, Dixit M, Nagini S. Astaxanthin inhibits JAK/STAT-3 signaling to abrogate cell proliferation, invasion and angiogenesis in a hamster model of oral cancer. PLoS One 2014;9(10). e109114. 36. Palozza P, Torelli C, Boninsegna A, Simone R, Catalano A, Mele MC, et al. Growth-inhibitory effects of the astaxanthin-rich alga Haematococcus pluvialis in human colon cancer cells. Cancer Lett 2009;283(1):108–17. 37. Brito AF, Ribeiro M, Abrantes AM, Pires AS, Teixo RJ, Tralha˜o JG, et al. Quercetin in cancer treatment, alone or in combination with conventional therapeutics? Curr Med Chem 2015;22(26):3025–39. 38. Srivastava S, Somasagara RR, Hegde M, Nishana M, Tadi SK, Srivastava M, et al. Quercetin, a natural flavonoid interacts with DNA, arrests cell cycle and causes tumor regression by activating mitochondrial pathway of apoptosis. Sci Rep 2016;6:24049. 39. Satyan KS, Swamy N, Dizon DS, Singh R, Granai CO, Brard L. Phenethyl isothiocyanate (PEITC) inhibits growth of ovarian cancer cells by inducing apoptosis: role of caspase and MAPK activation. Gynecol Oncol 2006;103(1):261–70. 40. Wattenberg LW. Inhibitory effects of benzyl isothiocyanate administered shortly before diethylnitrosamine or benzo[a]pyrene on pulmonary and forestomach neoplasia in A/J mice. Carcinogenesis 1987;8(12):1971–3. 41. Gaziano JM, Glynn RJ, Christen WG, Kurth T, Belanger C, MacFadyen J, et al. Vitamins E and C in the prevention of prostate and total cancer in men: the Physicians’ health study II randomized controlled trial. JAMA 2009;301(1):52–62. 42. Hercberg S, Galan P, Preziosi P, Bertrais S, Mennen L, Malvy D, et al. The SU.VI.MAX study: a randomized, placebo-controlled trial of the health effects of antioxidant vitamins and minerals. Arch Intern Med 2004;164(21):2335–42. 43. Lin J, Cook NR, Albert C, Zaharris E, Gaziano JM, Van Denburgh M, et al. Vitamins C and E and beta carotene supplementation and cancer risk: a randomized controlled trial. J Natl Cancer Inst 2009;101(1):14–23. 44. Klein EA, Thompson IM, Tangen CM, Crowley JJ, Lucia MS, Goodman PJ, et al. Vitamin E and the risk of prostate cancer: the selenium and vitamin E cancer prevention trial (SELECT). JAMA 2011;306(14):1549–56. 45. Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, et al. Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. N Engl J Med 1996;334(18):1150–5. 46. Alpha-Tocopherol, BtCCPSG. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J Med 1994;330(15):1029–35. 47. Mori N, Shimazu T, Sasazuki S, Nozue M, Mutoh M, Sawada N, et al. Cruciferous vegetable intake is inversely associated with lung cancer risk among current nonsmoking men in the Japan public health center (JPHC) study. J Nutr 2017;147(5):841–9. 48. Grosso G, Godos J, Lamuela-Raventos R, Ray S, Micek A, Pajak A, et al. A comprehensive meta-analysis on dietary flavonoid and lignan intake and cancer risk: level of evidence and limitations. Mol Nutr Food Res 2017;61(4). 49. Hua X, Yu L, You R, Yang Y, Liao J, Chen D. Association among dietary flavonoids, flavonoid subclasses and ovarian cancer risk: a meta-analysis. PLoS One 2016;11(3). e0151134. 50. Mandai M, Amano Y, Yamaguchi K, Matsumura N, Baba T, Konishi I. Ovarian clear cell carcinoma meets metabolism; HNF-1b confers survival benefits through the Warburg effect and ROS reduction. Oncotarget 2015;6(31):30704–14. 51. Kato N, Tamura G, Motoyama T. Hypomethylation of hepatocyte nuclear factor-1beta (HNF-1beta) CpG island in clear cell carcinoma of the ovary. Virchows Arch 2008;452(2):175–80. 52. Zhu JN, Jiang L, Jiang JH, Yang X, Li XY, Zeng JX, et al. Hepatocyte nuclear factor-1beta enhances the stemness of hepatocellular carcinoma cells through activation of the notch pathway. Sci Rep 2017;7(1):4793. 53. Chen C, Zhou Y, Hu C, Wang Y, Yan Z, Li Z, et al. Mitochondria and oxidative stress in ovarian endometriosis. Free Radic Biol Med 2019;136:22–34. 54. Hemachandra LP, Shin DH, Dier U, Iuliano JN, Engelberth SA, Uusitalo LM, et al. Mitochondrial superoxide dismutase has a protumorigenic role in ovarian clear cell carcinoma. Cancer Res 2015;75(22):4973–84. 55. Amano T, Chano T, Isono T, Kimura F, Kushima R, Murakami T. Abundance of mitochondrial superoxide dismutase is a negative predictive biomarker for endometriosis-associated ovarian cancers. World J Surg Oncol 2019;17(1):24.

Oxidative stress and ovarian cancers Chapter

8

85

56. Sugiyama T, Kamura T, Kigawa J, Terakawa N, Kikuchi Y, Kita T, et al. Clinical characteristics of clear cell carcinoma of the ovary: a distinct histologic type with poor prognosis and resistance to platinum-based chemotherapy. Cancer 2000;88(11):2584–9. 57. Goff BA, de la Sainz Cuesta R, Muntz HG, Fleischhacker D, Ek M, Rice LW, et al. Clear cell carcinoma of the ovary: a distinct histologic type with poor prognosis and resistance to platinum-based chemotherapy in stage III disease. Gynecol Oncol 1996;60(3):412–7. 58. Senkel S, Lucas B, Klein-Hitpass L, Ryffel GU. Identification of target genes of the transcription factor HNF1beta and HNF1alpha in a human embryonic kidney cell line. Biochim Biophys Acta 2005;1731(3):179–90. 59. Okamoto T, Mandai M, Matsumura N, Yamaguchi K, Kondoh H, Amano Y, et al. Hepatocyte nuclear factor-1b (HNF-1b) promotes glucose uptake and glycolytic activity in ovarian clear cell carcinoma. Mol Carcinog 2015;54(1):35–49. 60. Liu P, Khurana A, Rattan R, He X, Kalloger S, Dowdy S, et al. Regulation of HSulf-1 expression by variant hepatic nuclear factor 1 in ovarian cancer. Cancer Res 2009;69(11):4843–50. 61. Fujimura M, Katsumata N, Tsuda H, Uchi N, Miyazaki S, Hidaka T, et al. HER2 is frequently over-expressed in ovarian clear cell adenocarcinoma: possible novel treatment modality using recombinant monoclonal antibody against HER2, trastuzumab. Jpn J Cancer Res 2002;93(11):1250–7. 62. Kim HJ, Yoon A, Ryu JY, Cho YJ, Choi JJ, Song SY, et al. c-MET as a potential therapeutic target in ovarian clear cell carcinoma. Sci Rep 2016;6:38502. 63. Wakinoue S, Chano T, Amano T, Isono T, Kimura F, Kushima R, et al. ADP-ribosylation factor-like 4C predicts worse prognosis in endometriosisassociated ovarian cancers. Cancer Biomark 2019;24(2):223–9. 64. Konstantinopoulos PA, Brady WE, Farley J, Armstrong A, Uyar DS, Gershenson DM. Phase II study of single-agent cabozantinib in patients with recurrent clear cell ovarian, primary peritoneal or fallopian tube cancer (NRG-GY001). Gynecol Oncol 2018;150(1):9–13. 65. Farley JH, Brady WE, Fujiwara K, Nomura H, Yunokawa M, Tokunaga H, et al. A phase II evaluation of temsirolimus in combination with carboplatin and paclitaxel followed by temsirolimus consolidation as first-line therapy in the treatment of stage III-IV clear cell carcinoma of the ovary. J Clin Oncol 2016;34:5531. 66. Wiegand KC, Hennessy BT, Leung S, Wang Y, Ju Z, McGahren M, et al. A functional proteogenomic analysis of endometrioid and clear cell carcinomas using reverse phase protein array and mutation analysis: protein expression is histotype-specific and loss of ARID1A/BAF250a is associated with AKT phosphorylation. BMC Cancer 2014;14:120. 67. Bitler BG, Aird KM, Garipov A, Li H, Amatangelo M, Kossenkov AV, et al. Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated cancers. Nat Med 2015;21(3):231–8. 68. Berns K, Caumanns JJ, Hijmans EM, Gennissen AMC, Severson TM, Evers B, et al. ARID1A mutation sensitizes most ovarian clear cell carcinomas to BET inhibitors. Oncogene 2018;37(33):4611–25. 69. Ogiwara H, Takahashi K, Sasaki M, Kuroda T, Yoshida H, Watanabe R, et al. Targeting the vulnerability of glutathione metabolism in ARID1Adeficient cancers. Cancer Cell 2019;35(2):177–90. e8. 70. Le DT, Durham JN, Smith KN, Wang H, Bartlett BR, Aulakh LK, et al. Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science 2017;357(6349):409–13. 71. Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring AD, et al. PD-1 blockade in tumors with mismatch-repair deficiency. N Engl J Med 2015;372(26):2509–20. 72. Akbari MR, Zhang S, Cragun D, Lee JH, Coppola D, McLaughlin J, et al. Correlation between germline mutations in MMR genes and microsatellite instability in ovarian cancer specimens. Familial Cancer 2017;16(3):351–5. 73. Hamanishi J, Mandai M, Ikeda T, Minami M, Kawaguchi A, Murayama T, et al. Safety and antitumor activity of anti-PD-1 antibody, nivolumab, in patients with platinum-resistant ovarian cancer. J Clin Oncol 2015;33(34):4015–22. 74. Artandi SE, DePinho RA. Telomeres and telomerase in cancer. Carcinogenesis 2010;31(1):9–18. 75. Harley CB. Telomerase and cancer therapeutics. Nat Rev Cancer 2008;8(3):167–79. 76. Wu RC, Ayhan A, Maeda D, Kim KR, Clarke BA, Shaw P, et al. Shih IeM, Wang TL. Frequent somatic mutations of the telomerase reverse transcriptase promoter in ovarian clear cell carcinoma but not in other major types of gynaecological malignancy. J Pathol 2014;232(4):473–81. 77. Kawashima T, Kagawa S, Kobayashi N, Shirakiya Y, Umeoka T, Teraishi F, et al. Telomerase-specific replication-selective virotherapy for human cancer. Clin Cancer Res 2004;10(1 Pt 1):285–92. 78. Pujade-Lauraine E, Ledermann JA, Selle F, Gebski V, Penson RT, Oza AM, et al. Olaparib tablets as maintenance therapy in patients with platinumsensitive, relapsed ovarian cancer and a BRCA1/2 mutation (SOLO2/ENGOT-Ov21): a double-blind, randomised, placebo-controlled, phase 3 trial. Lancet Oncol 2017;18(9):1274–84. 79. Moore K, Colombo N, Scambia G, Kim BG, Oaknin A, Friedlander M, et al. Maintenance olaparib in patients with newly diagnosed advanced ovarian cancer. N Engl J Med 2018;379(26):2495–505. 80. Sanada S, Futami K, Terada A, Yonemoto K, Ogasawara S, Akiba J, et al. RECQL1 DNA repair helicase: a potential therapeutic target and a proliferative marker against ovarian cancer. PLoS One 2013;8(8). e72820. 81. Chan EM, Shibue T, McFarland JM, Bin Liu J, Lazaro JB, Gu P, et al. WRN helicase is a synthetic lethal target in microsatellite unstable cancers. Nature 2019;568(7753):551–6. 82. Arai A, Chano T, Futami K, Furuichi Y, Ikebuchi K, Inui T, et al. RECQL1 and WRN proteins are potential therapeutic targets in head and neck squamous cell carcinoma. Cancer Res 2011;71(13):4598–607. 83. He J, Wang S, Zhou M, Yu W, Zhang Y, He X. Phytoestrogens and risk of prostate cancer: a meta-analysis of observational studies. World J Surg Oncol 2015;13:231. 84. Jiang R, Botma A, Rudolph A, H€using A, Chang-Claude J. Phyto-oestrogens and colorectal cancer risk: a systematic review and dose-response metaanalysis of observational studies. Br J Nutr 2016;116(12):2115–28.

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85. Yamamoto S, Sobue T, Kobayashi M, Sasaki S, Tsugane S. Group JPHC-BPSoCCD. Soy, isoflavones, and breast cancer risk in Japan. J Natl Cancer Inst 2003;95(12):906–13. 86. Shimazu T, Inoue M, Sasazuki S, Iwasaki M, Sawada N, Yamaji T, et al. Isoflavone intake and risk of lung cancer: a prospective cohort study in Japan. Am J Clin Nutr 2010;91(3):722–8. 87. Wang LG, Chiao JW. Prostate cancer chemopreventive activity of phenethyl isothiocyanate through epigenetic regulation (review). Int J Oncol 2010;37(3):533–9. 88. Moy KA, Yuan JM, Chung FL, Wang XL, Van Den Berg D, Wang R, et al. Isothiocyanates, glutathione S-transferase M1 and T1 polymorphisms and gastric cancer risk: a prospective study of men in Shanghai, China. Int J Cancer 2009;125(11):2652–9. 89. Yang G, Gao YT, Shu XO, Cai Q, Li GL, Li HL, et al. Isothiocyanate exposure, glutathione S-transferase polymorphisms, and colorectal cancer risk. Am J Clin Nutr 2010;91(3):704–11. 90. London SJ, Yuan JM, Chung FL, Gao YT, Coetzee GA, Ross RK, et al. Isothiocyanates, glutathione S-transferase M1 and T1 polymorphisms, and lung-cancer risk: a prospective study of men in Shanghai, China. Lancet 2000;356(9231):724–9. 91. Yu D, Sekine-Suzuki E, Xue L, Fujimori A, Kubota N, Okayasu R. Chemopreventive agent sulforaphane enhances radiosensitivity in human tumor cells. Int J Cancer 2009;125(5):1205–11. 92. Yoshida T, Kageyama S, Isono T, Yuasa T, Kushima R, Kawauchi A, et al. Superoxide dismutase 2 expression can predict prognosis of renal cell carcinoma patients. Cancer Biomark 2018;22(4):755–61. 93. Isono T, Chano T, Yonese J, Yuasa T. Therapeutic inhibition of mitochondrial function induces cell death in starvation-resistant renal cell carcinomas. Sci Rep 2016;6:25669. 94. Choueiri TK, Escudier B, Powles T, Mainwaring PN, Rini BI, Donskov F, et al. Cabozantinib versus everolimus in advanced renal-cell carcinoma. N Engl J Med 2015;373(19):1814–23.

Chapter 9

Redox-dependent mechanisms of carcinogenesis in human papillomavirus infection Cesira Foppolia and Marzia Perluigib a

CNR Institute of Molecular Biology and Pathology, Sapienza University of Rome, Rome, Italy, b Department of Biochemical Sciences, Sapienza University

of Rome, Rome, Italy

List of abbreviations d ALA-D AP-1 CAT CDK c-IAP2 CIN COX-2 ERp57 GAPDH GPx GST HPV HR-HPV HSIL hTERT iNOS LCR LPO LR-HPV LSIL MAGUK MDA NHEK OS pRB Prx RNS ROS SOD TBARS URR

d-aminolevulinate dehydratase activator protein-1 catalase cyclin-dependent kinase cellular inhibitor of apoptosis protein 2 cervical intraepithelial neoplasia cycloxygenase2 endoplasmic reticulum protein 57 gliceraldehyde 3-phosphate dehydrogenase glutathione peroxidase glutathione S-transferase human papilloma virus high-risk HPV high-grade squamous intraepithelial lesion human telomerase reverse transcriptase inducible nitric oxide synthase long control region lipid peroxidation low-risk HPV low-grade squamous intraepithelial lesion membrane-associated guanylate kinase malondialdehyde normal human epithelial keratinocytes oxidative stress retinoblastoma protein peroxiredoxin reactive nitrogen species reactive oxygen species superoxide dismutase thiobarbituric acid reactive substances upstream regulatory region

Introduction Cervical cancer is the second most common cancer among women worldwide. At the beginning of the 1970s Harold zur Hausen1 hypothesized a correlation between cervical neoplasia and human papillomavirus (HPV) infection. After this first Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00009-2 © 2021 Elsevier Inc. All rights reserved.

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assumption, a plethora of laboratory and epidemiologic data indicated HPV as the major etiological factor in cervical carcinogenesis. HPVs are a group of DNA viruses highly species-specific and exclusively tissue tropic, undergoing a complete infectious cycle only in fully differentiating squamous epithelium. More than 100 HPV types are described,2 clinically classified as “low-risk” (LR-HPV) and “high-risk” (HR-HPV) based on the relative propensity to cause benign or cancerous lesions. LR-HPVs generate mild dysplasia and genital warts, while HR-HPVs are associated with high-grade lesions and invasive cancer. Virtually, all cervical cancers (over 99%) contain the genes of HR-HPV types, especially HPV 16, HPV 18, HPV 31, and HPV 33. HPV 16 is by far the most prevalent type, found in more than 50% of all cases. The oncogenic potential of HPV infection mostly depends on the activity of the viral oncogenes E6, E7, and to a lesser extent E5. E6 and E7 can modulate cellular proliferation and apoptosis by interfering with the function of two tumor suppressor proteins, p53 and Rb, respectively, and their expression is required for cell transformation and maintenance of the transformed state. In addition, E6 and E7 oncoproteins play key roles throughout the whole disease process by manipulating the function of a variety of host regulatory proteins. However viral oncogenes expression, although necessary, is not per se sufficient to induce cervical cancer and other factors are needed to drive the neoplastic progression. Despite the large number of studies on viral, host and environmental putative cofactors, results have been largely disappointing and our present understanding of cervical cancer progression remains largely unsatisfactory. Oxidative stress represents an interesting and underexplored candidate as a promoting factor in HPV-driven carcinogenesis. The role of reactive oxygen species (ROS) in tumor progression is well established,3 as well as the notion that oxidative stress perturbs the cellular redox status thus leading to alteration of gene expression responses and triggering a signaling cascade that affects both cell growth and cell death. In this chapter, we report results from several studies which support the role of oxidative stress in cervical cancer.

Cervical cancer Cervical cancer is a slow-evolution disease, arising from dysplastic lesions after long persistent infection. It is, in fact, a multistep process, in which progressive histologic and cytologic changes occur, that can be divided in early lesions, currently indicated as cervical intraepithelial neoplasia 1 (CIN 1) or low-grade squamous intraepithelial lesion (LSIL) and high-grade lesions, known as cervical intraepithelial neoplasia 2/3 (CIN 2/CIN3) or high-grade squamous intraepithelial lesion (HSIL).4 The majority of HPV infections (80%) is subclinical and transitory and is successfully cleared by an efficient cell-mediated immune response exerted by the host. The importance of the immune system in counteracting HPV infection is also demonstrated by the fact that regressing warts are infiltrated by T lymphocytes and macrophages and that in immunosuppressed individuals a higher prevalence of HPV-induced lesions and HPV-related tumors is observed.4, 5 However, in case the infection is not properly cleared in those individuals with “less efficient” immune response, it progresses to mild dysplasia, CIN 1. Also, CIN 1 lesions typically regress spontaneously, but a few lesions progress to moderate and then severe dysplasia (CIN 2/CIN 3) and eventually to carcinoma in situ and invasive carcinoma.6 The rates of regression, persistence or progression vary according to the different CIN grades and these data are shown in Fig. 1.

HPV structure The viral genome is constituted by a circular double-strand DNA containing 6000–8000 base pairs and comprises eight gene sequences, codifying for proteins with early (E) or late (L) functions. L region encodes the two capsid proteins L1 and L2. E region contains six genes expressed in the initial phase of the replicative cycle, codifying for the E1, E2, E4, E5, E6, E7 proteins. In addition, the viral genome contains a region, named Long Control Region (LCR) or Upstream Regulatory Region (URR), where the site for plasmide replication origin, several sequences implied in viral replication and numerous binding sites for cellular transcription factors have been identified. This specific region is crucial in determining the host range and tissue tropism of each HPV type and also regulates viral gene expression upon infection. E6, E7, and in minor grade E5 are the oncoproteins, well-recognized as responsible for cell transformation events. Molecular mechanisms of oncogenic action of E6 and E7 proteins from HR-HPVs are summarized in Fig. 2.

E6 oncoprotein The most characterized role of HR-HPV E6 is the induction of tumor suppressor protein p53 degradation. p53 represents a major defense to viral replication since once activated, it can promote cell cycle arrest or apoptosis of the infected cell. p53

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80%

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5% HPV infection

CIN 3

15% PERSISTENCE 10%

57%

CIN 3

CIN 1 1%

R E G R E S S I O N

Invasive carcinoma

32% PERSISTENCE 17%

43%

CIN 3

CIN 2 Invasive carcinoma

5% 35%

P R O G R E S S I O N

PERSISTENCE 32%

12% Invasive carcinoma

CIN 3

56% PERSISTENCE

FIG. 1 Rates of regression, persistence or progression of different grades of cervical intraepithelial neoplasia (CIN). Only a minor part of cervical HPV infections tend to progress to cancer.

Tumor suppression

Bak

Apoptosis

E6

Tumor suppression Repressiion of E2Fdependent transcription

E6 E6-AP Bak

E6 AP

Ubiquitin proteasome

E7

Degraded Bak E2F pRb

E6 E6-AP

p53

Degraded p53

E6 E6-AP p53

Ubiquitin proteasome

E7 pRb Active E2F

Apoptosis arrest

Ubiquitin proteasome

Apoptosis Transcription activation Tumor suppression

Degraded pRb

No Tumor suppression

Cell proliferation FIG. 2 Oncogenic activities of E6 and E7 proteins of high-risk human papillomavirus. E6 oncogenic power is due to its ability to bind the tumor suppressor protein p53 and to induce its degradation through the ubiquitin pathway. Therefore, apoptosis is inhibited. Apoptosis arrest can also be provoked by E6 through the inactivation of Bak. E7 binds to the retinoblastoma (Rb) family of tumor suppressor proteins and induces its degradation through the ubiquitin pathway. In this way, the Rb/E2F complexes are disrupted, E2F-dependent transcription is no more repressed and cell division is driven.

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is considered the “guardian of the genome,” by maintaining its integrity through the inhibition of cell division if DNA damage occurs or eventually reprogramming it once the damage has been eliminated. In case the replication is already active even in the presence of damaged DNA, p53 can modulate cell cycle by forcing it into the apoptotic pathway. By doing this, it prevents the selection of potentially transformed progeny. A major strategy employed by the HR-HPV E6 protein to abrogate the oncosuppressive function of p53 is to induce its degradation through the ubiquitin-proteasome pathway. HPV-16 E6 binds to E6-associated protein (E6AP), a cellular protein with ubiquitin-protein ligase function. The E6/E6AP complex then binds p53, which is targeted for degradation. E6 from HR-HPV types can also interact with other additional ubiquitin ligases that may be involved in HPV-mediated p53 degradation.7 Since p53 plays a key role in preserving genome integrity, HPV infected cells tend to accumulate chromosomal abnormalities, thus increasing the probability of a malignant evolution. Exclusively the E6 proteins from the HR-HPV types have the ability to induce degradation of p53, while no p53 degradation was observed in cells expressing E6 from LR-HPVs.8 HPV E6 can modulate the apoptotic pathways independently of p53, through the proapoptotic effector Bak that displays E6-binding affinity. E6 inhibits Bak-induced apoptosis and this is mediated by an interaction between the E6 and Bak proteins resulting in degradation of the Bak protein.9 In addition, HPV E6 upregulates the expression of the inhibitors of apoptosis proteins, as c-IAP2 and survivin, through a mechanism that involves NF-kB (discussed below). Another characteristic feature of E6 from HR-HPV types is the ability to bind members of the membrane-associated guanylate kinase (MAGUK) family, large proteins localized in the cytoplasmic membrane involved in cell-cell contact. These kinase family members contain multiple protein/protein interaction domains, including also PDZ motifs which are those interacting with E6, at his four amino acid PDZ-binding motif.10 The E6/MAGUK association is detrimental because it causes the loss of cell–cell contact and cell polarity. HPV16 E6 has also the ability to activate the transcription of the hTERT (human telomerase reverse transcriptase) gene encoding for one of the catalytic subunits of the telomerase complex. In somatic cells telomerase activity is usually very low or completely lacking, conversely, HPV 16-infected cells show high levels of telomerase activity, which allows telomere length maintenance and indefinite proliferation. The induction of hTERT activation by E6 is accomplished through the degradation of transcriptional repressors, such as NFX1-91, or the activation of hTERT promoter, such as Myc.11 In contrast to E6, E7 alone has no effect on hTERT expression; however, the concomitant expression of E7 and E6 significantly enhances E6-induced h-TERT transcription.12

E7 oncoprotein E7, one of the most efficient cell cycle deregulators, is a small nuclear phosphoprotein known to bind to the retinoblastoma protein (pRb) promoting its degradation via the ubiquitin/proteasome route.13 pRb is an inhibitory protein that negatively regulates, via direct association, the activity of several transcription factors, including members of the E2F family, stimulators of the expression of multiple genes involved in the progression of cell cycle and DNA synthesis. In quiescent cells, pRb is present in a hypophosphorylated form and associates with E2F molecules, thereby inhibiting their transcriptional activity. When quiescent cells are exposed to mitogenic signals, pRb undergoes phosphorylation in mid-G1 phase, through cyclin-dependent kinase (CDK) activity. Phosphorylation of pRb leads to the disruption of pRb/E2F complexes, with consequent activation of E2F, which in turn activates the transcription of a group of genes encoding proteins essential for cell cycle progression, such as cyclin E and cyclin A. pRB remains phosphorylated until late M when it again becomes hypophosphorylated through the action of a specific phosphatase. E7 binding to pRb mimics its phosphorylation. Thus, E7 expressing cells can enter S phase in the absence of mitogenic signals. E7 has the ability to alter the cell cycle via several mechanisms. First, HPV 16 E7 can associate with the CDK inhibitors p21WAF1/CIP1 and p27KIP1, thus neutralizing their inhibitory effects on the cell cycle or can also directly and/or indirectly interact with the cyclin A/CDK2 complex.14, 15 Second, it has been suggested that E7 may modulate kinase complexes thus favoring phosphorylation of a different set of substrates involved in progression/completion of the viral life cycle. The correlation between pRb-binding efficiency and E7 transforming activity vary consistently among HPV types. For example, E7s from certain noncarcinogenic HPV types, such as the cutaneous HPV 1 and the mucosal HPV 32, strongly associate with pRb but do not display any in vitro transforming activities.16 Although HPV 1, 32 and 16 E7s associate with pRb with similar affinity, the consequences of these interactions can be different. Exclusively in the case of HPV 16 E7, the association is responsible for pRb degradation via a ubiquitin/proteasome-dependent mechanism,17 while HPV 1 and 32 E7 proteins do not. Thus, only the HR-HPV types are able to promote pRb degradation, with all its downstream effects for cell replication and survival.

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Cancer cell is characterized by the accumulation of genetic damage, and also by alteration of intracellular pH, that is, alkalinization. It has been demonstrated that HPV 16 E7 has the ability to induce Na +/H + exchanger NHE-1 in NIH3T3 cells and primary human keratinocytes.18 This effect is blocked by treatment with specific NHE-1 inhibitors thus inhibiting the development of the transformed phenotype, supporting the importance of this event in HPV-mediated carcinogenesis. HR-HPV oncoproteins also cause mitotic abnormalities, which result in genomic instability and contribute to malignant progression. Several evidences showed that cervical cancer cells accumulated a plethora of numerical and structural chromosomal aberrations.19 In detail, HR- but not LR-HPV infections are responsible for genomic instability that can be already evidenced in premalignant lesions prior to the integration of HPV genomes into host chromosomes.20 These data led to hypothesize a causal role for HR-HPV in the subversion of genomic stability. Accordingly, several cytogenetic abnormalities have been detected in HPV immortalized keratinocytes which confirm that HR-HPV oncogene expression may promote genomic destabilization.

E5 oncoprotein Compared with E6 and E7, the oncotic role of E5 protein has not been fully elucidated. During HR-HPV infection steps, the HPV E5 protein is expressed in precancerous stages but not after viral integration. It is reasonable that E5 plays a role mostly in the early stage of cervical carcinogenesis. The finding supporting this hypothesis is that E5 gene is frequently deleted when the HPV genome is integrated during the progression from low-grade to malignant disease. Further, it is likely that E5 may also contribute to cell transformation by modulating cellular signaling pathways in addition to potentiating the immortalizing ability of E6 and E7. Recent evidences have shown that HPV 16 E5 may contribute to cervical carcinogenesis in part via stimulation of IFN-b. This effect is mediated by the induction of interferon regulatory factor 1 (IRF-1) which, in turn, induces transcriptional activation of IRF-1-targeted interferon-stimulated genes (ISGs).21

HPV oncoproteins and transcription factors A number of transcription factors have been linked to the development of human tumors. E6 and E7 oncoproteins stimulate proliferation by manipulating the function of a variety of host regulatory proteins. In turn, the binding of transcription factors to URR of HPV genome facilitates the expression of viral oncoproteins; the carcinogenic power is also dependent on the availability of a defined set of transcription factors derived from the infected host cell. Intracellular signaling cascade occurs through a sequence of molecular events initiated by the specific interaction of an extracellular ligand with its receptor. This complex series of signals are further amplified by the action of second messengers that are able to modulate a number of target proteins ultimately affecting several cellular functions. Within this frame, ROS/RNS have been proposed as second messengers able to activate several signaling pathways leading to mitogenesis or apoptosis. It is well established that gene expression is significantly regulated in response to oxidative stress and require the activation of specific redox-sensitive transcription factors, such as Nrf2, NF-kB, AP-1, and MAPKs, necessary to ensure cell survival. In response to changes in the activities of these transcription factors, most likely through oxidation of critical cysteine residues found on these proteins, the cellular redox status is modified.22

Activator protein-1 Activator protein-1 (AP-1) is closely linked with proliferation and transformation of tumor cells. This factor, normally consisting of a heterodimer between c-Fos and c-Jun, seems to play a central role in transcriptional regulation of viral oncogene expression. In fact, while AP-1 binding was very low or absent in normal as well as in premalignant lesions, AP-1 transcription and binding activity were found to be very high in malignant tissues.23 It was also shown that antioxidant-induced changes of the AP-1 transcription complex were paralleled by selective suppression of HPV transcription.24 The AP-1 is a direct target for the E7 oncogene, which post translationally interacts with c-Jun in elevating its transactivation activity.25 AP-1 in response to low levels of oxidants binds to specific DNA sequence thus resulting in activation of gene expression. Once activated, AP-1, in turn, induces JNK activity which phosphorylates the c-Jun transactivation domain. Conversely, when ROS are produced at high concentrations, AP-1 and AP-1-induced gene expression is inhibited. ROS cause oxidation of specific cysteine residues in c-Jun’s DNA-binding region, thus blocking AP-1/DNA interactions.

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NF-kB Similarly, NF-kB contains a redox-sensitive critical cysteine residue that is involved in DNA binding.22 NF-kB is a protein complex that controls the transcription of DNA and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens. NF-kB plays a key role in regulating the immune response to infection and its incorrect regulation has been linked to several diseases, including cancer. This transcription factor is a dimeric complex composed of different members of the Rel/NF-kB family of polypeptides. NF-kB is normally located in the cytosol complexed with the inhibitory protein IkBa, but under oxidative conditions, IkBa sequentially undergoes phosphorylation by IkB kinase (IKK), ubiquitination, and subsequent degradation by the proteasome. Once it is dissociated by IkBa, the activated NF-kB is then translocated into the nucleus and binds to specific sequences of DNA adjacent to the genes that it regulates. Though ROS production is necessary to promote the initial events leading to the dissociation of the NF-kB/IkB complex, excessive ROS levels result in the oxidation of cysteine residues without affecting its translocation to the nucleus, but rather its DNA binding. Activated NF-kB suppresses apoptosis in a wide variety of tumor cells26 and several proliferative, proinflammatory, and proangiogenic factors associated with aggressive tumor growth are regulated by NF-kB. Among these is cyclooxygenase-2 (COX-2), enzyme regulating prostaglandins production, that has been implicated in participating to both carcinogenesis and cancer progression in various malignancies, including cervical cancer.27 COX-2 transcription was shown to be stimulated by HPV 16 E6 and E728 and also by E529 through NF-kB and AP-1. COX-2 expression found an increase from LSIL to HSIL, the highest score being noted in HSIL corresponding to CIN3 lesions, suggesting that this enzyme may have a role in the development and progression of cervical squamous intraepithelial lesions.30 Results from different groups demonstrated that COX-2 expression promotes the progression of cervical cancer by increasing lymphonode metastasis and resistance to radiation therapy. It is worth to mentioning that several naturally occurring compounds are used against cervical cancer for their antioxidant and antiinflammatory properties. In fact, their protective effects rely on the ability to modulate the activation/ repression of multiple redox-sensitive transcription factors.31, 32

Oxidative stress and cervical cancer Despite the evidence that HPV is strongly implicated as the causative agent of cervical cancer, oncogenic HPV infection alone is not sufficient for tumor development. Other factors have to be involved in the progression of infected cells to the full neoplastic phenotype. Oxidative stress appears a good candidate as cancer-promoting factor. It is known that cancer cells are characterized by enhanced oxidative stress. Elevated rates of ROS have been detected in almost all cancers, where they act as second messengers in intracellular signaling cascades, promoting many aspects of tumor development and progression. It is well documented that some risk factors known to be implicated in cervical cancer development, such as tobacco smoking and chronic inflammation, determine oxidative stress increase and several evidences depose for the occurrence of oxidative stress in cervical cancer.33–35

Oxidative/nitrosative stress markers Studies performed on blood from patients with lesions diagnosed as CIN or cervical cancer, evidenced significant changes in the levels of oxidative and nitrosative stress indicators (Table 1).

Lipid peroxidation products An important role in the control of cell division is played by lipid peroxidation (LPO). Oxidative stress and LPO associated with infections and chronic inflammation may induce several human cancers. LPO products are reactive to nucleic acids, proteins and cellular thiols, and have profound mutagenic potential. Modifications of proteins with LPO products may regulate cellular processes like apoptosis, cell signaling, and senescence. A significant increase of plasma levels of malondialdheyde (MDA), a product of lipid peroxidation initiated by ROS, was found in women with invasive cervical cancer36, 37 and CIN37, 38 compared to those of healthy subjects. Also, erythrocyte MDA level was found significantly more elevated in patients with cervical cancer respect to controls.36 An estimation of LPO, as indexed by the measurement of thiobarbituric acid reactive substances (TBARS) indicated increased TBARS levels in plasma from patients with cervical carcinoma.39 Higher TBARS than control subjects were also

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TABLE 1 Changes in oxidative stress markers levels in samples from patients with lesions at different grades or cervical carcinoma respect to samples from control healthy subjects. Oxidative stress marker a

Human sample

Lesion b

c

Pathological sample vs. control

References

MDA

Plasma

CC , CIN

Increase

36–38

MDAa

Erythrocyte

CCb

Increase

36

TBARSb

Plasma

CCd

Increase

39

TBARSb

Erythrocyte

LSILe, HSILf, CCb

Increase

40

NO products

Plasma

CCb

Increase

36

d ALA-Dg

Blood

LSILb, HSILc, CCb

Increase

40

Protein carbonyls

Cervical tissue

CINc, CCb

Increase

41

a

MDA, malondialdheyde. CC, cervical carcinoma. CIN, cervical intraepithelial neoplasia. d TBARS, thiobarbituric acid reactive substances. e LSIL, low-grade squamous intraepithelial lesion. f HSIL, high-grade squamous intraepithelial lesion. g d ALA-D, d-amino levulinate dehydratase. b c

demonstrated in the erythrocytes of LSIL, HSIL and carcinoma patients,40 indicating that oxidative stress occurs precociously with premalignant states.

d-Aminolevulinate dehydratase As an index of overproduction of free radicals, the reactivation index of d-aminolevulinate dehydratase (d ALA-D) in the blood of premalignant and malignant stages of cervical cancer was also determined.40 This enzyme plays a fundamental role in most aerobic organisms by participating in heme biosynthesis pathway and its inhibition can lead to d-ALA accumulation, which in turn can enhance the generation of free radicals, aggravating oxidative damage to cellular components. The reactivation index of d ALA-D activity is a good tool to evaluate oxidative stress due to the high sensitivity of the enzyme to oxidation of its SH groups. Results obtained by Gonc¸alves et al.40 indicated that the enzyme is more oxidized in LSIL, HSIL, and carcinoma patients compared to control group.

Nitric oxide Nitrosative stress was also evidenced in patients with cervical carcinoma, as shown by increased plasmatic nitric oxide (NO) levels.36 NO is a crucial factor in the regulation of many homeostatic mechanisms. However, besides its regulatory effects, it can react with superoxide anion, forming peroxynitrite, a reactive and oxidant compound able to induce nitration and nitrosation of proteins, so altering their functions and therefore causing severe oxidative damage to the cell structures. NO and its derivatives, owing their cytotoxicity and capacity to cause direct or indirect genetic damage, have been suggested to play role in carcinogenetic processes. NO can stimulate ROS-induced LPO; in fact, a direct correlation between NO levels and erythrocyte MDA in cervical cancer was reported by Beevi et al.36

Antioxidant systems The cells counteract the oxidative stress by an array of many different defense mechanisms, including free radical scavengers and antioxidant enzymes to protect themselves from the potentially deleterious effects of ROS/RNS. However, in pathological conditions, despite this wide set of protecting mechanisms oxidative damage eventually accumulates contributing to a number of degenerative diseases including cancer. Oxidative burden attenuates the antioxidant defense systems and reduction in antioxidant defense mechanisms correlates with the emergence of the malignant phenotype. In cervical cancer, a reduction of both enzymatic and nonenzymatic antioxidant defense has been shown to occur (Table 2).

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TABLE 2 Changes in antioxidants levels in samples from patients with lesions at different grades or cervical carcinoma respect to samples from control healthy subjects. Enzymatic antioxidants a

SOD

c

CAT

d

Human sample Plasma Plasma

Lesion b

CC

b

CC

b

e

GPX

Plasma

CC , CIN

Nonenzymatic antioxidants

Human sample

Lesion f

g

Pathological sample vs. control

References

Decrease

36, 39, 42

Decrease

36, 42

Decrease

36, 37, 39

Pathological sample vs. control

References

Vitamin C

Plasma

LSIL , HSIL , CCb

Decrease

38–40

Vitamin E

Plasma

CINe, CCb

Decrease

37–39, 43

e

b

Vitamin A, lutein, lycopene, zeaxanthin

Plasma

CIN , CC

Decrease

37

GSHh

Plasma

CCb

Decrease

39

Decrease

44

Coenzyme Q10

Plasma

e

b

CIN , CC

a

SOD, superoxide dismutase. CC, cervical carcinoma. CAT, catalase. d GPX, glutathione peroxidase. e CIN, cervical intraepithelial neoplasia. f LSIL, low-grade squamous intraepithelial lesion. g HSIL, high-grade squamous intraepithelial lesion. h GSH, reduced glutathione. b c

A significant depletion in the activities of primary antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), was observed in patients with cervical cancer36, 39, 42 and CIN37 as compared to healthy subjects. Also, plasma levels of antioxidant vitamins and compounds, such as a-tocopherol,37–39, 43 vitamin C,38–40 lutein, beta-carotene, lycopene, and zeaxanthin37 were all significantly lower in women with cervical CIN or invasive cervical cancer, compared to those of the normal control group. A decrease in the level of other two potent antioxidants, that is, reduced glutathione and coenzyme Q10, was also found in the plasma of patients with various grades of CIN and cervical cancer compared to controls.39, 44 However, some contradictory results were also reported correlating the increased expression of other antioxidant enzymes with the dysplastic/neoplastic phenotype of cell lines or histological lesions.45, 46

Protein oxidation Oxidative stress in HPV-infected cells is also assessed by the oxidation of proteins that represent important targets of the deleterious effect of this condition. Several studies demonstrated that protein oxidation is frequently associated with a decrease or complete loss of protein catalytic functions and triggers the formation of high molecular, potentially cytotoxic aggregates. In order to elucidate the molecular events and mechanisms associated with cervical carcinogenesis and to identify prognostic or predictive markers, alterations of protein profiles were investigated. For this purpose, a proteomics approach was utilized, because this technique gives qualitative and quantitative information not only about the native proteins but also about their posttranslation modifications. Among these, the formation of carbonyl groups is the most widely studied index of protein oxidation. In particular, redox proteomics allows to investigate protein oxidative modifications and is revealed to be a useful method to identify those proteins resulting specifically altered in pathological conditions. Protein oxidation in cervical cancer has been recently investigated by studies on cell cultures and patients’ tissues.

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Studies on cell cultures A convenient experimental method to study the molecular mechanisms of tumor progression is represented by cell lines containing HPV genome, models of preneoplastic and neoplastic stages of cervical cancer. Perluigi et al.46, 47 applied a proteomic approach to study the effects of oxidative stress induced by UVB irradiation on HK-168, a keratinocyte cell line transfected with the whole genome of the high-risk type HPV-16, in comparison with normal human epithelial keratinocytes (NHEK). Significant changes in expression profile and specific protein oxidation were evidenced in both cell types. Proteins involved in folding (glucose-regulated protein 78, HSPs, glucosidase II), protein synthesis (elongation factors), maintenance of cytoskeleton integrity (cytokeratins, actin-related protein 3), cell-cell interaction (protein disulfide isomerase) resulted more oxidized upon UVB irradiation. On the other hand, proteins implicated in defense systems were not efficiently activated in HK-168, thus making the cells more susceptible to accumulate damaged and toxic proteins, by driving the virus-transformed cell toward a neoplastic phenotype.

Studies on patients’ tissues In a published study performed on tissues from high-grade dysplastic HPV-16 lesions (CIN 2/3) and invasive squamous cervical carcinoma tissues,41 it was shown that protein carbonyls were significantly increased in histological samples of dysplastic tissues, while levels detected in neoplastic tissues were not significantly different from controls (normal cervical tissue specimens). This unexpected trend was also paralleled by the extent of oxidative DNA damage, which clearly increased in dysplastic tissues with respect to both cancer and controls. By redox proteomic approach five proteins having increased carbonyls levels in dysplastic samples respect to controls were identified: cytokeratin 6, actin, cornulin, retinal dehydrogenase, and GAPDH. Comparison of cancer with dysplastic tissues evidenced that in carcinoma samples five proteins (peptidyl-prolyl cis-trans isomerase A, ERp57, serpin B3, annexin 2, and GAPDH) showed lower carbonylation than in dysplastic tissues. Interestingly, GAPDH was found more oxidized in dysplastic lesion versus either controls or neoplastic tissue. GAPDH-increased oxidation was paralleled by decreased enzymatic activity in dysplastic tissue with respect to control and, conversely, a reduced oxidation level in cancer tissue was accompanied by a considerable retrieval of enzymatic activity. Besides the well-known function in aerobic metabolism of glucose, GAPDH has been implicated in several cell pathways and its role in cell death regulation and carcinogenesis has been suggested.48 These redox proteomics results indicate the occurrence—in dysplastic lesions—of selective oxidation of specific proteins involved in cell signaling/division, morphogenesis and differentiation, leading to cytoskeleton derangement and suppression of terminal differentiation. In addition, a reduced control on viral oncogenes activity in dysplasia can also derive from the oxidative modification of protein implied in the synthesis of compounds able to operate a suppressive modulation of E6/E7 oncogenes, such as the increased carbonylation of retinal dehydrogenase, the enzyme catalyzing the formation of retinoic acid, a negative regulator of AP-1, and a transcription factor which plays a pivotal role in initiating and maintaining the expression of viral oncoproteins. Conversely, cancer tissue seems to attain an improved control on oxidative damage, as shown by lower protein oxidation respect to dysplasia (Fig. 3). This control could be explained by the activation—triggered by viral oncogenes—of some antioxidant/detoxifying mechanisms allowing the host cell to achieve adaptation to oxidative stress.

O X I D A T I V E S T R E S S

Increased protein oxidation

Dysplasia

Decreased protein oxidation

Control

Carcinoma

TUMOR GROWTH

FIG. 3 Interplay between tumor growth and oxidative stress. In dysplastic lesions, a selective oxidation of specific proteins occurs. Conversely, cancer tissue seems to attain an improved control on oxidative damage, as shown by lower protein oxidation. It is likely to hypothesize that tumor cells have the ability to withstand high levels of ROS by protecting cellular components from oxidative insult.

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Redox control and adaptive mechanisms in HPV-infected cells Tumor cells are characterized by a rather efficient control on ROS production and oxidative damage due to the increasing oxidant micro-environment along the steps of carcinogenesis. Such regulation of redox homeostasis enables cancer cells to survive, proliferate, and metastasize. In the viral carcinogenesis, in order to ensure the host cell and its survival, HPV confers to the infected cell the ability to resist and adapt to the oxidant environment. The HPV-induced control of redox homeostasis and the enhancement of host cell adaptation capabilities is achieved through different mechanisms, such as the modulation of stress and antioxidant response and the inhibition of oxidative stress-induced apoptosis.

Modulation of stress response markers Modulation of activity and/or expression of some stress response markers in the different types of lesions has been detected. An increase of the activity of glutathione S-transferase (GST) was observed in erythrocytes of cervical cancer patients36 and also studies on patients’ tissues evidenced that GST expression level was sharply increased in dysplastic and in neoplastic histological samples compared with controls.41 GST catalyzes the nucleophilic addition of the thiol of GSH to a variety of electrophiles and plays a prominent role in the protection of cells against many cytotoxic and carcinogenic chemicals. Increased expression of various GST isoenzymes has been found in human tumor cell lines of different histological origin, and increased GST activity has been observed in most of the human cancers, including cervical cancer.49 GSTP1 was identified as one of the major partners of HPV-16 E7. It was shown that the levels of oxidized GSTP1, which occurs under oxidizing conditions is associated with enzyme inactivation, and strongly decreases by HPV-16 E7. A direct link between the HPV-16 E7 and GSTP1 has been proposed50: the viral factor interacts with the enzyme through specific regions of the E7 sequence, and this interaction protects GSTP1 against inactivation via oxidative attacks at Cys 47 and/or Cys 101. The enzyme stabilization in its active reduced state allows protection against the cytotoxic effects of oxidative stress conditions and also accounts for the GSTP1 antiapoptotic JNK-dependent properties.51 In fact, the reduced form of the enzyme interacts with the c-Jun N-terminal kinase (JNK), negatively regulating the ability of this latter to phosphorylate the Jun protein. Since JNK-mediated signal transduction leads to apoptosis, this can be a mechanism by which HPV16 E7transformed cells escape apoptosis. In this scenario, the direct link observed between the HPV-16 E7 viral factor and the GSTP1 protein confers a pivotal role to this interaction promoting the survival of HPV 16 E7-expressing cancer cells. Notably, the recent study of Checa-Rojas et al.52 further suggests GSTP1 as a novel player driving tumor progression in cervical cancer. Also, the expression of endoplasmic reticulum protein 57 (ERp57), a protein assisting the maturation and transport of unfolded secretory proteins by facilitating disulfide bond formation, was significantly higher in neoplastic tissue compared with both dysplastic and controls tissues. On the contrary, a progressive downregulation of the inducible form of nitric oxide synthase (iNOS) expression during the different stages of cervical carcinogenesis—from the control to dysplastic and to neoplastic samples—was evidenced by different authors.41, 53 This trend—which is divergent from that observed in most cancer types—suggests that low iNOS expression may address toward a malignant phenotype, promoting tumor progression rather than an antitumor response. However, the role of this enzyme in carcinogenesis is far from being completely clear.

Modulation of antioxidant response: Peroxiredoxins Peroxiredoxins (Prxs) represent one of the most important protective mechanisms against ROS. These cysteine-rich proteins participate to the regulation of redox balance by degrading hydrogen peroxide, organic hydroperoxides, and peroxynitrite, involved in signal transduction pathways in response to both physiological and oxidative stimuli and play a fundamental role in carcinogenesis and tumor progression. Overexpression of various Prx isoforms has been suggested to be responsible for tumor progression, prognosis, and resistance to chemo- and radiotherapy.54 About cervical cancer, a direct correlation between Prx2 and 3 expression and severity of CIN at any grade was shown and in the study of Hu et al.55 on histological samples from invasive cervical carcinomas, Prx3 was found highly expressed in cancer areas, while only a slight positivity was observed in the basal layer of adjacent nonneoplastic tissue. Moreover, the pattern of Prx3 expression in cervical cancer cells was positively correlated with that of both HPV16 E6/E7 and the nuclear protein Ki67, marker of tumor proliferation. Proteomic analysis of Lee et al.56 identified Prx2 among the proteins that were upregulated by HPV16 E7 and recently Prx2 overexpression was detected in the secretome of cervical cancer cell lines.57

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Suppression of oxidative stress-induced apoptosis HPV-infected cells are resistant to programmed cell death induced by oxidant conditions. In addition to the mentioned antiapoptotic JNK-dependent properties of GSTP1, this goal is achieved by regulation of some other apoptosis inhibitors, such as survivin and c-IAP2 (cellular inhibitor of apoptosis protein 2). Survivin has been shown to suppress apoptosis and control cell division, besides its capacity to act as an antioxidant compound. It is interesting to note that this protein is completely absent in terminally differentiated cell, while it is highly expressed in fetal tissue and in most malignant tumors, including cervical cancer. Recent results revealed a higher expression level of survivin in cervical cancer than in CIN lesions and normal cervical tissues and the increment was correlated with the grade and the stage of the disease.58 It has been shown by experiments performed in different cancer cell lines and with different E6 mutants that HPV-16 E6 significantly transactivates the survivin promoter and that this effect is largely dependent on p53 status. Survivin regulation seems, in fact, to be linked to the p53 tumor suppressor protein, that downregulates its expression. The ability of E6 oncoprotein to interact and degrade p53 lead to the upregulation of surviving factors, decreased rate of apoptosis, and resistance to apoptotic stimuli. Some studies have shown that the expression of survivin may be considered as a prospective novel anticancer therapeutic target and a recent study has suggested that targeting of surviving expression might be an ideal strategy for cervical cancer treatment, as it would decrease viable cell number and enhance apoptosis sensitivity.59 c-IAPs are well known for their ability to inhibit caspase activation and permit cell survival in stress conditions. Upregulation of c-IAP2 expression was shown to confer resistance to oxidative stress-mediated apoptosis in various cell types. The transcription of c-IAP2 was found significantly upregulated by E6 and E7 oncoproteins in cells infected with high-risk type HPV 16, but not in cells infected with low-risk type HPV 6, and c-IAP2 depletion in these cells led to apoptosis.60 The c-IAP induction was found to require NF-kB activity and it has been proved that E6-induced expression of cIAP-2 was directly associated with the oncogene ability to induce NF-kB binding to the cIAP-2 promoter, thus increasing cIAP-2 transcript level. On the whole, these findings support the view that dysplastic state is highly vulnerable to oxidative damage, a major factor of genetic instability, providing the conditions for the neoplastic evolution of transformed cells. Thus, oxidative injury is a prominent factor even in the early steps of carcinogenesis. Conversely, tumor cells adapt their metabolism in order to support their growth and survival, seemingly creating a paradox of high ROS production in the presence of high antioxidant levels, seeming to fit well with stress conditions (Fig. 4). The fine mechanisms activated by cancer cells to counteract oxidative burden, allowing to escape ROS-induced cytotoxicity, are anyway responsible for ROS-mediated mutagenic events and may favor the accumulation of genetic damages and the malignant progression.

FIG. 4 From HPV infection to cancer: role of oxidative stress. Oxidative injury is a prominent factor even in the early steps of carcinogenesis. Conversely, tumor cells adapt their metabolism in order to support their growth and survival, through upregulation of (1) stress response (GSTP1, ERp57) and antioxidant response (Prx); (2) apoptosis inhibitors (GSTP1, survivin, c-IAP2). The HPV-induced control of redox homeostasis and apoptosis inhibition provides the condition to turn the oxidative stress from a potentially harmful and limiting condition into a positive factor for virus survival and proliferation.

HPV OS LSIL CIN1 Immune response, cell antioxidant response, apoptosis

OS HSIL CIN2/3

Cell death, virus death Infection regression

OS Infection regression

HPV 1 Antioxidant and stress response induction

Cervical cancer

2 Apoptosis inhibition

OS adaptation cell proliferation, virus proliferation

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Conclusion Elevated rates of ROS have been detected in almost all cancers, where they act as second messengers in intracellular signaling cascades, promoting many aspects of tumor development and progression. Indeed, it is well established that some risk factors implicated in cervical cancer development, such as tobacco smoking and chronic inflammation, are responsible for increased oxidative stress conditions. However, the molecular events associated with oxidative damage remain still unclear. The present poor knowledge about the mechanisms involved in neoplastic progression has dramatic consequences on the clinical side. Because it is not possible to predict the clinical outcome of precancerous lesions, while just a very minor part of them tend to progress, all of them have to be regarded as a potentially progressive. Further research should be directed to evaluate if the impairment of protein function, consequent to oxidative modifications, may correlate with a specific state of neoplastic transformation/progression. In addition, because of the bimodal effect of oxidative stress on cervical cancer initiation and progression, development of novel strategies for cancer treatment relies on a better understanding of molecular mechanisms allowing cancer cells to cope with oxidant conditions. Future studies should explore alternative therapeutic strategies which can effectively target oxidative stress-related pathways during various phases of viral infection and its oncogenic expression in order to reduce the risk of HPV-mediated cancer.

Summary points l

l

l

l

l

l l

l

The infection with human papillomavirus (HPV) is the primary and causative event in the development of cervical cancer, one of the most diffuse virus-caused neoplastic transformations. E6, E7, and E5 are recognized as the oncogenic proteins of high-risk HPV types, involved in cell transformation and cancer promotion. They play roles throughout the whole disease process, from early lesion to invasive carcinoma. The progression of infected cells to the full neoplastic phenotype is supported by a number of factors, most importantly, oxidative stress. In samples from patients with lesions at different grades or cervical carcinoma, the increase of the levels of oxidative stress indicators and oxidative damage to proteins has been detected. Viral oncogenes selectively interact with redox-sensitive transcription factors and a subset of intracellular proteins is mainly involved in cell division, differentiation, and morphogenesis. HPV oncogenes confer to the infected cells the ability to adapt to oxidant environment. The modulation of host cell redox-homeostasis promotes the survival of transformed cells and may favor tumor development. Knowledge of the mechanisms activated by viral oncogenes that confer to the infected cells the ability to survive in an increasing oxidant environment can provide a rationale for the development of novel strategies for cancer prevention and treatment.

Acknowledgments We thank Dr. Fabio Di Domenico and Dr. Federico De Marco for their critical review of the manuscript.

References 1. zur Hausen H, Meinhof W, Scheiber W, Bornkamm GW. Attempts to detect virus-specific DNA in human tumors. I. Nucleic acid hybridizations with complementary RNA of human wart virus. Int J Cancer 1974;13:650–6. 2. De Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H. Classification of papillomaviruses. Virology 2004;324:17–27. 3. Storz P. Reactive oxygen species in tumor progression. Front Biosci 2005;10:1881–96. 4. Burd EM, Dean CL. Human papillomavirus. Microbiol Spectr 2016;4(4):1–17. 5. Reusser NM, Downing C, Guidry J, Tyring SK. HPV carcinomas in immunocompromised patients. J Clin Med 2015;4:260–81. 6. Baak JP, Kruse AJ, Robboy SJ, Janssen EA, van Diermen B, Skaland I. Dynamic behavioural interpretation of cervical intraepithelial neoplasia with molecular biomarkers. J Clin Pathol 2006;59:1017–28. 7. Massimi P, Shai A, Lambert P, Banks L. HPV E6 degradation of p53 and PDZ containing substrates in an E6AP null background. Oncogene 2008;27:1800–4. 8. Hiller T, Poppelreuther S, Stubenrauch F, Iftner T. Comparative analysis of 19 genital human papillomavirus types with regard to p53 degradation, immortalization, phylogeny, and epidemiologic risk classification. Cancer Epidemiol Biomarkers Prev 2006;15:1262–7. 9. Jackson S, Harwood C, Thomas M, Banks L, Storey A. Role of BAK in UV-induced apoptosis in skin cancer and abrogation by HPV E6 proteins. Genes Dev 2000;14:3065–73.

Oxidative stress and viral carcinogenesis Chapter

9

99

10. Watson RA, Thomas M, Banks L, Roberts S. Activity of the human papillomavirus E6 PDZ-binding motif correlates with an enhanced morphological transformation of immortalized human keratinocytes. J Cell Sci 2003;116:4925–34. 11. Veldman T, Liu X, Yuan H, Schlegel R. Human papillomavirus E6 and Myc proteins associate in vivo and bind to and cooperatively activate the telomerase reverse transcriptase promoter. Proc Natl Acad Sci U S A 2003;100:8211–6. 12. Liu X, Roberts J, Dakic A, Zhang Y, Schlegel R. HPV E7 contributes to the telomerase activity of immortalized and tumorigenic cells and augments E6-induced hTERT promoter function. Virology 2008;375:611–23. 13. M€ unger K, Basile JR, Duensing S, Eichten A, Gonzalez SL, Grace M, et al. Biological activities and molecular targets of the human papillomavirus E7 oncoprotein. Oncogene 2001;20:7888–98. 14. Zerfass-Thome K, Zwerschke W, Mannhardt B, Tindle R, Botz JW, Jansen-D€urr P. Inactivation of the CDK inhibitor p27KIP1 by the human papillomavirus type 16 E7 oncoprotein. Oncogene 1996;13:2323–30. 15. He W, Staples D, Smith C, Fisher C. Direct activation of cyclin-dependent kinase 2 by human papillomavirus E7. J Virol 2003;77:10566–74. 16. Caldeira S, Dong W, Tomakidi P, Paradiso A, Tommasino M. Human papillomavirus type 32 does not display in vitro transforming properties. Virology 2002;301:157–64. 17. Jones DL, M€ unger K. Analysis of the p53-mediated G1 growth arrest pathway in cells expressing the human papillomavirus type 16 E7 oncoprotein. J Virol 1997;71:2905–12. 18. Reshkin SJ, Bellizzi A, Caldeira S, Albarani V, Malanchi I, Poignee M, et al. Na+/H+ exchanger-dependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformation-associated phenotypes. FASEB J 2000;14:2185–97. 19. Mitelman F, Johansson B, Mertens F. Mitelman database of chromosome aberrations in cancer. http://cgap.nci.nih.gov/Chromosomes/Mitelman; 2007. 20. Bulten J, Poddighe PJ, Robben JC, Gemmink JH, de Wilde PC, Hanselaar AG. Interphase cytogenetic analysis of cervical intraepithelial neoplasia. Am J Pathol 1998;152:495–503. 21. Muto V, Stellacci E, Lamberti AG, Perrotti E, Carrabba A, Matera G, et al. Human papillomavirus type 16 E5 protein induces expression of beta interferon through interferon regulatory factor 1 in human keratinocytes. J Virol 2011;85:5070–80. 22. Matthews JR, Kaszubska W, Turcatti G, Wells TN, Hay RT. Role of cysteine62 in DNA recognition by the P50 subunit of NF-kappa B. Nucleic Acids Res 1993;21:1727–34. 23. Prusty BK, Das BC. Constitutive activation of transcription factor AP-1 in cervical cancer and suppression of human papillomavirus (HPV) transcription and AP-1 activity in HeLa cells by curcumin. Int J Cancer 2005;113:951–60. 24. R€ osl F, Das BC, Lengert M, Geletneky K, zur Hausen H. Antioxidant-induced changes of the AP-1 transcription complex are paralleled by a selective suppression of human papillomavirus transcription. J Virol 1997;71:362–70. 25. Antinore MJ, Birrer MJ, Patel D, Nader L, McCance DJ. The human papillomavirus type 16 E7 gene product interacts with and transactivates the AP1 family of transcription factors. EMBO J 1996;15:1950–60. 26. Wang CY, Mayo MW, Kornelul RG, Goeddel DV, Baldwin Jr. AS. NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and cIAP2 to suppress caspase-8 activation. Science 1998;281:1680–3. 27. Kim MH, Seo SS, Song YS, Kang DH, Park IA, Kang SB, et al. Expression of cyclooxygenase-1 and -2 associated with expression of VEGF in primary cervical cancer and at metastatic lymphnodes. Gynecol Oncol 2003;90:83–90. 28. Subbaramaiah K, Dannenberg AJ. Cyclooxygenase-2 transcription is regulated by human papillomavirus 16 E6 and E7 oncoproteins: evidence of a corepressor/coactivator exchange. Cancer Res 2007;67:3976–85. 29. Kim SH, Oh JM, No JH, Bang YJ, Juhnn YS, Song YS. Involvement of NF-kB and AP-1 in COX-2 upregulation by human papillomavirus 16 E5 oncoprotein. Carcinogenesis 2009;30:753–7. 30. Sarian LO, Derchain SF, Yoshida A, Vassallo J, Pignataro F, De Angelo A, et al. Expression of cyclooxygenase-2 (COX-2) and Ki67 as related to disease severity and HPV detection in squamous lesions of the cervix. Gynecol Oncol 2006;102:537–41. 31. Prusty BK, Bhudev CD. Constitutive activation of transcription factor AP-1 in cervical cancer and suppression of human papillomavirus (HPV) transcription and AP-1 activity in HeLa cells by curcumin. Int J Cancer 2004;113:951–60. 32. Di Domenico F, Foppoli C, Coccia R, Perluigi M. Antioxidants in cervical cancer: chemopreventive and chemotherapeutic effects of polyphenols. Biochim Biophys Acta 2012;1822:737–47. 33. Moktar A, Singh R, Vadhanam MV, Ravoori S, Lillard JW, Gairola CG, et al. Cigarette smoke condensate-induced oxidative DNA damage and its removal in human cervical cancer cells. Int J Oncol 2011;39:941–7. 34. Georgescu SR, Mitran CI, Mitran MI, Caruntu C, Sarbu MI, Matei C, et al. New insights in the pathogenesis of HPV infection and the associated carcinogenic processes: the role of chronic inflammation and oxidative stress. J Immunol Res 2018;2018:5315816. 35. De Marco F. Oxidative stress and HPV carcinogenesis. Viruses 2013;5:708–31. 36. Beevi SS, Rasheed MH, Geetha A. Evidence of oxidative and nitrosative stress in patients with cervical squamous cell carcinoma. Clin Chim Acta 2007;375:119–23. 37. Kim YT, Kim JW, Choi JS, Kim SH, Choi EK, Cho NH. Relation between deranged antioxidant system and cervical neoplasia. Int J Gynecol Cancer 2004;14:889–95. 38. Lee GJ, Chung HW, Lee KH, Ahn HS. Antioxidant vitamins and lipid peroxidation in patients with cervical intraepithelial neoplasia. J Korean Med Sci 2005;20:267–72. 39. Manju V, Kalaivani Sailaja J, Nalini N. Circulating lipid peroxidation and antioxidant status in cervical cancer patients: a case-control study. Clin Biochem 2002;35:621–5.

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40. Gonc¸alves TL, Erthal F, Corte CL, M€uller LG, Piovezan CM, Nogueira CW, et al. Involvement of oxidative stress in the pre-malignant and malignant states of cervical cancer in women. Clin Biochem 2005;38:1071–5. 41. De Marco F, Bucaj E, Foppoli C, Fiorini A, Blarzino C, Filipi K, et al. Oxidative stress in HPV-driven viral carcinogenesis: redox proteomics analysis of HPV-16 dysplastic and neoplastic tissues. PLoS One 2012;7:e34366. 42. Manju V, Balasubramanian V, Nalini N. Oxidative stress and tumor markers in cervical cancer patients. J Biochem Mol Biol Biophys 2002;6:387–90. 43. Palan PR, Woodall AL, Anderson PS, Mikhail MS. Alpha-tocopherol and alpha-tocopheryl quinone levels in cervical intraepithelial neoplasia and cervical cancer. Am J Obstet Gynecol 2004;190:1407–10. 44. Palan PR, Mikhail MS, Shaban DW, Romney SL. Plasma concentrations of coenzyme Q10 and tocopherols in cervical intraepithelial neoplasia and cervical cancer. Eur J Cancer Prev 2003;12:321–6. 45. Del Nonno F, Pisani G, Visca P, Signore F, Grillo LR, Baiocchini A, et al. Role and predictive strength of transglutaminase type 2 expression in premalignant lesions of the cervix. Mod Pathol 2011;24:855–65. 46. Termini L, Filho AL, Maciag PC, Etlinger D, Alves VA, Nonogaki S, et al. Deregulated expression of superoxide dismutase-2 correlates with different stages of cervical neoplasia. Dis Markers 2011;30:275–81. 47. Perluigi M, Giorgi A, Blarzino C, De Marco F, Foppoli C, Di Domenico F, et al. Proteomics analysis of protein expression and specific protein oxidation in human papillomavirus transformed keratinocytes upon UVB irradiation. J Cell Mol Med 2009;13:1809–22. 48. Colell A, Green DR, Ricci JE. Novel roles for GAPDH in cell death and carcinogenesis. Cell Death Differ 2009;16:1573–81. 49. Chatterjee A, Gupta S. The multifaceted role of glutathione S-tranferases in cancer. Cancer Lett 2018;433:33–42. 50. Mileo AM, Abbruzzese C, Mattarocci S, Bellacchio E, Pisano P, Federico A, et al. Human papillomavirus-16 E7 interacts with glutathione S-transferase P1 and enhances its role in cell survival. PLoS One 2009;4:e7254. 51. Wang T, Arifoglu P, Ronai Z, Tew KD. Glutathione S-transferase P1-1 (GSTP1-1) inhibits c-Jun N-terminal kinase (JNK1) signaling through interaction with the C terminus. J Biol Chem 2001;276:20999–1003. 52. Checa-Rojas A, Delgadillo-Silva LF, Velasco-Herrera MDC, Andrade-Domı´nguez A, Gil J, Santilla´n O, et al. GSTM3 and GSTP1: novel players driving tumor progression in cervical cancer. Oncotarget 2018;9:21696–714. 53. Mazibrada J, Ritta M, Mondini M, De Andrea M, Azzimonti B, Borgogna C, et al. Interaction between inflammation and angiogenesis during different stages of cervical carcinogenesis. Gynecol Oncol 2008;108:112–20. 54. Kim B, Kim YS, Ahn HM, Lee HJ, Jung MK, Jeong HY, et al. Peroxiredoxin 5 overexpression enhances tumorigenicity and correlates with poor prognosis in gastric cancer. Int J Oncol 2017;51:298–306. 55. Hu JX, Gao Q. Peroxiredoxin 3 is a novel marker for cell proliferation in cervical cancer. Biomed Rep 2013;1:228–30. 56. Lee KA, Kang JW, Shim JH, Kho CW, Park SG, Lee HG, et al. Protein profiling and identification of modulators regulated by human papillomavirus 16 E7 oncogene in HaCaT keratinocytes by proteomics. Gynecol Oncol 2005;99:142–52. 57. Kontostathi G, Zoidakis J, Makridakis M, Lygirou V, Mermelekas G, Papadopoulos T, et al. Cervical cancer cell line secretome highlights the roles of transforming growth factor-beta-induced protein ig-h3, peroxiredoxin-2, and NRF2 on cervical carcinogenesis. Biomed Res Int 2017;2017:4180703. 58. Fan Y, Chen J. Clinicopathological significance of survivin expression in patients with cervical cancer: a systematic meta-analysis. Bioengineered 2017;8:511–23. 59. Lee JP, Chang KH, Han JH, Ryu HS. Survivin, a novel anti-apoptosis inhibitor, expression in uterine cervical cancer and relationship with prognostic factors. Int J Gynecol Cancer 2005;15:113–9. 60. Yuan H, Fu F, Zhuo J, Wang W, Nishitani J, An DS, et al. Human papillomavirus type 16 E6 and E7 oncoproteins upregulate c-IAP2 gene expression and confer resistance to apoptosis. Oncogene 2005;24:5069–78.

Chapter 10

Polymorphisms, antioxidant genes, and cancer Mazhar Al Zoubia and Alaa Aljabalib a

Department of Basic Medical Sciences, Faculty of Medicine, Yarmouk University, Irbid, Jordan, b Department of Pharmaceutical Sciences,

Faculty of Pharmacy, Yarmouk University, Irbid, Jordan

Introduction Oxidative stress is linked to many cell injuries including DNA damage; and enormous pieces of evidence showed an association between oxidative stress and the risk of cancers.1,2 Therefore, antioxidative mechanisms are crucial for the cellular integrity and avoidance of diseases development.3 Human antioxidant enzymes, proteins, and molecules play an important role in this defense mechanism. Thus, conformational changes or the levels of expression of these genes are expected to have a significant influence on their functions and may lead to serious diseases and disorders such as cancers.4–9 Polymorphic alterations in the coded genes of the antioxidant enzymes or proteins have shown—as expected—a substantial alteration in the activity of these enzymes (Fig. 1).10–14 For instance, Lena Forsberg et al. demonstrated the functional effect of C> T substitution in the
262 bp position in the promoter region of the catalase gene (CAT) and its influence on the binding of transcription factors, reporter gene transcription, and catalase level expression.14 In another example, a polymorphic site was identified in the signal sequence of human superoxide dismutase type 2 (MnSOD) (Ala16Val) with a prediction to form an amphiphilic helix with higher efficiency.12 The Ala16Val polymorphism of the MnSOD is located in the mitochondrial targeting sequence, recent studies pointed out that MnSOD-Ala is targeted into the mitochondria, while the MnSOD-Val is detained in the inner mitochondrial membrane (Fig. 2).4 Moreover, in the MnSOD Ala16Val polymorphism, it has been hypothesized that the less efficient T allele (Ala) is associated with higher levels of ROS expression, which may lead to cancer’s development. However, it has been found that C polymorphism (Val) is associated with a higher risk of breast,15,16 prostate,17 and bladder,18 but not lung19 cancer, assuming that the C allele might be associated with other mechanisms, such as protein-protein interactions and subsequent disruption of MnSOD despite efficient localization to the mitochondrion. In another example, it has been reported that in in vivo and in vitro study, MnSOD has higher activity in Ile58 allele expressing cells than Thr58 allele expressing cells.20 As well, the myeloperoxidase (MPO) 463 A allele showed lower expression levels compared to the G allele in vitro.21,22 The 63 antioxidant genes were classified into three groups including superoxide dismutases (SODs), peroxidases, thiolredox proteins.23 Polymorphisms in certain antioxidant genes have been proposed to be associated with many pathological conditions such as cancers, vitamins deficiencies, infertility, and other metabolic disorders.24–26 In this context, polymorphic variants in antioxidant genes have been extensively studied to illustrate their role in the development of different types of cancers.27–30 The MnSOD Val16Ala (rs4880) genotype is one of the most studied variants in many cancers due to the structural and functional impact of the MnSOD Val16Ala polymorphism on the intracellular fate of the MnSOD enzyme. The Val16Ala polymorphism is located in the signal sequence of the MnSOD peptide, which alters its destination from the mitochondrial matrix to be arrested in the inner mitochondrial membrane as well as a reduction in the Val-MnSOD activity as shown in Fig. 1.4 It has been hypothesized that polymorphic variations in MnSOD, MPO, and CAT genes might be associated with anticytotoxicity activity and, in that way improves survival rates in women receiving radiation and chemotherapy for breast cancer.31 For instance, a recent metaanalysis study that included 88 studies with a total population of 33,098 cases and 37,831 controls; the findings suggested that the influence of the MnSOD Val16Ala polymorphism is to increase the overall cancer risk and increased risk of prostate cancer among Asians and Caucasians populations reported in this study.32 However, the polymorphic studies showed contradictory results in a different population of the studied cancers. Herein, we will discuss the role of this genotype in different cancers as well as the other studied polymorphisms.

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00010-9 © 2021 Elsevier Inc. All rights reserved.

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FIG. 1 A representative diagram of the SOD1, SOD2, SOD3, CAT, and MPO genes, mentioning the number of exons and the promotor site for the binding of transcription factors.

Ala MnSOD

Val MnSOD

MnSOD

MnSOD

O2–.

H2O2

CATT

MPOG HOCl

D

SO

Zn

H2O+O2

FIG. 2 A representative diagram of the polymorphic impact on the action of MnSOD, MPO, and CAT enzymes. The C allele of MnSOD or Ala variant produces more active MnSOD and matrix localization in the mitochondria. T allele of the CAT enzyme produces less active CAT and higher ROS. The G allele of the MPO causes higher transcription of the MPO and higher ROS. The ZnSOD enzyme is localized in the intermembrane space.

Breast cancer Various polymorphic variants in the oxidative genes have been inspected to elucidate the role of these variants in the development or susceptibility of breast cancer. Not limited to, the most studied genes included superoxide dismutase 1 (SOD1) (CuZn-SOD), superoxide dismutase 2 (SOD2) (MnSOD), glutathione peroxidase 1 (GPX1), GPX4, glutathione reductase (GSR), myeloperoxidase (MPO), thioredoxin (TXN), thioredoxin reductase 1 (TXNRD1), thioredoxin reductase 2 (TXNRD2), and catalase (CAT). In a multiple loci approach, Oestergaard et al. did not find any statistically significant association between TXN (t2715c), TXNRD2 (g23524a), GSR (c39396t), and TXNRD2 (a442g) polymorphisms and their

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susceptibility of breast cancer development.28 Another study included more than 100 polymorphisms in 22 candidate genes including CAT, GPX1, GSR, MPO, metallothionein 2A (MT2A), peroxiredoxin 1 (PRDX1), peroxiredoxin 6 (PRDX6), SOD1, SOD2, TXN, thioredoxin 2 (TXN2), TXNRD1, and TXNRD2. The researchers reported a lack of association between the variants in MPO, SOD1 or SOD2 and breast cancer risk.33 Similar results reported by Ahn et al. showed that genotypes of CAT, MnSOD, and MPO were not associated with the risk of radiotoxicity in Caucasian women with breast cancer who received radiotherapy following lumpectomy.34 In addition, in another large case-control study conducted in the United Kingdom, the researchers identified 54 single nucleotide polymorphisms in 10 well-known genes. In their study, the researchers reported an absence of significant association between SOD1, SOD2, GPX1, GPX4, GSR, TXNRD1, and TXN2. However, a borderline association was proposed for a group of variants in CAT g27168a, TXN t2715c, and TXNRD2 A66S and TXNRD2 g23524a.35 In the following sections, we categorized the most studied antioxidant genes in breast cancer.

MnSOD It has been found that MnSOD secondary structure is altered by Val16Ala polymorphic transformation and consequently affects the mitochondrial import of the MnSOD enzyme as well as the activity levels of the enzyme.4,12,36 Therefore, intensive research was conducted to elucidate the role of this polymorphism in the susceptibility of breast cancer as well as other cancers. Some studies showed a statistically significant association between MnSOD (Val16Ala) (AT! C) (rs4880) polymorphism and the risk of breast cancer development with the impact of other factors such as smoking.15,16 In a population-based case-control study conducted in Chinese women, Cai et al. have demonstrated a nonsignificant association between MnSOD Ala/Ala (Val16Ala) (AT ! C) genotype and elevated risk of breast cancer.37 However, this Chinese study found a slight increase in the risk of breast cancer among Chinese women with high levels of oxidative stress or low intake of antioxidants. In another study of the MnSOD (Val16Ala) polymorphism, the researchers reported an association between the homozygous A allele and the increased risk of breast cancer by fourfolds in comparison to those with V alleles without the ignorance of antioxidant diets role in minimizing the negative impact of the MnSOD polymorphism.15 Interestingly, Bergman et al. found a significant association between Val/Val and Val/Ala genotypes and the risk of breast cancer.38 A recent metaanalysis study reported a significant association between MnSOD (Val16Ala) polymorphism and the risk of breast cancer.39 In addition, in an interesting Greek study, the researchers showed an association between the diet habit and the genetic predisposition of certain polymorphisms including MnSOD and CAT genes. In that study, it has been reported that the high vegetable intake lowered breast cancer risk in women with at least one MnSOD Val allele or one CAT262C allele. While the high fish intake conferred a decreased breast cancer risk of CAT-262CC women compared with the CAT-262TT women and low fish quantity intake.40 On the other hand, some other studies reported a lack of association between MnSOD (Val16Ala) polymorphism and the risk of breast cancer.41,42 In addition, Silva et al. reported a lack of significant association between Val16Ala polymorphism and the susceptibility of breast cancer.43 Similar results were reported by Gaudet et al. as they did not find any risk of the Val16Ala polymorphism on breast cancer development.44 Moreover, in 2017, Wang et al. reported a lack of relationship between MnSOD (Val16Ala) polymorphism and breast cancer risk or survival in a metaanalysis study.45 Another metaanalysis study has also reported a lack of association between breast cancer risk and SOD2 (Val16Ala) polymorphism.46 Nevertheless, Mitrunen et al. found that postmenopausal women with past smoking history and an Ala allele had a higher risk of cancer development.16 Another study suggested that the Ala allele of the MnSOD (Ala16Val) polymorphism is not an autonomous risk factor but might alter breast cancer risk among smokers.47 In another supporting study, Millikan et al. observed moderate joint effects between smoking and Ala-containing genotypes.48 Cai et al. showed evidence of higher-risk Ala women who were in the premenopausal phase.37 Although, Egan et al. did not observe any significant risk of Ala carrier or homozygous genotypes and breast cancer.42 Despite the controversial results, most studies support the lack of association between MnSOD (Ala16Val) polymorphism and the susceptibility of breast cancer. However, some of these studies emphasized the importance of the antioxidant diet and smoking as another factor that needs to be considered in the study design.

CAT The C-262T (rs1001179) polymorphism of the CAT gene was found to affect the catalase levels. Specifically, the T allele carriers showed a higher level of catalase activity in comparison with C allele.14 Therefore, CAT C-262T (rs1001179) gene polymorphism has been investigated in breast cancer patients in many populations. A metaanalysis study reported a lack of association between C-262T (rs1001179) polymorphism of the CAT and the risk of breast cancer.49 These findings were

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supported by another systematic review and metaanalysis study.50 In an interesting study of the interaction of food diet and genetic polymorphisms, Ahn et al. reported that there is about 17% reduction in breast cancer risk among CAT-CC genotype carriers with a positive influence of fruit consumption to decrease breast cancer risk in those patients.51 Additionally, Quick et al. demonstrated a lack of association between CAT genotype alone with the development of breast cancer. Instead, they reported an association between hormone replacement therapy (HRT) and increased risk of breast cancer. However, they suggested that the CAT genotype modifies the effect of HRT use on breast cancer risk.52 In general, most studies support the lack of association between CAT C-262T (rs1001179) polymorphism and the risk of breast cancer. At the same time, antioxidants are expected to influence the impact of C-262T (rs1001179) polymorphism on breast cancer development.

MPO The MPO (G463A) (rs2333227) polymorphism has been proposed to be associated with cancer risk in many studies.18,53–57 A functional study revealed the presence of polymorphic site at the MPO promoter area (Alu element) (G463A) (rs2333227) that affect the binding capacity of SP-1 transcription factor which may decrease in MPO expression (Fig. 1).21 The same study suggested the important role of this polymorphic element in regulating the MPO gene in AML cases.21 Some studies reported a lack of significant decrease in breast cancer risk among AA genotype patients.58–60 However, London et al. reported a reduction in the risk of lung cancer among Caucasians and AfricanAmericans with the MPO A/A genotype.53 Interestingly, a large case-control study demonstrated a reduction of breast cancer risk (13%) among A allele (low expression) carrier and the influence of fruit and vegetable consumption on the reduction of the risk of breast cancer.58 These findings were supported by another study that proposed the role of antioxidant dietary intake and antioxidant as a modulator of the association between the MPO polymorphism and susceptibility of breast cancer.61 Christine et al. proposed that MPO (-463 GA), MnSOD 16 Val-T/Ala-C (Ala9 Val), CAT (-262C > T) variants modify prognosis after treatment for breast cancer treated with radiation and chemotherapy. In brief, they reported that the homozygous G allele of the MPO gene is associated with increased transcription and better survival compared to the other genotypes. However, the study showed that both CAT TT and MnSOD CC genotypes were associated with nonsignificant reduced hazard of death but the MnSOD CC and MPO GG genotypes had a threefold decrease in hazard of death.

GPXs The GPXs group includes seven enzymes that are classified in a group of peroxidases. GPX1 and GPX4 gene polymorphisms are the most studied genotypes under study genotypes in cancers. The GPX4 rs713041 is located near the selenocysteine insertion sequence element in the 30 -UTR end.62 Udler et al. surveyed 54 polymorphisms of variants in 10 antioxidative genes including (CAT, SOD1, SOD2, GPX1, GPX4, GSR, TXN, TXN2, TXNRD1, and TXNRD2) in association with the survival of breast cancer patients. Their results concluded a lack of association between all tested variants except for the GPX4 rs713041 and rs757229 polymorphisms and breast cancer patients survival.63 Furthermore, gene-gene interaction was studied in order to elucidate the possible association of these genes in the development or risk of breast cancer, however, the 10 studied genes (SOD1, SOD2, GPX1, GPX4, GSR, CAT, TXN, TXN2, TXNRD1, and TXNRD2) including 52 SNPs did not find any gene-gene interaction that may influence the development of breast cancer.28 In a European study that considered genetic polymorphisms in five redox genes included GPX1 (rs1050450), GPX4 (rs713041), SOD2 (rs4880), SEPP1 (rs3877899), and SEP15 (rs5859), the researchers reported a significant association between breast cancer risk and GPX1 rs1050450 (Pro198Leu) polymorphism but not the other polymorphisms. They reported a protective effect of Leu variant and interestingly showed increased activity of GPx1 correlated with lipid peroxidation in the breast cancer among the cancer patients having GPX1 Pro/Pro genotype.64

Prostate cancer Like many other cancers, it has been proposed that some antioxidant genes polymorphisms are associated with the risk of prostate cancer. For instance, SODs and GPX1 polymorphisms were studied in different populations. In a study of the three main isoforms of SODs, SOD1 (CuZn-SOD; IVS3-251A > G), SOD2 [MnSOD; Ex2 +24T > C (V16A)], and SOD3 (ECSOD; IVS1 + 186C > T, Ex3-631C > G, Ex3-516C > T, and Ex3-489C > T), genetic polymorphisms in the functional variant (Val16Ala) of SOD2 has been found to be associated with prostate cancer risk but not lungs, colorectal, and ovarian cancer.65 Some studies reported that the low antioxidant status with the MnSOD Ala/Ala genotype may be associated with an increased risk of aggressive prostate cancer.66–70 In addition, Woodson et al. found that men homozygous for Ala allele had a 70% increased risk for prostate cancer over the Val/Val men17 and Ala allele was found with a statistically higher risk for prostate cancer in a study on Turkish population.71 Moreover, Sun et al. conducted a large metaanalysis study to

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investigate the role of MnSOD polymorphism in prostate, esophageal and lung cancers, in conclusion, the result showed that the Ala allele of the Ala16Val-MnSOD polymorphism was associated with increased risk of prostate and esophageal cancers, but with decreased risk of lung cancer.72 These findings were supported by a recent metaanalysis study.32 Besides, Oskina et al. did not find any association between rs1695 and rs4880 in the MnSOD gene and rs1050450 in the GPX1 gene, and the risk of prostate cancer.29 Moreover, in a large metaanalysis study, Mao et al. suggested that the Ala allele of the MnSOD gene has a low-penetrance in prostate cancer development, especially in the Caucasian population.73 These findings were supported by another metaanalysis study by Liwei et al. where they reported a lack of significant association between the Ala16Val polymorphism in MnSOD and the susceptibility prostate cancer.74 In a cross-sectional study, Abe et al. investigated the role of certain polymorphisms in central antioxidant enzymes including glutathione peroxidases GPX1, GPX4, SOD1, SOD2, and SOD3 in association with the risk of aggressive prostate cancer. In conclusion, Abe et al. reported that rs17884057 (–/AGA) and rs4816407 (A/G) intronic polymorphisms in SOD1 genes were significantly associated with the risk of aggressive prostate cancer. Moreover, they found an association between low level of selenium and aggressive prostate cancer in A allele carriers at rs2842958 (A/G) polymorphism in SOD2 gene which also modified by SOD1 rs10432782 (G/T) variant and SOD2 rs2758330 (G/T) variant. While GPX1 and GPX4 polymorphisms did not show any association with the aggressiveness of prostate cancer.75 Another study provided evidence of the impact of the GPx1 Pro198Leu polymorphism on the development and progression of prostate cancer.76 In order to investigate the associations of antioxidant genes polymorphisms with prostate cancer patients who are smokers or at an exposure of asbestos, a case-control study investigated the three common polymorphisms (MnSOD Ala16Val, CAT C-262T, and GPX1 Pro200Leu). The researchers did not find any significant association between these polymorphic variants and the risk of prostate cancer patients who are smokers or exposed to asbestos.67 Polymorphisms of CAT enzyme have been studied in prostate cancer patients as well. A metaanalysis study of a group of cancers showed a significant association between CAT C-262T polymorphism and cancer risk. Moreover, the subgroup analyses proposed an association between the CAT C-262T polymorphism and increased risk of prostate cancer (TT vs CT + CC) but not associated with breast cancer.50

Gastric, colorectal, and colon cancer A group of polymorphisms in the most common antioxidant genes have been studied in gastric cancer patients in different populations. In a Chinese case-control study, Yi et al. reported ZnSOD (SOD1) G7958A (rs4998557) and MnSOD (SOD2) Val16Ala (rs4880) polymorphisms are associated with the risk of gastric cancer.77 Additionally, in another report, both rs2758339 (A/C) and rs5746136 (A/G 30 -UTR) polymorphisms of the MnSOD showed a significant association with the risk of gastric cancer.78 Whereas Stoehlmacher et al. linked the Ala allele of Val16Ala polymorphism of the MnSOD gene with an increased risk to develop colorectal cancer.79 However, Levine et al. identified a protective role of Ala for distal colorectal adenomas, known precursor lesions for colorectal cancers.80 In another study, a case-control study reported a significant association between the presence of G allele of SOD1 A251G polymorphism and the risk of colorectal cancer (CRC) as well as a lack of association between CAT C-262T polymorphism and susceptibility to CRC. However, the combination of both genotypes AG + GG (SOD1) and CC (CAT) were associated with the susceptibility of colorectal cancer.81 In a large cohort study, the researchers suggested a contribution of SOD2 rs4880 and GSTP1 rs1695 genotypes to gastric cancer progression and aggressiveness in Chinese patients. Specifically, Xu et al. demonstrated that SOD2 rs4880 CT + CC genotypes are associated with the lymph node metastasis.82 A study that explored the polymorphic impact of the SOD1 A251G and CAT C-262T variants, the researchers proposed a protective effect of the G allele of the SOD1 A251G polymorphism and lack of association between CAT C-262T polymorphism and gastric cancer susceptibility.83 In another study, the results suggested that Hispanics with A allele might be associated with an increased risk to develop colorectal cancer at a young age compared to the non-Hispanic whites.65 In a Chinese study, the researchers reported a significant association between MPO-463 G to A variant and the decreased susceptibility of gastric cancer, where the (GA and AA) individuals had a 44% reduced risk of gastric cancer.84 GPX-Pro198Leu polymorphism was also investigated in cancers, in a large metaanalysis study, the researchers found a significant association between the presence of Leu allele and increased risk of cancer.85 On the other hand, another metaanalysis study did not support the association between GPX-Pro198Leu polymorphism and gastric cancer development.86 Moreover, in a Polish study, Martin et al. reported no significant association between the MnSOD polymorphisms at -102 C> T (promoter site) and the -9 T > C (Val16Ala) and the risk of gastric cancer.87 In addition, a recent report did not show an association between SOD1 Ins/Del and SOD3 rs2536512 (A/G/T) polymorphisms and the risk of gastric cancer risk.78 Moreover, in a case-control study, the researchers reported a lack of a significant link between GPX-Pro198Leu variant and risk of colorectal cancer.88 In addition, Bermano et al. suggested that T allele of the GPX4 T718C SNP is associated with a lower risk of colorectal cancer.89

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Lung cancer Different studies demonstrated an association between certain polymorphisms of some antioxidant genes and the risk of lung cancer. For instance, Titov et al. showed a significant protective effect of the major C allele in the glutathione peroxidase-1 GPx1 (599C > T) rs1050450 locus in lung cancer patients.27 In another study, the researchers reported an association between the codon Leu198Pro (C593T) (rs869025667) of glutathione peroxidase hGPX1 and increased risk of lung cancer. In brief, the researchers reported that (leu/leu) and (leu/Pro) genotypes have a greater risk for lung cancer compared to (pro/pro) genotype.90 Additionally, Wang et al. found significantly increased risks of lung cancer for individuals heterozygous or homozygous for the MnSOD Val allele of codon 16.19 Interestingly, a metaanalysis study proposed that the MPO G-463A (G to A) polymorphism has a protective function in lung cancer.91 In addition, a study showed that homozygous A allele individuals of the myeloperoxidase MPO gene may be at a decreased risk of lung cancer.53 The researcher’s explanations based on the low expression of the MPO enzymes due to the lower affinity of binding between the SP1 transcription factor and the A allele at Alu promotor sequence. On the other hand, MnSOD (47Т > С) rs4880, CAT (-262С > T) rs1001179, and GSTP1 (341C > T, rs1695, 313A > G, rs1138272 did not show any significant relation with the presence of lung cancer in the studied population in Russia.27 In another study, Lin et al. showed a lack of association between MnSOD polymorphisms and an increased risk of lung cancer.92

Other cancers It has been proposed that polymorphic GPX1 Pro198Leu genotype significantly increases the risk of bladder cancer while the Ala16Val and the Ile56Thr polymorphisms of MnSOD were not associated with the risk of bladder cancer.93 In addition, a systematic review and metaanalysis study showed that the GPX1 Pro198Leu polymorphism significantly increased susceptibility to bladder cancer, while the MnSOD Ala-9Val polymorphism is not associated with the risk of bladder cancer.94 These results were supported by another study that reported a lack of association between MnSOD polymorphism and the incidence of bladder cancer.95 Conversely, it has been reported that Val/Val genotype of the MnSOD polymorphism is associated with the risk of bladder cancer.18 The same study evaluated the effect of environmental factors by a case-control study. The study concluded that the MPO G-463A homozygous variant was associated with a reduced risk of bladder cancer. While the homozygous MnSOD Val genotype is associated with increased risk of bladder cancer and modulate the susceptibility to bladder cancer.18 Acute myeloid leukemia (AML) was also investigated by a case-control study that inspected CAT C262T, GPX1 Pro198Leu, MnSOD Ala16Val, GSTM1, GSTT1, and GSTP1 Ile105Val genes polymorphisms. The results did not show an association between AML and CAT, MnSOD, GSTM1, and GSTT1 polymorphisms. While the data have shown a statistically significant association between GPX1 Pro198Leu and GSTP1 Ile105Val variants and AML development.96 Four SNPs; (rs4880, rs5746136, rs1804450, and rs11556620) in the SOD1 and SOD2 were investigated in Oral squamous cell carcinoma (OSCC) patients. But the results did not show any significant difference in the rs5746136, rs1804450, and rs11556620 between the patients and control group. However, the results suggested rs4880 of MnSOD as a potential genetic marker for OSCC risk.97 In a small case-control study, the results demonstrated a protective effect of T allele of CAT C262T polymorphism against the risk of ovarian cancer.98 While in another study, the researchers did not find any association between the most common antioxidant genes polymorphisms (MnSOD, CAT, GPX1, and MPO) and the risk of ovarian cancer, however, their results showed an association between CAT C/T polymorphism and the patients’ survival.99 These results were supported by another study that did not find any association between MnSOD Val16Ala polymorphism and the susceptibility or survival of ovarian cancer.100 A brain tumor study evaluated nine SNPs in seven antioxidant genes (CAT, GPX1, NOS3, PON1, SOD1, SOD2, and SOD3). The results showed an increased risk of glioma and meningioma with the C variant of SOD3 rs699473 and increased risk of acoustic neuroma among SOD2 rs4880 Ala variant carriers and decreased risk of acoustic neuroma among CAT rs1001179 T allele carriers.31 On the other hand, another study did not find a significant association between the Ala16Val variants of the SOD2 gene and primary brain tumor.101 Nevertheless, in cervical cancer study, the researchers showed that the GG genotype of the MPO (G463A) (rs2333227) polymorphism had a lower risk for cervical cancer compared to the heterozygous genotype GA.102 Interestingly, up to our literature search, there were no studies of the association with gastrointestinal cancer with the polymorphisms of antioxidants genes.

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This chapter discusses the most studied polymorphisms in the antioxidant genes (SOD1, SOD2, SOD3, GPX1, CAT, and MPO). CuZn-SOD, a superoxide dismutase 1 (SOD1) polymorphic variants include G7958A (rs4998557), (IVS3-251A > G), rs17884057 (–/AGA), rs10432782 (G/T), A251G, and rs4816407 (A/G). MnSOD, a superoxide dismutase 2 (SOD2) polymorphisms are the most studied variants including (Val16Ala) (AT ! C) (rs4880) polymorphism and rs2758330 (G/T). MnSOD Val16Ala polymorphism alters the secondary structure of the MnSOD, affecting the mitochondrial localization and the activity of the MnSOD enzyme. And showed a significant association with the development of breast cancer and some other cancer. GPX1, a peroxidase gene, showed an association between GPX1 (rs1050450) (Pro198Leu) polymorphism and their relation to the development of breast cancer in certain populations. C > T substitution in the
262 bp (rs1001179) position in the promoter region of the catalase gene (CAT) influences the binding of transcription factors, consequently, altering the expression of CAT enzyme. The polymorphic site at the MPO promoter area (Alu element) (G463A) (rs2333227) affecting the binding capacity of SP-1 transcription factor which may alter the expression of MPO enzyme, proposes its role in the development of certain cancers.

References 1. Sander CS, Chang H, Hamm F, Elsner P, Thiele JJ. Role of oxidative stress and the antioxidant network in cutaneous carcinogenesis. Int J Dermatol 2004;43(5):326–35. 2. Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO, et al. Dysfunctional KEAP1–NRF2 interaction in non-small-cell lung cancer. PLoS Med 2006;3(10):e420. 3. Levine RL, Moskovitz J, Stadtman ER. Oxidation of methionine in proteins: roles in antioxidant defense and cellular regulation. IUBMB Life 2000;50 (4–5):301–7. 4. Sutton A, Khoury H, Prip-Buus C, Cepanec C, Pessayre D, Degoul F. The Ala16Val genetic dimorphism modulates the import of human manganese superoxide dismutase into rat liver mitochondria. Pharmacogenet Genomics 2003;13(3):145–57. 5. Baker AM, Oberley LW, Cohen MB. Expression of antioxidant enzymes in human prostatic adenocarcinoma. Prostate 1997;32(4):229–33. 6. Wang S, Wang F, Shi X, Dai J, Peng Y, Guo X, et al. Association between manganese superoxide dismutase (MnSOD) Val-9Ala polymorphism and cancer risk—a meta-analysis. Eur J Cancer 2009;45(16):2874–81. 7. Lee W-H, Morton RA, Epstein JI, Brooks JD, Campbell PA, Bova GS, et al. Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc Natl Acad Sci 1994;91(24):11733–7. 8. Leone A, Roca MS, Ciardiello C, Costantini S, Budillon A. Oxidative stress gene expression profile correlates with cancer patient poor prognosis: identification of crucial pathways might select novel therapeutic approaches. Oxidative Med Cell Longev 2017;2017. 9. Khan MA, Tania M, Zhang D-z, Chen H-c. Antioxidant enzymes and cancer. Chin J Cancer Res 2010;22(2):87–92. 10. Wan XS, Devalaraja MN, St. Clair DK. Molecular structure and organization of the human manganese superoxide dismutase gene. DNA Cell Biol 1994;13(11):1127–36. 11. Hirono A, Sasaya-Hamada F, Kanno H, Fujii H, Yoshida T, Miwa S. A novel human catalase mutation (358 T_del) causing Japanese-type acatalasemia. Blood Cells Mol Dis 1995;21(3):232–4. 12. Shimoda-Matsubayashi S, Matsumine H, Kobayashi T, Nakagawa-Hattori Y, Shimizu Y, Mizuno Y. Structural dimorphism in the mitochondrial targeting sequence in the human manganese superoxide dismutase gene: a predictive evidence for conformational change to influence mitochondrial transport and a study of allelic association in Parkinson’s disease. Biochem Biophys Res Commun 1996;226(2):561–5. 13. Young RP, Hopkins R, Black PN, Eddy C, Wu L, Gamble GD, et al. Functional variants of antioxidant genes in smokers with COPD and in those with normal lung function. Thorax 2006;61(5):394–9. 14. Forsberg L, Lyren€as L, Morgenstern R, de Faire U. A common functional CT substitution polymorphism in the promoter region of the human catalase gene influences transcription factor binding, reporter gene transcription and is correlated to blood catalase levels. Free Radic Biol Med 2001;30 (5):500–5. 15. Ambrosone CB, Freudenheim JL, Thompson PA, Bowman E, Vena JE, Marshall JR, et al. Manganese superoxide dismutase (MnSOD) genetic polymorphisms, dietary antioxidants, and risk of breast cancer. Cancer Res 1999;59(3):602–6. 16. Mitrunen K, Sillanp€a€a P, Kataja V, Eskelinen M, Kosma V-M, Benhamou S, et al. Association between manganese superoxide dismutase (MnSOD) gene polymorphism and breast cancer risk. Carcinogenesis 2001;22(5):827–9. 17. Woodson K, Tangrea JA, Lehman TA, Modali R, Taylor KM, Snyder K, et al. Manganese superoxide dismutase (MnSOD) polymorphism, a-tocopherol supplementation and prostate cancer risk in the alpha-tocopherol, beta-carotene cancer prevention study (Finland). Cancer Causes Control 2003;14(6):513–8.

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18. Hung RJ, Boffetta P, Brennan P, Malaveille C, Gelatti U, Placidi D, et al. Genetic polymorphisms of MPO, COMT, MnSOD, NQO1, interactions with environmental exposures and bladder cancer risk. Carcinogenesis 2004;25(6):973–8. 19. Wang LI, Miller DP, Sai Y, Liu G, Su L, Wain JC, et al. Manganese superoxide dismutase alanine-to-valine polymorphism at codon 16 and lung cancer risk. J Natl Cancer Inst 2001;93(23):1818–21. 20. Zhang HJ, Yan T, Oberley TD, Oberley LW. Comparison of effects of two polymorphic variants of manganese superoxide dismutase on human breast MCF-7 cancer cell phenotype. Cancer Res 1999;59(24):6276–83. 21. Piedrafita FJ, Molander RB, Vansant G, Orlova EA, Pfahl M, Reynolds WF. An Alu element in the myeloperoxidase promoter contains a composite SP1-thyroid hormone-retinoic acid response element. J Biol Chem 1996;271(24):14412–20. 22. Feyler A, Voho A, Bouchardy C, Kuokkanen K, Dayer P, Hirvonen A, et al. Point: myeloperoxidase
463G!a polymorphism and lung cancer risk. Cancer Epidemiol Biomark Prev 2002;11(12):1550–4. 23. Gelain DP, Dalmolin RJ, Belau VL, Moreira JC, Klamt F, Castro M. A systematic review of human antioxidant genes. Front Biosci 2009; 14(12):4457–63. 24. Al Zoubi MS. X-ray repair cross-complementing protein 1 and 3 polymorphisms and susceptibility of breast cancer in a Jordanian population. Saudi Med J 2015;36(10):1163. 25. Zoubi MSA, Zavaglia K, Mazanti C, Hamad MA, Batayneh KA, Aljabali AA, et al. Polymorphisms and mutations in GSTP1, RAD51, XRCC1 and XRCC3 genes in breast cancer patients. Int J Biol Markers 2017;32(3):337–43. 26. Al-Zoubi MS, Mazzanti CM, Zavaglia K, Al Hamad M, Armogida I, Lisanti MP, et al. Homozygous T172T and heterozygous G135C variants of homologous recombination repairing protein RAD51 are related to sporadic breast cancer susceptibility. Biochem Genet 2016;54(1):83–94. 27. Titov R, Minina V, Soboleva O, Ryzhkova A, Kulemin YE, Voronina E. Polymorphism of genes of the antioxidant system in the development of predispositions to lung cancer. Russ J Genet 2017;53(8):903–9. 28. Oestergaard M, Tyrer J, Cebrian A, Shah M, Dunning A, Ponder B, et al. Interactions between genes involved in the antioxidant defence system and breast cancer risk. Br J Cancer 2006;95(4):525. 29. Oskina N, Еrmolenko N, Boyarskih U, Lazarev А, Petrova V, Ganov D, et al. Associations between SNPs within antioxidant genes and the risk of prostate cancer in the Siberian region of Russia. Pathol Oncol Res 2014;20(3):635–40. 30. Jansen RJ, Robinson DP, Stolzenberg-Solomon RZ, Bamlet WR, Tan X, Cunningham JM, et al. Polymorphisms in metabolism/antioxidant genes may mediate the effect of dietary intake on pancreatic cancer risk. Pancreas 2013;42(7):1043. 31. Ambrosone CB, Ahn J, Singh KK, Rezaishiraz H, Furberg H, Sweeney C, et al. Polymorphisms in genes related to oxidative stress (MPO, MnSOD, CAT) and survival after treatment for breast cancer. Cancer Res 2005;65(3):1105–11. 32. Wang P, Zhu Y, Xi S, Li S, Zhang Y. Association between MnSOD Val 16Ala polymorphism and cancer risk: evidence from 33,098 cases and 37,831 controls. Dis Markers 2018;2018. 33. Seibold P, Hein R, Schmezer P, Hall P, Liu J, Dahmen N, et al. Polymorphisms in oxidative stress-related genes and postmenopausal breast cancer risk. Int J Cancer 2011;129(6):1467–76. 34. Ahn J, Ambrosone CB, Kanetsky PA, Tian C, Lehman TA, Kropp S, et al. Polymorphisms in genes related to oxidative stress (CAT, MnSOD, MPO, and eNOS) and acute toxicities from radiation therapy following lumpectomy for breast cancer. Clin Cancer Res 2006;12(23):7063–70. 35. Cebrian A, Pharoah PD, Ahmed S, Smith PL, Luccarini C, Luben R, et al. Tagging single-nucleotide polymorphisms in antioxidant defense enzymes and susceptibility to breast cancer. Cancer Res 2006;66(2):1225–33. 36. Rosenblum JS, Gilula NB, Lerner RA. On signal sequence polymorphisms and diseases of distribution. Proc Natl Acad Sci U S A 1996;93(9):4471–3. 37. Cai Q, Shu X-O, Wen W, Cheng J-R, Dai Q, Gao Y-T, et al. Genetic polymorphism in the manganese superoxide dismutase gene, antioxidant intake, and breast cancer risk: results from the Shanghai Breast Cancer Study. Breast Cancer Res 2004;6(6):R647. 38. Bergman M, Ahnstr€om M, Wegman PP, Wingren S. Polymorphism in the manganese superoxide dismutase (MnSOD) gene and risk of breast cancer in young women. J Cancer Res Clin Oncol 2005;131(7):439–44. 39. Balci T, Gunduz C. Meta-analysis of the relationship between MnSOD polymorphism and cancer in the Turkish and Cypriot population. Turk J Biochem 2018;43(2):184–96. 40. Kakkoura MG, Demetriou CA, Loizidou MA, Loucaides G, Neophytou I, Malas S, et al. MnSOD and CAT polymorphisms modulate the effect of the Mediterranean diet on breast cancer risk among Greek-Cypriot women. Eur J Nutr 2016;55(4):1535–44. 41. Knight JA, Onay UV, Wells S, Li H, Shi EJ, Andrulis IL, et al. Genetic variants of GPX1 and SOD2 and breast cancer risk at the Ontario site of the Breast Cancer Family Registry. Cancer Epidemiol Biomark Prev 2004;13(1):146–9. 42. Egan KM, Thompson PA, Titus-Ernstoff L, Moore JH, Ambrosone CB. MnSOD polymorphism and breast cancer in a population-based case–control study. Cancer Lett 2003;199(1):27–33. 43. Silva SN, Cabral MN, Bezerra de Castro G, Pires M, Azevedo AP, Manita I, et al. Breast cancer risk and polymorphisms in genes involved in metabolism of estrogens (CYP17, HSD17b1, COMT and MnSOD): possible protective role of MnSOD gene polymorphism Val/Ala and Ala/Ala in women that never breast fed. Oncol Rep 2006;16(4):781–8. 44. Gaudet MM, Gammon MD, Santella RM, Britton JA, Teitelbaum SL, Eng SM, et al. MnSOD Val-9Ala genotype, pro-and anti-oxidant environmental modifiers, and breast cancer among women on Long Island, New York. Cancer Causes Control 2005;16(10):1225–34. 45. Wang C, Liu Y, Zhou J, Ye L, Chen N, Zhu M, et al. There is no relationship between SOD2 Val-16Ala polymorphism and breast cancer risk or survival. Mol Clin Oncol 2017;7(4):579–90. 46. Bag A, Bag N. Target sequence polymorphism of human manganese superoxide dismutase gene and its association with cancer risk: a review. Cancer Epidemiol Biomark Prev 2008;17(12):3298–305.

Polymorphisms, antioxidant genes, and cancer Chapter

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47. Tamimi RM, Hankinson SE, Spiegelman D, Colditz GA, Hunter DJ. Manganese superoxide dismutase polymorphism, plasma antioxidants, cigarette smoking, and risk of breast cancer. Cancer Epidemiol Biomark Prev 2004;13(6):989–96. 48. Millikan RC, Player J, de Cotret AR, Moorman P, Pittman G, Vannappagari V, et al. Manganese superoxide dismutase Ala-9Val polymorphism and risk of breast cancer in a population-based case–control study of African Americans and whites. Breast Cancer Res 2004;6(4):R264. 49. Saadat M, Saadat S. Genetic polymorphism of CAT C-262 T and susceptibility to breast cancer, a case–control study and meta-analysis of the literatures. Pathol Oncol Res 2015;21(2):433–7. 50. Shen Y, Li D, Tian P, Shen K, Zhu J, Feng M, et al. The catalase C-262T gene polymorphism and cancer risk: a systematic review and meta-analysis. Medicine 2015;94(13):679–86. 51. Ahn J, Gammon MD, Santella RM, Gaudet MM, Britton JA, Teitelbaum SL, et al. Associations between breast cancer risk and the catalase genotype, fruit and vegetable consumption, and supplement use. Am J Epidemiol 2005;162(10):943–52. 52. Quick SK, Shields PG, Nie J, Platek ME, McCann SE, Hutson AD, et al. Effect modification by catalase genotype suggests a role for oxidative stress in the association of hormone replacement therapy with postmenopausal breast cancer risk. Cancer Epidemiol Biomark Prev 2008;17(5):1082–7. 53. London SJ, Lehman TA, Taylor JA. Myeloperoxidase genetic polymorphism and lung cancer risk. Cancer Res 1997;57(22):5001–3. 54. Schabath MB, Spitz MR, Hong WK, Delclos GL, Reynolds WF, Gunn GB, et al. A myeloperoxidase polymorphism associated with reduced risk of lung cancer. Lung Cancer 2002;37(1):35–40. 55. Pakakasama S, Chen TTL, Frawley W, Muller C, Douglass EC, Tomlinson GE. Myeloperoxidase promotor polymorphism and risk of hepatoblastoma. Int J Cancer 2003;106(2):205–7. 56. Larsen JE, Colosimo ML, Yang IA, Bowman R, Zimmerman PV, Fong KM. CYP1A1 Ile462Val and MPO G-463A interact to increase risk of adenocarcinoma but not squamous cell carcinoma of the lung. Carcinogenesis 2005;27(3):525–32. 57. Olson S, Carlson M, Ostrer H, Harlap S, Stone A, Winters M, et al. Genetic variants in SOD2, MPO, and NQO1, and risk of ovarian cancer. Gynecol Oncol 2004;93(3):615–20. 58. Ahn J, Gammon MD, Santella RM, Gaudet MM, Britton JA, Teitelbaum SL, et al. Myeloperoxidase genotype, fruit and vegetable consumption, and breast cancer risk. Cancer Res 2004;64(20):7634–9. 59. Lin S, Chou Y, Wu M, Wu C, Lin W, Yu C, et al. Genetic variants of myeloperoxidase and catechol-O-methyltransferase and breast cancer risk. Eur J Cancer Prev 2005;14(3):257–61. 60. Yang J, Ambrosone CB, Hong C-C, Ahn J, Rodriguez C, Thun MJ, et al. Relationships between polymorphisms in NOS3 and MPO genes, cigarette smoking and risk of post-menopausal breast cancer. Carcinogenesis 2007;28(6):1247–53. 61. He C, Tamimi RM, Hankinson SE, Hunter DJ, Han J. A prospective study of genetic polymorphism in MPO, antioxidant status, and breast cancer risk. Breast Cancer Res Treat 2009;113(3):585–94. 62. Berry MJ, Banu L, Chen Y, Mandel SJ, Kieffer JD, Harney JW, et al. Recognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 30 untranslated region. Nature 1991;353(6341):273. 63. Udler M, Maia A-T, Cebrian A, Brown C, Greenberg D, Shah M, et al. Common germline genetic variation in antioxidant defense genes and survival after diagnosis of breast cancer. J Clin Oncol 2007;25(21):3015–23. 64. Jablonska E, Gromadzinska J, Peplonska B, Fendler W, Reszka E, Krol MB, et al. Lipid peroxidation and glutathione peroxidase activity relationship in breast cancer depends on functional polymorphism of GPX1. BMC Cancer 2015;15(1):657. 65. Kang D, Lee K-M, Park SK, Berndt SI, Peters U, Reding D, et al. Functional variant of manganese superoxide dismutase (SOD2 V16A) polymorphism is associated with prostate cancer risk in the prostate, lung, colorectal, and ovarian cancer study. Cancer Epidemiol Biomark Prev 2007; 16(8):1581–6. 66. Mikhak B, Hunter DJ, Spiegelman D, Platz EA, Wu K, Erdman Jr. JW, et al. Manganese superoxide dismutase (MnSOD) gene polymorphism, interactions with carotenoid levels and prostate cancer risk. Carcinogenesis 2008;29(12):2335–40. 67. Choi J-Y, Neuhouser ML, Barnett M, Hudson M, Kristal AR, Thornquist M, et al. Polymorphisms in oxidative stress–related genes are not associated with prostate cancer risk in heavy smokers. Cancer Epidemiol Biomark Prev 2007;16(6):1115–20. 68. Giovannucci E, Rimm EB, Liu Y, Stampfer MJ, Willett WC. A prospective study of tomato products, lycopene, and prostate cancer risk. J Natl Cancer Inst 2002;94(5):391–8. 69. Wu K, Erdman JW, Schwartz SJ, Platz EA, Leitzmann M, Clinton SK, et al. Plasma and dietary carotenoids, and the risk of prostate cancer: a nested case-control study. Cancer Epidemiol Biomark Prev 2004;13(2):260–9. 70. Etminan M, Takkouche B, Caamano-Isorna F. The role of tomato products and lycopene in the prevention of prostate cancer: a meta-analysis of observational studies. Cancer Epidemiol Biomark Prev 2004;13(3):340–5. 71. Ergen HA, Narter F, Timirci O, Isbir T. Effects of manganase superoxide dismutase Ala-9Val polymorphism on prostate cancer: a case-control study. Anticancer Res 2007;27(2):1227–30. 72. Sun G-G, Wang Y-D, Lu Y-F, Hu W-N. Different association of manganese superoxide dismutase gene polymorphisms with risk of prostate, esophageal, and lung cancers: evidence from a meta-analysis of 20,025 subjects. Asian Pac J Cancer Prev 2013;14(3):1937–43. 73. Mao C, Qiu L-X, Zhan P, Xue K, Ding H, Du F-B, et al. MnSOD Val 16 Ala polymorphism and prostate cancer susceptibility: a meta-analysis involving 8,962 subjects. J Cancer Res Clin Oncol 2010;136(7):975–9. 74. Liwei L, Chunyu L, Ruifa H. Association between manganese superoxide dismutase gene polymorphism and risk of prostate cancer: a meta-analysis. Urology 2009;74(4):884–8. 75. Abe M, Xie W, Regan MM, King IB, Stampfer MJ, Kantoff PW, et al. Single-nucleotide polymorphisms within the antioxidant defence system and associations with aggressive prostate cancer. BJU Int 2011;107(1):126–34.

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76. Kucukgergin C, Gokpinar M, Sanli O, Tefik T, Oktar T, Seckin S. Association between genetic variants in glutathione peroxidase 1 (GPx1) gene, GPx activity and the risk of prostate cancer. Minerva Urol Nefrol 2011;63(3):183–90. 77. Yi J-F, Li Y-M, Liu T, He W-T, Li X, Zhou W-C, et al. Mn-SOD and CuZn-SOD polymorphisms and interactions with risk factors in gastric cancer. World J Gastroenterol 2010;16(37):4738. 78. Eftekhari A, Peivand Z, Saadat I, Saadat M. Association between genetic polymorphisms in superoxide dismutase gene family and risk of gastric cancer. Pathol Oncol Res 2018;26(1):335–9. 79. Stoehlmacher J, Ingles SA, Park DJ, Zhang W, Lenz H-J. The-9Ala/-9Val polymorphism in the mitochondrial targeting sequence of the manganese superoxide dismutase gene (MnSOD) is associated with age among Hispanics with colorectal carcinoma. Oncol Rep 2002;9(2):235–8. 80. Levine AJ, Elkhouly E, Diep AT, Lee ER, Frankl H, Haile RW. The MnSOD A16V mitochondrial targeting sequence polymorphism is not associated with increased risk of distal colorectal adenomas: data from a sigmoidoscopy-based case control study. Cancer Epidemiol Biomark Prev 2002;11 (10):1140–1. 81. Jamhiri I, Saadat I, Omidvari S. Genetic polymorphisms of superoxide dismutase-1 A251G and catalase C-262T with the risk of colorectal cancer. Mol Biol Res Commun 2017;6(2):85. 82. Xu Z, Zhu H, Luk JM, Wu D, Gu D, Gong W, et al. Clinical significance of SOD2 and GSTP1 gene polymorphisms in Chinese patients with gastric cancer. Cancer 2012;118(22):5489–96. 83. Ebrahimpour S, Saadat I. Association of CAT C-262T and SOD1 A251G single nucleotide polymorphisms susceptible to gastric cancer. Mol Biol Res Commun 2014;3(4):223. 84. Zhu H, Yang L, Zhou B, Yu R, Tang N, Wang B. Myeloperoxidase G–463A polymorphism and the risk of gastric cancer: a case–control study. Carcinogenesis 2006;27(12):2491–6. 85. Chen J, Cao Q, Qin C, Shao P, Wu Y, Wang M, et al. GPx-1 polymorphism (rs1050450) contributes to tumor susceptibility: evidence from metaanalysis. J Cancer Res Clin Oncol 2011;137(10):1553. 86. Wang J, Sun T, Yang M, Lin D, Tan W, Li K, et al. Association of genetic polymorphisms in selenoprotein GPX1 and TXNRD2 with genetic susceptibility of gastric cancer. Zhonghua Yu Fang Yi Xue Za Zhi 2008;42(7):511–4. 87. Martin RC, Lan Q, Hughes K, Doll MA, Martini BD, Lissowska J, et al. No apparent association between genetic polymorphisms (
102 C >T) and (
9 T >C) in the human manganese superoxide dismutase gene and gastric cancer1. J Surg Res 2005;124(1):92–7. 88. Hansen R, Sæbø M, Skjelbred CF, Nexø BA, Hagen PC, Bock G, et al. GPX Pro198Leu and OGG1 Ser326Cys polymorphisms and risk of development of colorectal adenomas and colorectal cancer. Cancer Lett 2005;229(1):85–91. 89. Bermano G, Pagmantidis V, Holloway N, Kadri S, Mowat N, Shiel R, et al. Evidence that a polymorphism within the 30 UTR of glutathione peroxidase 4 is functional and is associated with susceptibility to colorectal cancer. Genes Nutr 2007;2(2):225–32. 90. Ratnasinghe D, Tangrea JA, Andersen MR, Barrett MJ, Virtamo J, Taylor PR, et al. Glutathione peroxidase codon 198 polymorphism variant increases lung cancer risk. Cancer Res 2000;60(22):6381–3. 91. Zou S, Pan X, Hua C, Wu M, He B, Chen Z. Myeloperoxidase-463 G/A polymorphism is associated with lung cancer risk: a meta-analysis with 7420 cases and 9132 controls. J Cancer Res Ther 2018;14(9):282. 92. Lin P, Hsueh Y-M, Ko J-L, Liang Y-F, Tsai K-J, Chen C-Y. Analysis of NQO1, GSTP1, and MnSOD genetic polymorphisms on lung cancer risk in Taiwan. Lung Cancer 2003;40(2):123–9. 93. Ichimura Y, Habuchi T, Tsuchiya N, Wang L, Oyama C, Sato K, et al. Increased risk of bladder cancer associated with a glutathione peroxidase 1 codon 198 variant. J Urol 2004;172(2):728–32. 94. Cao M, Mu X, Jiang C, Yang G, Chen H, Xue W. Single-nucleotide polymorphisms of GPX1 and MnSOD and susceptibility to bladder cancer: a systematic review and meta-analysis. Tumour Biol 2014;35(1):759–64. 95. Cengiz M, Ozaydin A, Ozkilic AC, Dedekarginoglu G. The investıgatıon of GSTT1, GSTM1 and SOD polymorphism in bladder cancer patıents. Int Urol Nephrol 2007;39(4):1043–8. 96. Ba˘nescu C, Iancu M, Trifa AP, C^andea M, Benedek Lazar E, Moldovan VG, et al. From six gene polymorphisms of the antioxidant system, only GPX Pro198Leu and GSTP1 Ile105Val modulate the risk of acute myeloid leukemia. Oxid Med Cell Longev 2016;2016. 97. Liu Y, Zha L, Li B, Zhang L, Yu T, Li L. Correlation between superoxide dismutase 1 and 2 polymorphisms and susceptibility to oral squamous cell carcinoma. Exp Ther Med 2014;7(1):171–8. 98. Moradi M-T, Khazaei M, Khazaei M. The effect of catalase C262T gene polymorphism in susceptibility to ovarian cancer in Kermanshah province, Western Iran. J Obstet Gynaecol 2018;38(4):562–6. 99. Belotte J, Fletcher NM, Saed MG, Abusamaan MS, Dyson G, Diamond MP, et al. A single nucleotide polymorphism in catalase is strongly associated with ovarian cancer survival. PLoS One 2015;10(8):e0135739. 100. Johnatty SE, Nagle CM, Spurdle AB, Chen X, Australian Breast Cancer Family Study, Webb PM, et al. The MnSOD Val9Ala polymorphism, dietary antioxidant intake, risk and survival in ovarian cancer (Australia). Gynecol Oncol 2007;107(3):388–91. 101. Tas¸ A, Silig Y, Pinarbas¸ i H, G€urelik M. Role of SOD2 Ala16Val polymorphism in primary brain tumors. Biomed Rep 2019;10(3):189–94. ˆ , Bicho M, Medeiros R, et al. Association of myeloperoxidase polymorphism (G463A) with cervix 102. Castela˜o C, Da Silva AP, Matos A, Ina´cio A cancer. Mol Cell Biochem 2015;404(1–2):1–4.

Chapter 11

The interconnection of high-fat diets, oxidative stress, the heart, and carcinogenesis Bianka Bojkova´a, Natalia Kurhalukb, and Pawel J. Winklewskic a

Department of Animal Physiology, Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Sˇafa´rik University in Kosˇice, Kosˇice, Slovak

Republic, b Department of Zoology and Animal Physiology, Institute of Biology and Earth Sciences, Pomeranian University, Slupsk, Poland, c Department of Human Physiology, Medical University of Gdansk, Gdansk, Poland

List of abbreviations AT CAT CI CLA CVDs GPx GR HIF-1a HR HSP70 LPO MUFAs NOS PA PUFAs ROS RR SFAs SOD TFAs TNFa

adipose tissue catalase confidence interval conjugated linoleic acid cardiovascular diseases glutathione peroxidase glutathione reductase hypoxia-inducible factor 1a hazard ratio heat shock protein 70 lipid peroxidation monounsaturated fatty acids nitric oxide synthase palmitic acid polyunsaturated fatty acids reactive oxygen species relative risk saturated fatty acids superoxide dismutase trans-fatty acids tumor necrosis factor a

Oxidative stress: General introduction Reactive oxygen species (ROS) are one of the most important factors involved in the regulation of animal metabolism. They play a role as mediators in growth, maturation, and apoptosis.1 In living organisms, ROS are produced under both physiological and pathological conditions.2, 3 Natural ROS production in the body is associated with metabolic changes, cellular respiration, phagocyte activity, cytochrome P-450, and autoxidation of biologically active compounds such as epinephrine, hemoglobin, thiol compounds, and many others. Under physiological conditions, ROS do not accumulate in tissues but are permanently inactivated by local enzymatic and nonenzymatic antioxidant mechanisms. The term “oxidative stress” was introduced into scientific terminology and defined as an imbalance between oxidants and antioxidants in favor of the former.4 The additional definition is “a condition when [a] temporarily or chronically increased concentration of ROS disrupts cellular metabolism and its regulation and then causes damage to cellular components.”2 Oxidative stress plays a crucial role in the development of neurodegenerative and cardiovascular diseases Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00011-0 © 2021 Elsevier Inc. All rights reserved.

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(CVDs), as well as diabetes, cancer, and infectious diseases. Collectively, these disorders take millions of lives each year.3 In the simplest case, the pathological process develops as a result of simultaneous transformations of free radicals, both in the course of their formation and in the process of their elimination. The use of antioxidant therapy may be promising, but it is important to know whether oxidative stress is the primary cause that underlies the development of the disease, or if its manifestation is due to the clinical course of the disease.5 Antioxidants are substances of natural or synthetic origin that interact with lipid radicals (direct antioxidants) and inhibit oxidative stress by influencing one or more stages of ROS formation, thus reactivating antioxidant enzymes (Tables 1 and 2). Despite more than 30 years of research into the role of radical processes in the pathogenesis of various diseases, the search for the optimal antioxidant agent is still ongoing. From the point of view of the antioxidant mechanism TABLE 1 Classification, basic mechanism of action and localization of antioxidants. Classification

Primary (radical scavengers) vs secondary antioxidants (e.g., singlet oxygen quenchers, peroxide decomposers, metal chelators) Enzymatic vs nonenzymatic Preventative (block/capture radicals that are formed) vs repair antioxidant systems (repair/remove damaged macromolecules) Hydrosoluble vs liposoluble Endogenous vs exogenous (natural vs synthetic)6

Mechanism of action

Prevention of the formation of new radicals Scavenging of already formed radicals

Localization

Destruction of radicals and their precursors in different parts of the cell: Inside the cell membrane (hydrophobic, lipid-soluble), e.g., alpha-tocopherol, ubiquinone, beta-carotene At the interface of mediums (hydrophilic, water-soluble), e.g., ascorbic acid, carnosine, acetylcysteine

TABLE 2 Antioxidant types. Antioxidant group

Select representatives

Primary pathway inhibitors of reactive oxygen species (ROS) formation

Xanthine-oxidase inhibitors; Nitri oxide synthase (NOS) inhibitors

ROS scavengers

Superoxide radical scavengers (urea, thiotriazolin, thiourea, ceruloplasmine, nicotine acid and its derivatives); Hydroxyl radical scavengers (mannitol, ethanol, dimethyl sulfoxide, albumin, tryptophan, copper suspension, L-methionine); Single oxygen scavengers (histidine, phenylalkylamine derivatives); NO scavengers and their derivatives (glutathione, methionine)

Direct antioxidants: Scavengers of free fatty acid radicals and lipid hydroperoxides

a-Tocopherol; Phenol derivatives and polyphenolic compounds, Plant flavonoids from various species (thyme, carnation, oregano, etc.): e.g., quercetin, rutin, etc. Aliphatic and aromatic sulfur-containing compounds (methionine, glutathione, acetylcysteine); Derivatives of oxy acids (gallic, chlorogenic, caffeine, p-oxybenzoic, ascorbic and other acids); Ubiquinones (ubiquinone, coenzyme Q10); Selenites (sodium selenite, Se-methionine, Se-glutathione); Retinoids and b-carotene

Microelement chelators

Trillon B, pectins as a high-molecular-weight carbohydrate polymers present in virtually all plants

Antioxidant enzymes

Catalase, superoxide dismutase, glutathione peroxidase

Factors that regulate the expression of endogenous antioxidants

HSP70, HIF (hypoxia-inducible factor - the factor that induces hypoxia)-1a

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of action, the most effective are agents that prevent the formation or directly interact with active metabolites, bind catalysts and reduce the intensity of free radical processes, interact with lipid hydroperoxides and inhibit terminal stages of lipid peroxidation (LPO), and promote synthesis and formation of endogenous antioxidants. The most reactive ROS present in biological systems are the hydroxyl radical (OH) and the superoxide radical (O2 ); both can react with all biological macromolecules (proteins, lipids, nucleic acids, and carbohydrates). The initial reaction generates a second radical that can react with a second macromolecule and induce a chain reaction.1 Organisms possess effective mechanisms against the destructive effects of oxidative stress: enzymatic [superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx)] as well as nonenzymatic, low-molecular-weight substances, including glutathione, vitamins C, E, and K, and carotenoids.3 By developing the antioxidant defense, organisms protect their cells against oxidation of proteins, DNA, and steroid components, as well as peroxidation of unsaturated lipids in cell membranes. When choosing a drug for antioxidant therapy, it is also necessary to consider that no universal compound can block all ROS generation mechanisms or interrupt all types of ROS reactions. Proceeding from its chemical structure and mechanism of action, each antioxidant more or less effectively influences individual stages of free radical generation without being a universal remedy. Numerous experimental and clinical studies testify to the greater therapeutic effectiveness of the complex application of several antioxidants with different mechanisms of action compared to a single agent.7, 8 New approaches in nutrient genetics consider individual genetic variability in the endogenous protective antioxidant systems, the relationship between genetic variability in endogenous antioxidant enzymes and biomarkers of oxidative stress that affect the level of exposure of target cells to antioxidants.7 Many studies have confirmed that a diet rich in antioxidants reduces the risk of chronic diseases. Common exogenous antioxidants are found in fruits and vegetables, e.g., ascorbic acid (vitamin C) in bell peppers, strawberries, kiwi, and Brussels sprouts; tocopherols, e.g., vitamin E in vegetable oils, nuts, and seeds; carotenoids, zeaxanthin, lutein, and lycopene in carrots, tomatoes, apricots, plums, spinach, and kale; polyphenols in apples, berries, grapes, celery, kale, onions, beans, soybeans, nuts, wine, tea, coffee, and cocoa; micro- and macroelements in seafood, meat, and grains. Natural water-soluble antioxidants are important for the prevention of CVDs, atherosclerosis, cancer, diabetes, rheumatoid arthritis, post-ischemic reperfusion injury, myocardial infarction, chronic inflammation, and stroke. Therefore, they should be included as a part of a well-balanced diet. These components can be found in plants, e.g., phenolic substances, catechins, sterols, and aliphatic acids—citric, malic, fumaric, lactic, phytic acid, melanoid compounds, procyanidins, cyclodextrins, and essential oils. Antioxidants are found in many plants, e.g., carob pods (Сеratonia), bark and birch branches (Веtula), eucalyptus (Eucalyptus), mountain ash fruits (Sоrbus), cranberries (Vaccinium), black currant (Ribes), mint (Mentha), thyme (Thymus), oregano grass (Origanum), and many others.8, 9

Obesity, oxidative stress, and inflammation Obesity is one of the most common diseases of modern civilization. According to the WHO, at the end of the 20th century, 30% of the world’s populations (1.7 billion people) were overweight. “Westernization” of the diet and poor diet combined with low physical activity lead to the development of a “pandemic” of alimentary or diet-induced obesity. A high-fat diet is a risk factor for insulin resistance and obesity. Obesity impairs the metabolic, immune, and endocrine functions of adipose tissue (AT), which leads to the release of fatty acids, proinflammatory cytokines (tumor necrosis factor a [TNFa], interleukin-6, interleukin-1, etc.], and adipokines (leptin, adiponectin, resistin, etc.) and promotes the development of inflammation in the AT. In conditions of oxidative stress, there is a disruption of the production of both cytokines and adipokines in AT, which is one of the pathogenetic links of diseases associated with obesity. TNFa can play a key role in modulating energy consumption and fat deposition.10–12 A high-fat diet causes lipid accumulation in adipocytes, their hypertrophy, and activation of intracellular systems of ROS production. These mechanisms lead to changes in metabolic processes and secretion of biologically active adipokines. Additionally, exo- and endogenous saturated fatty acids can activate congenital immunity receptors localized on the adipocytic membrane and initiate a cascade of intracellular inflammatory reactions. This activation stimulates cytokine secretion and significantly influences the production of proinflammatory adipokines.13 This phenomenon is the basis for the concept that inflammation in AT is a self-sustaining process: once initiated, it progresses without the presence of additional factors. This fact deserves attention due to the role of iron in the activation of LPO as well as its multidirectional influence on adipogenesis—stimulation in young growing animals and restriction in an adult body. The data obtained with regard to the relationship between oxidative stress, changes in adipokine regulation, and the presence of signs of inflammation in AT, especially those that appear during young age, are very important for the clarification of obesity pathogenesis with respect to age.

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Diet-induced oxidative stress in relation to noncommunicable diseases A high-fat diet is regarded as a crucial factor in developing CVDs, cancer, and other noncommunicable diseases. Both animal (reviewed in Ref. 14) and human data15 showed adverse metabolic effects of high-fat diet even after short-term administration. The major negative impact of caloric overnutrition is obesity-induced low-grade systemic inflammation, which triggers metabolic and hormonal disturbances. Data indicate that systemic inflammation is most probably a result of a complex network of signals that interconnect several organs. Alterations in gut microbiota induced by high-fat diet seem to be the initial link in the chain of events, followed by elevated release of intestinal lipopolysaccharides and/or free fatty acids that promote production of proinflammatory cytokines in the gut. Increased delivery of lipopolysaccharides, free fatty acids, and proinflammatory cytokines into the systemic and portal circulation leads to systemic inflammation.14, 16 Overnutrition increases ROS production during mitochondrial oxidative metabolism and leads to redox imbalances and induction of oxidative stress, both of which contribute to inflammation. High levels of ROS and proinflammatory cytokines, as mentioned previously, are also produced in hypertrophic adipocytes. Furthermore, apart from interfering with metabolic signaling pathways, ROS negatively impact surrounding tissues such as perivascular endothelium, and, vice versa, vascular damage and inflammation contribute to ROS generation, thus maintaining a vicious cycle.17 Chronic low-grade inflammation induced by high-fat diet supports the development of numerous diseases including type-2 diabetes, CVDs, intestinal diseases, osteoporosis, chronic kidney diseases, central nervous system disorders, and cancer14 (Fig. 1).

Dietary fat in relation to cancer and CVD risk A recent comprehensive meta-analysis based on 43 relevant studies did not confirm the impact of the intake of total fat, saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) on CVD risk. Higher trans-fatty acid (TFA) intake, however, was associated with greater risk of CVDs in a dose-response manner 18. Therefore, the subsequent paragraphs focus predominantly on the impact of different fats on cancer risk. Tumor-promoting effects of a high-fat diet in experimental cancer have long been known and confirmed by vast data. However, tumor promotion/progression is modulated by myriad factors, both dietary and lifestyle, which cannot be fully controlled in population studies. Human data indicate that obesity (most likely induced by both fat- and carbohydrate-rich diets), particularly central adiposity that is also strongly associated with CVD risk,19 rather than the total dietary fat contribution influences cancer risk.20 Obesity induces redox and hormonal imbalances that promote tumor growth17, 21 (Table 3). Current experimental and epidemiologic data show that it is important to focus on fat composition rather than total fat intake. The fatty-acid spectrum differs from one fat/food source to another, and fatty acids differ in their biochemical properties and their physiological and metabolic effects. Thus, the impact of the fatty-acid spectrum on signaling pathways that are involved in cancer progression must be elucidated.

High-fat diet/overnutrition Metabolic dysregulation

Oxidative stress

Inflammation

Obesity

Cardiovascular disease Cancer Diabetes Other noncommunicable diseases FIG. 1 Major factors that interconnect noncommunicable diseases.

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TABLE 3 Key obesity-induced changes related to cancer. Underlying cause

Consequence

Induced pathologies

Sustained production of reactive oxygen species in adipocytes (mediated by free fatty acids, leptin and angiotensin II)

Increased oxidative stress Decrease in adiponectin secretion Impaired insulin response Chronic hyperinsulinaemia

DNA damage Insulin resistance Activation of mitogenic and proangiogenic pathways

Decreased antioxidant defense in obese individuals

Increased production of proinflammatory cytokines Aromatase production in adipose tissue

Increased estrogen production

Recruitment of inflammatory cells (which produce additional reactive oxygen species) Chronic inflammation Promotion of estrogen-dependent cancers

Summarized according to reviews by Refs. 17, 21.

Total fat The discussion on the role of high-fat diets in cancer development is still ongoing. Plant-derived fat has generally been considered healthier due to the content of PUFAs, which include essential omega-3 and omega-6 fatty acids. Animalderived products are usually high in saturated fat but some are also high in MUFAs and PUFAs, including essential fatty acids (Table 4). Thus, excluding them completely from the diet may not be a prudent choice. Experimental data show that certain fatty acids may promote cancer independent of obesity, e.g., through enhancement of stemness and tumorigenicity, while some others show protective effect.22

Saturated fat Concerns regarding the impact of SFA intake on cancer risk persists. High saturated fat intake (but not total, monounsaturated, or polyunsaturated fat intake) was associated with increased risk of breast cancer and also overall cancer risk, but the source of saturated fat was not considered.23, 24 Factors other than saturated fat content per se must be considered, e.g., the formation of carcinogenic heterocyclic amines and polycyclic aromatic hydrocarbons in red meat during the cooking process and the generation of lipid oxidation products and nitroso compounds catalyzed by heme iron during digestion.25 Saturated fat in dairy products does not apparently increase the cancer risk. Fermented dairy products consumption was inversely related with total mortality even if not with cancer mortality.26 Research into the role of specific fatty acids in cancer development is ongoing and, among SFAs, palmitic acid (PA), which is the most abundant SFA in human body, has drawn great attention. Preclinical data indicated that PA promotes tumor growth,27 and human studies reported a positive association between circulating PA levels and cancer risk.28 However, under physiological conditions, the changes in PA intake do not significantly alter its tissue concentration, which is maintained by endogenous biosynthesis. The homeostatic balance of PA may be disrupted by positive energy balance, excessive intake of carbohydrates, and a sedentary lifestyle, all of which would lead to overaccumulation of PA in tissues. This phenomenon would result in dyslipidaemia, hyperglycaemia, increased ectopic fat accumulation, and elevated inflammatory tone. An imbalance in the PA/PUFAs ratio in the diet may contribute to these pathologies and promote cancer growth.29 There is current agreement that the consumption of palm oil within a balanced diet does not pose a health risk (regarding cancers or CVDs) if the SFA intake is kept under 10% of total energy.30 The isolated effect of other SFAs in human pathology remains to be elucidated.

Unsaturated fat MUFAs Oleic acid is the most abundant MUFA in common oils and fats. The intake of MUFAs from animal sources is associated with higher total mortality. Isocaloric replacement of MUFAs from animal sources with MUFAs from plant sources

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TABLE 4 Fatty acid composition of common vegetable and animal fats and oils. Oil/fat

% of saturated fatty acids

% of monounsaturated fatty acids

% of polyunsaturated fatty acids

Beef tallow

48

49

4

Butterfat

66

31

3

Canola

6

62

32

Cocoa butter

62

34

3

Coconut

92

6

2

Corn

13

28

59

Flaxseed

9

20

70

Lard

42

48

10

Olive

17

72

11

Palm kernel

82

15

2

Palm

50

40

10

Palm olein (IV 56)

46

42

11

Palm stearin (IV 32)

64

27

8

Peanut

19

50

32

Safflower

9

13

78

Soybean

15

24

61

Sunflower

12

19

69

Fatty acid composition was determined by gas-liquid chromatography. Total percentage of saturated, mono- and polyunsaturated fatty acids was calculated using the data in the original source. Component fatty acids may not add up to 100% due to rounding. IV, iodine value. Adapted from Institute of Shortening and Edible Oils (ISEO). IX. Products prepared from fats and oils. Washington, DC: Institute of Shortening Edible Oils (ISEO), 2004. Available at: http://iseo.org/FFO/page7.html Accessed 6 July 2019.

decreased both cancer mortality and mortality ascribed to other causes including cardiovascular mortality.31 Similarly, decreased cancer and CVD risk was observed when part of the saturated fat intake was replaced with plant MUFAs.32 Therefore, plant MUFA sources should be preferred. Olive oil appears to be suitable MUFA source; epidemiologic data support its protective role in cancer development, but it is unclear whether the beneficial effects of olive oil may be attributed to the MUFA or antioxidant content.33 Nuts, another source of MUFAs, may be beneficial in decreasing cancer risk.34 However, the beneficial effects might be attributed to antioxidants and other phytochemicals, including the fiber.

PUFAs Among fatty acids, two PUFAs are essential for humans: alpha-linolenic (an omega-3 fatty acid and the precursor to eicosapentaenoic and docosahexaenoic acid) and linoleic acid (an omega-6 fatty acid and dominant PUFA in most commonly used fats and oils). During the million years of human evolution, the ratio of omega-6 to omega-3 fatty acids equalled approximately 1, but this ratio markedly rose in recent millennia. This change is considered to be one of the reasons for increase in diseases of civilization.35 High omega-6 PUFA intake and a very high omega-6/omega-3 ratio is involved in many pathologies, including cancer, CVDs, and inflammatory and autoimmune disease. Comparatively, increased omega-3 PUFA intake appears to be protective.36 Omega-3 and omega-6 PUFAs play a significant role in inflammation. Omega-6 PUFAs are often described as proinflammatory and omega-3 PUFAs as anti-inflammatory agents. However, not enough data support this simple hypothesis,

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and the interaction of omega-3 and omega-6 fatty acids and their lipid mediators in the context of inflammation is complex and not fully understood.37 The role of omega-3 PUFAs in cancer development and progression was evaluated in a number of preclinical studies. Most of them reported preventive/therapeutic effects that result from the regulation of several processes, including cell proliferation, survival, differentiation, invasion, and angiogenesis.35 The evidence from human studies is not convincing, most likely due to total omega-6 PUFA consumption, which counteracts the beneficial effects of omega-3 PUFAs.38 Other factors, such as PUFA bioavailability, may also have an impact. Modification in dietary fat composition may be beneficial; isocaloric replacement of as little as 2% of energy from SFAs with linoleic acid decreased the cancer mortality by 8% [hazard ratio (HR) 0.92, 95% confidence interval (CI) 0.90–0.93] and also reduced CVD mortality by 6% (HR 0.94, 95% CI 0.92–0.96).32

TFAs TFAs are unsaturated fatty acids (monounsaturated or polyunsaturated) with at least one bond in the trans configuration. TFAs may be found in small amounts in meat and dairy products from ruminants, but industrial partially hydrogenated vegetable oils, which may contain up to 50% TFAs, are considered to be the major dietary TFA source. Hydrogenated fish oils are another source of TFAs. Partially hydrogenated oils are widely used in many food products to increase their shelf life (Table 5). TFA intake is positively associated with an increase in total and low-density lipoprotein (LDL) cholesterol and inversely associated with high-density lipoprotein (HDL) cholesterol. Thus, TFA intake contributes to CVD risk. Dose-response analysis found that the risk of CVDs increased by 16% [relative risk (RR) 1.16, 95% CI 1.07–1.25] for an increment of 2% energy/day of TFA intake 18. Research data also indicate a positive association between TFAs intake and cancer.39

TABLE 5 Typical trans-fatty acid content of foods produced or prepared with partially hydrogenated vegetable oils in the USA. Type of food

Trans-fatty acid content (g/100 g)

% of total fatty acids

French fries

4.2–5.8

28–36

Breaded fish burger

3.4

28

Breaded chicken nuggets

4.9

25

French fries frozen

2.5

30

Enchilada

1.1

12

Burrito

0.9

12

Pizza

0.5

9

Tortilla (corn) chips

5.8

22

Granola bar

3.7

18

Popcorn, microwave

3

11

Cookies

5.9

26

Doughnuts

5.7

25

Brownie

3.4

21

Muffin

1.3

14

Fast/frozen foods

Packaged snacks

Bakery products

Adapted from Mozaffarian D, Katan MB, Ascherio A, Stampfer MJ, Willett WC. Trans fatty acids and cardiovascular disease. N Engl J Med 2006;354:1601–1613.

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According to World Health Organization (WHO) estimates, every year trans-fat intake leads to more than 500,000 deaths from CVDs. However, it is necessary to emphasize that industrial—not ruminant—TFAs are responsible for this increased risk.40 Therefore, in May 2018, WHO released REPLACE, a step-by-step guide for the elimination of industrially produced TFAs from the global food supply. The total trans-fat intake should be limited to less than 1% of total energy intake, which translates to less than 2.2 g/day with a 2000-cal diet.41 Evaluation of relevant data from 29 countries shows that the energy intake from trans-fat exceeds the recommended value in several countries including Canada and the United States. However, a positive trend has been observed as a substantial decline in industrial trans-fat intake since 1995 was reported in most countries.42

TFAs and cancer risk Naturally occurring conjugated linoleic acid (CLA), an omega-6 PUFA, may have a protective effect on cancer development. CLA inhibits the growth of experimental mammary and colon tumors. A dietary level of 1% CLA appears to be the optimal dose for the suppression of cancer growth in animal studies.43 However, anticarcinogenic properties of CLA were not proved by a recent meta-analysis of prospective studies, most probably due to limited intake of CLA in human-based studies that did not reach the level of an effective dose. There was no significant relationship between dietary intake of total TFAs and the risk of breast cancer, but serum TFA levels were positively associated with an increased risk of breast cancer among postmenopausal women.44 Ruminant TFAs are suggested to increase the risk of postmenopausal breast cancer, according to the results of a large Norwegian cohort study, but the authors supposed that this increase was linked to saturated fat.45 CLA effectively suppresses experimental gastrointestinal cancers, but not all studies confirmed this protective effect (which may also depend on the type of the isomer used).43 Human data indicate a positive association between trans-fat intake and colon cancer.46 However, in another study, neither trans-fat nor total or other fat types (animal, vegetable, saturated, monounsaturated fat, and polyunsaturated fat) impacted cancer recurrence or survival in colon cancer patients.47 Data on the effects of TFAs on other cancers are scant. One study reported a positive association between total TFA intake and prostate cancer,48 but another found a decreased risk between vegetable TFA intake and pancreatic cancer in men and non-Hodgkin lymphoma in both genders. An inverse trend was also observed for a cancer of the central nervous system in women. A reduced risk was also observed between fish TFA intake and prostate cancer. On the other hand, fish TFA intake was positively associated with the risk of rectal cancer and multiple myeloma. There was also a positive trend observed for stomach cancer. Ruminant TFAs were associated with increased risk of cancer of mouth/pharynx and decreased risk of multiple myeloma.45 These results support the idea that the impact of TFAs on cancer risk is source-dependent. Nevertheless, further studies are needed to confirm this association and elucidate the role of TFAs in tumor progression.

Cancer and heart disease CVDs and cancer are the two major causes of death worldwide.49 There are several common pathophysiological mechanisms that link cancer and heart disease: inflammation, neurohormonal activation, oxidative stress, and immune system dysfunction.50 Activation of the renin-angiotensin-aldosterone system, one of the eminent features of CVDs, reinforces increased ROS production.51, 52 ROS play an important role in both heart disease and cancer.53, 54 The heart heavily relies on mitochondrial oxidative phosphorylation, which is also a source of ROS that plays an essential role in cancer progression.55 Furthermore, downstream effects of cardiac-derived inflammatory factors potentially support tumor growth.56 Inflammation, even if of low intensity, appears to be at the crossroads of CVDs and cancer. In the recently published phase III CANTOS trial, the interleukin 1b blocker canakinumab administered in patients with myocardial infarction reduced major adverse cardiovascular events by 25% in comparison to placebo (HR 0.75, 95% CI 0.66–0.85).57 Awareness of the cardio-oncology interactions is growing among practitioners, but there is still a need for extensive preclinical studies in this field. In particular, there is no data regarding specific circulating factors or pathophysiological mechanisms linked to specific forms of cancer. New insights and proposals for combined heart and cancer surveillance strategies are clearly and urgently needed.

Summary points l

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Oxidative stress, which may also be triggered by high caloric intake, plays a key role in the progression of most noncommunicable diseases, including CVDs and cancer. Obesity-associated low-grade systemic inflammation potentiates the oxidative stress and thus contributes to the development of noncommunicable diseases.

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A plethora of antioxidants may be used as dietary supplements, but the ideal combination of dietary antioxidants to alleviate oxidative stress must be established. Total fat intake does not appear to have a larger impact on either CVD or cancer risk compared to central obesity. Replacement of saturated fat with unsaturated fat, particularly of plant origin, decreases the risk of CVDs and cancer. Reduction in TFA intake is beneficial for CVD management, but additional studies are needed to clarify the role of TFAs in cancer progression. A link between CVDs and cancer remains to be elucidated.

References 1. Giustarini D, Dalle-Donne I, Tsikas D, Rossi R. Oxidative stress and human diseases: origin, link, measurement, mechanisms, and biomarkers. Crit Rev Clin Lab Sci 2009;46:241–81. 2. Marnett LJ. Oxyradicals and DNA damage. Carcinogenesis 2000;21:361–70. 3. Halliwell B. Oxidative stress and cancer: have we moved forward? Biochem J 2007;401:1–11. 4. Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C. Chronic inflammation and oxidative stress in human carcinogenesis. Int J Cancer 2007;121:2381–6. 5. Purohit V, Simeone DM, Lyssiotis CA. Metabolic regulation of redox balance in cancer. Cancers 2019;11:955. 6. Pisoschi AM, Pop A. The role of antioxidants in the chemistry of oxidative stress: a review. Eur J Med Chem 2015;97:55–74. 7. Da Costa LA, Garcı´a-Bailo B, Badawi A, El-Sohemy A. Genetic determinants of dietary antioxidant status. Prog Mol Biol Transl Sci 2012;108:179–200. 8. Elvira-Torales LI, Garcı´a-Alonso J, Periago-Casto´n MJ. Nutritional importance of carotenoids and their effect on liver health: a review. Antioxidants 2019;8:229. 9. Piccolella S, Crescente G, Candela L, Pacifico S. Nutraceutical polyphenols: new analytical challenges and opportunities. J Pharm Biomed Anal 2019;175:112774. 10. Bullo´-Bonet M, Garcı´a-Lorda P, Lo´pez-Soriano FJ, Argiles JM, Salas-Salvado´ J. Tumour necrosis factor, a key role in obesity? FEBS Lett 1999;451:215–9. 11. Suzuki M, Ding Q, Muranaka S, Kigure M, Kojima M, Terada M, et al. Correlation between body weight (Epididymal fat) and permeation rate of serum leptin through the blood-brain barrier (BBB) in male rats aged 8 months. Exp Anim 2008;57:485–8. 12. Bradley RL, Fisher FM, Maratos-Flier E. Dietary fatty acids differentially regulate production of TNF-a and IL-10 by murine 3T3-L1 adipocytes. Obesity 2008;16:938–44. 13. McClung JP, Karl JP. Iron deficiency and obesity: the contribution of inflammation and diminished iron absorption. Nutr Rev 2009;67:100–4. 14. Duan Y, Zeng L, Zheng C, Song B, Li F, Kong X, et al. Inflammatory links between high fat diets and diseases. Front Immunol 2018;9:2649. 15. Guay V, Lamarche B, Charest A, Tremblay AJ, Couture P. Effect of short-term low- and high-fat diets on low-density lipoprotein particle size in normolipidemic subjects. Metabolism 2012;61:76–83. 16. Bleau C, Karelis AD, St-Pierre DH, Lamontagne L. Crosstalk between intestinal microbiota, adipose tissue and skeletal muscle as an early event in systemic low-grade inflammation and the development of obesity and diabetes. Diabetes Metab Res Rev 2015;31:545–61. 17. Le Lay S, Simard G, Martinez MC, Andriantsitohaina R. Oxidative stress and metabolic pathologies: from an adipocentric point of view. Oxidative Med Cell Longev 2014;2014:1–18. 18. Zhu Y, Bo Y, Liu Y. Dietary total fat, fatty acids intake, and risk of cardiovascular disease: a dose-response meta-analysis of cohort studies. Lipids Health Dis 2019;18:91. 19. Casanueva FF, Moreno B, Rodrı´guez-Azeredo R, Massien C, Conthe P, Formiguera X, et al. Relationship of abdominal obesity with cardiovascular disease, diabetes and hyperlipidaemia in Spain. Clin Endocrinol 2009;73:35–40. 20. Barberio AM, Alareeki A, Viner B, Pader J, Vena JE, Arora P, et al. Central body fatness is a stronger predictor of cancer risk than overall body size. Nat Commun 2019;10:383. 21. Nieman KM, Romero IL, Van Houten B, Lengyel E. Adipose tissue and adipocytes support tumorigenesis and metastasis. Biochim Biophys Acta 2013;1831:1533–41. 22. Beyaz S, Mana MD, Roper J, Kedrin D, Saadatpour A, Hong S-J, et al. High-fat diet enhances stemness and tumorigenicity of intestinal progenitors. Nature 2016;531:53–8. 23. Gonzalez CA, Riboli E. Diet and cancer prevention: contributions from the European prospective investigation into cancer and nutrition (EPIC) study. Eur J Cancer 2010;46:2555–62. 24. Sellem L, Srour B, Gueraud F, Pierre F, Kesse-Guyot E, Fiolet T, et al. Saturated, mono- and polyunsaturated fatty acid intake and cancer risk: results from the French prospective cohort NutriNet-Sante. Eur J Nutr 2019;58:1515–27. 25. Demeyer D, Mertens B, De Smet S, Ulens M. Mechanisms linking colorectal cancer to the consumption of (processed) red meat: a review. Crit Rev Food Sci Nutr 2016;56:2747–66. 26. Mazidi M, Mikhailidis DP, Sattar N, Howard G, Graham I, Banach M, et al. Consumption of dairy product and its association with total and cause specific mortality—a population-based cohort study and meta-analysis. Clin Nutr 2018;38:2833–45 S0261-5614. 27. Pascual G, Avgustinova A, Mejetta S, Martı´n M, Castellanos A, Attolini CS, et al. Targeting metastasis-initiating cells through the fatty acid receptor CD36. Nature 2017;541:41–5.

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28. Bassett JK, Hodge AM, English DR, MacInnis RJ, Giles GG. Plasma phospholipids fatty acids, dietary fatty acids, and breast cancer risk. Cancer Causes Control 2016;27:759–73. 29. Carta G, Murru E, Banni S, Manca C. Palmitic acid: physiological role, metabolism and nutritional implications. Front Physiol 2017;8:902. 30. Marangoni F, Galli C, Ghiselli A, Lercker G, La Vecchia C, Maffeis C, et al. Palm oil and human health. Meeting report of NFI: nutrition foundation of Italy symposium. Int J Food Sci Nutr 2017;68:643–55. 31. Guasch-Ferre M, Zong G, Willett WC, Zock PL, Wanders AJ, Hu FB, et al. Associations of monounsaturated fatty acids from plant and animal sources with total and cause-specific mortality in two US prospective cohort studies. Circ Res 2019;124:1266–75. 32. Zhuang P, Zhang Y, He W, Chen X, Chen J, He L, et al. Dietary fats in relation to total and cause-specific mortality in a prospective cohort of 521 120 individuals with 16 years of follow-up. Circ Res 2019;124:757–68. 33. Psaltopoulou T, Kosti RI, Haidopoulos D, Dimopoulos M, Panagiotakos DB. Olive oil intake is inversely related to cancer prevalence: a systematic review and a meta-analysis of 13800 patients and 23340 controls in 19 observational studies. Lipids Health Dis 2011;10:127. 34. Lee J, Shin A, Oh JH, Kim J. The relationship between nut intake and risk of colorectal cancer: a case control study. Nutr J 2018;17:37. 35. Weylandt KH, Serini S, Chen YQ, Su H-M, Lim K, Cittadini A, et al. Omega-3 polyunsaturated fatty acids: the way forward in times of mixed evidence. Biomed Res Int 2015;2015:1–24. 36. Simopoulos AP. The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases. Exp Biol Med 2008;233:674–88. 37. Innes JK, Calder PC. Omega-6 fatty acids and inflammation. Prostaglandins Leukot Essent Fat Acids 2018;132:41–8. 38. Witte TR, Hardman WE. The effects of omega-3 polyunsaturated fatty acid consumption on mammary carcinogenesis. Lipids 2015;50:437–46. 39. Islam MA, Amin MN, Siddiqui SA, Hossain MP, Sultana F, Kabir MR. Trans fatty acids and lipid profile: a serious risk factor to cardiovascular disease, cancer and diabetes. Diabetes Metab Syndr Clin Res Rev 2019;13:1643–7. 40. Ferlay A, Bernard L, Meynadier A, Malpuech-Bruge`re C. Production of trans and conjugated fatty acids in dairy ruminants and their putative effects on human health: a review. Biochimie 2017;141:107–20. 41. World Health Organization. WHO plan to eliminate industrially-produced trans-fatty acids from global food supply. Available at: https://www.who. int/news-room/detail/14-05-2018-who-plan-to-eliminate-industrially-produced-trans-fatty-acids-from-global-food-supply; 2018. (Accessed 5 July 2019). 42. Wanders A, Zock P, Brouwer I. Trans fat intake and its dietary sources in general populations worldwide: a systematic review. Nutrients 2017;9:840. 43. Bhattacharya A, Banu J, Rahman M, Causey J, Fernandes G. Biological effects of conjugated linoleic acids in health and disease. J Nutr Biochem 2006;17:789–810. 44. Anjom-Shoae J, Sadeghi O, Larijani B, Esmaillzadeh A. Dietary intake and serum levels of trans fatty acids and risk of breast cancer: a systematic review and dose-response meta-analysis of prospective studies. Clin Nutr 2019;39:755–64. S0261-5614. 45. Laake I, Carlsen MH, Pedersen JI, Weiderpass E, Selmer R, Kirkhus B, et al. Intake of trans fatty acids from partially hydrogenated vegetable and fish oils and ruminant fat in relation to cancer risk. Int J Cancer 2013;132:1389–403. 46. Vinikoor LC, Millikan RC, Satia JA, Schroeder JC, Martin CF, Ibrahim JG, et al. Trans-fatty acid consumption and its association with distal colorectal cancer in the North Carolina Colon Cancer Study II. Cancer Causes Control 2010;21:171–80. 47. Van Blarigan EL, Ou F-S, Niedzwiecki D, Zhang S, Fuchs CS, Saltz L, et al. Dietary fat intake after colon cancer diagnosis in relation to cancer recurrence and survival: CALGB 89803 (Alliance). Cancer Epidemiol Biomark Prev 2018;27:1227–30. 48. Liss MA, Al-Bayati O, Gelfond J, Goros M, Ullevig S, DiGiovanni J, et al. Higher baseline dietary fat and fatty acid intake is associated with increased risk of incident prostate cancer in the SABOR study. Prostate Cancer Prostatic Dis 2019;22:244–51. 49. Crespo-Leiro MG, Anker SD, Maggioni AP, Coats AJ, Filippatos G, Ruschitzka F, et al. European society of cardiology heart failure long-term registry (ESC-HF-LT): 1-year follow-up outcomes and differences across regions. Eur J Heart Fail 2016;18:613–25. 50. Boer RA, Meijers WC, Meer P, Veldhuisen DJ. Cancer and heart disease: associations and relations. Eur J Heart Fail 2019. https://doi.org/10.1002/ ejhf.153918. 51. Winklewski PJ, Radkowski M, Wszedybyl-Winklewska M, Demkow U. Brain inflammation and hypertension: the chicken or the egg? J Neuroinflammation 2015;12:85. 52. Mowry FE, Biancardi VC. Neuroinflammation in hypertension: the renin-angiotensin system versus pro-resolution pathways. Pharmacol Res 2019;144:279–91. 53. Takimoto E, Kass DA. Role of oxidative stress in cardiac hypertrophy and remodeling. Hypertension 2007;49:241–8. 54. Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med 2010;49:1603–16. 55. Maiuri MC, Kroemer G. Essential role for oxidative phosphorylation in cancer progression. Cell Metab 2015;21:11–2. 56. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell 2010;140:883–99. 57. Ridker PM, MacFadyen JG, Everett BM, Libby P, Thuren T, Glynn RJ, et al. Relationship of C-reactive protein reduction to cardiovascular event reduction following treatment with canakinumab: a secondary analysis from the CANTOS randomised controlled trial. Lancet 2018;391:319–28.

Chapter 12

Cancer during pregnancy. Maternal, placenta, and fetal damage. Nutrition, antioxidant defenses, and adult offspring tumor-bearing Carla de Moraes Salgadoa,c, Natalia Angelo da Silva Miyagutia,c, Sarah Christine Pereira de Oliveiraa,c, Bianca Cristine Favero-Santosb,c, Laı´s Rosa Vianaa,c, Melina de Moraes Santos Oliveiraa,c, and Maria Cristina Cintra Gomes-Marcondesa,c a

Laboratory of Nutrition and Cancer, Department of Structural and Functional Biology, Institute of Biology, University of Campinas, Campinas, SP,

Brazil, b Obesity and Comorbidities Research Centre, Department of Structural and Functional Biology, Institute of Biology, University of Campinas, Campinas, SP, Brazil

Introduction: Cancer and pregnancy Cancer during pregnancy incidence The simultaneous occurrence of cancer and pregnancy is approximately once per 1000 pregnancies annually.1, 2 The most common malignancies associated with pregnancy include melanoma, lymphomas, leukemia, and cancers of the cervix, breast, ovary, colon, and thyroid; this also includes other types of tumors that could also metastasize to the placenta and fetus. Despite the low incidence, this association is considered a delicate and critical situation because the life of the mother and the fetus are affected.3

Cancer-induced metabolic changes in pregnancy evolution The neoplastic growth changes the pregnancy’s physiological processes, and these alterations lead to a severe reduction in nutrient supply to fetal growth. This is probably caused by anorexia and the deviation of specific nutrient substrates to neoplastic cells, leading to a catabolic state in both the mother and the fetus. Under the occurrence of cancer and pregnancy, the first two anabolic trimesters of a healthier pregnancy are changed to an impaired metabolic process, whereas the catabolic phase in the third gestational trimester was exacerbated, compromising the adequate supply for fetal growth. The accelerated cell proliferation—for both the fetus and the tumor—may be partly provided by nutrients coming from the mobilization of maternal storage. This conducts to a severe decrease of gestational hyperphagia due to the anorexia state, which causes more adverse effects in the mother and especially over the fetal tissues. As previously shown in experimental studies, the 50% reduction of food intake in pregnant tumor-bearing rats promoted a significant changes in the metabolism of carbohydrates, lipids, and proteins. In this peculiar condition as cancer during pregnancy, the inappropriate maternal energy reserves lead to impairment of the course of pregnancy. Besides, proinflammatory cytokines produced by the tumor and maternal cells may compromise the physiological state in gestation, which will affect the pregnancy evolution and put in danger the maternal and fetal viability and prognosis.4–6

Carbohydrate metabolic changes The carbohydrate maternal metabolism is severely changed during the association with cancer evolution. During pregnancy, the hormonal milieu leads to the anabolism process, mainly stimulated by insulin.7 In a pregnancy associated with cancer, the insulin level decreases, causing a reduction of maternal nutrient stores; these are intensely catabolized because c. All authors have equal contribution to write, research, and develop data from experimental procedures for this work. Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00012-2 © 2021 Elsevier Inc. All rights reserved.

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of the tumor growth impairing the healthier development of the fetus to benefit cancer cell proliferation.4, 8, 9 Furthermore, during cancer, the actions of the placental lactogen hormone, which is already present 5–10 days after implantation, are intensified to stimulate lipolysis and to mobilize hepatic glycogen storage. In these early months of pregnancy with cancer, there is a failure to release progesterone and insulin associated with a higher level of estrogen, which compromises the anabolic processes that these hormones would stimulate. The altered anabolic processes lead to additional impairment since the higher serum levels of proinflammatory cytokines such as TNF a, IFN g IL-6, or IL-1, increase maternal waste of adipose tissue and, mainly, skeletal muscle.4, 9, 10 Similarly, during fasting, pregnant women with cancer have a rapid and pronounced decrease in serum levels of glucose.9 This is in addition to the fact that cancer cells consume high rates of glucose, producing increased lactate concentrations during the anaerobic metabolic process.10 This hypoglycemia caused by cancer harms the placenta, especially during the first months of pregnancy. This consequently changes the hormonal responses such as higher adrenaline and glucocorticoid release, which induces the mobilization of substrates for gluconeogenesis to maintain glycemia.10, 11Therefore, this high energy demand of cancer cells leads to a lower supply of substrates for the fetal/placental unit.12 Our experimental studies show that during the first 2 weeks of pregnancy in tumor-bearing rats, when the tumor growth was approximately 7% of the body mass, the blood glucose slightly decreased and the serum insulin was unchanged. However, during the third week of pregnancy when the tumor growth was higher (more than 10%), severe hypoglycemia and low insulin levels resulted.11, 13, 14 At the end of gestation with cancer, the glycogenolysis is high due to the intense activity of glycogen phosphorylase associated with the increased content of counteracting hormones, such as glucagon and glucocorticoids.15, 16 Therefore, the competition for nutrients provided by the maternal stores mainly damages the fetal growth and perhaps the neoplastic development.

Lipid metabolic changes Lipid metabolism is also intensively changed in the presence of cancer and pregnancy. During pregnancy, the predominant catabolic phase is also associated with a higher cancer evolution, where lipid spoliation increases due to the release of the lipid mobilizing factor (LMF17). Besides the insulin resistance, the placental lactogen hormone also contributes to intense maternal lipolysis in the case of neoplastic evolution.18 During pregnancy with cancer, the adipose tissue is depleted by 32% in pregnant rats, which promotes hypertriglyceridemia and elevated serum free fatty acids that are associated with increased ketone body levels.9, 10, 19 Parallel to the higher triacylglycerol mobilization at the end of pregnancy, the cancer development induces a decrease in fatty acid uptake by adipocytes and lower lipoprotein lipase activity. The progression of cancer increases the maternal liver energy demand for free fatty acids due to excessive gluconeogenesis.20 Glycerol from lipid mobilization constitutes an additional mechanism that ensures the continuous availability of glucose to the fetus and also to the neoplastic cells by gluconeogenesis. In an experimental model of cachexia, our previous studies showed that fetal growth was mainly impaired, increasing the rate of resorption and consequently stopping the pregnancy course not only by fetal death but also by the maternal tissue host wasting.6, 8, 16, 21

Protein metabolic changes One of the most compromised metabolic changes during cancer evolution is the protein metabolism, which did not differ during the association between cancer and pregnancy, and which is worse than in other patients. In being pregnant with cancer, the presence of anorexia can intensify the decreased feeding efficiency due to reduced intestinal absorption, especially for some amino acids, as shown in one of our previous studies.13 In addition to this point, the waste of lean body mass of the pregnant increases, as a result of a negative nitrogen balance and the imbalance of circulating nutrients, mainly amino acids. These alterations in the nutrient’s availability directly affect placental activities and the consequent fetal energy supply. Tumor and fetal cells use substrates such as alanine, glutamine, lactate, and ketone bodies as structural or energetic sources. Because amino acids are essential for protein formation, in the case of nutritional competition, both the tumor and the fetus can be affected. The cancer-induced depletion and the increased availability of specific amino acids in maternal plasma, such as glutamine and alanine, favor gluconeogenesis that provides sources for tumor cell proliferation. Furthermore, the inefficient energy process accounts for the inability of the maternal host to adapt the protein synthesis processes, leading to reduced fetal development.6, 22, 23 In this line, in a tumor-bearing mother, the possible nutritional competition results in impaired growth and metabolic changes in both the mother and fetus.5 However, in our previous work, the fetal and placental damage noted was also due to indirect effects, more specifically by substances synthesized by the tumor; as the proteolysis-inducing factor, which intensified the mother metabolic changes, causing fetal death and resorption as verified in pregnant rats.6, 17 Likewise, the peripheral insulin resistance present in the last third of pregnancy with cancer further contributes to the imbalance of the amino acid profile, which enhanced the skeletal muscle

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catabolism.24, 25 Therefore, cancer during pregnancy led to several metabolic changes that increased peripheral insulin resistance, compromised the amino acid and glucose cell uptake, and forced a higher lean body mass wasting, including the fetus tissues.

Oxidative stress in pregnancy associated with cancer Placental and fetal changes and viability associated with cancer As in other systems, the reproductive physiology usually involves the production of reactive oxygen species (ROS) as well as the responses of the antioxidant system to control the levels of oxidative stress.26 The physiological levels of the ROS have an important role in the regulation of the pathways related to uterine function, embryonic implantation, embryogenesis, and fetus-placental development.27 During pregnancy, there is normal mild oxidative stress that is essential to maintain uterine vascularization, being controlled by estrogen action.28 Oxidative stress increases during early pregnancy due to an increased placental metabolic rate, leading to intense ROS production.26 On the other hand, increased antioxidant activity leads the placenta to adapt to a higher oxygen-rich environment.29 However, the increased oxidative stress has been related to many disorders during pregnancy, such as embryonic resorption, recurrent pregnancy loss, preeclampsia, intrauterine growth restriction, and fetal death.30 Therefore, in all these cases, changes in placental formation and activity modify the nutrient exchange between the mother and the fetus, leading to cellular changes that interfere with ROS production and antioxidant balance. As a result, there is a loop that leads to a nonfunctional placenta and then to fetal malformation, less viability, and even postpartum disease. In some pathologies, the higher production of these oxidative elements might promote several cellular issues such as damage to DNA and organelles.31 In pregnancy, the placenta DNA damage might promote gene malfunction leading to p53 failure. This could promote some pathologies such as preeclampsia as a consequence of the activation or not of specific genes, even in placental tissue, as well as increasing the impairment of fetal development.29 Placental impairment and irregular blood pressure can promote hypoxia status, leading the organ and the fetus to oxidative stress. Also, the pregnancy hormones such as estrogen regulate the oxygen uptake by the placenta, which also produces estrogen. Then, when this organ is somehow injured, it can promote a reduction in aromatase concentration and estrogen synthesis, leading to damage in the mobility of trophoblastic cells, angiogenesis, and uterine vascular tone.29 Studies have shown that the mitochondrial cristae morphology changed in cases where a placental impairment occurred by differences between the antioxidant levels. In cases where an imbalance of antioxidant levels occurs,29, 30 as happens during the association with cancer evolution, the products of conception could suffer damage by cellular impairment by deregulated hormonal production. One of our previous studies showed that higher glycogen content in the placentas of Walker-256 tumor-bearing rats caused placental impairment, leading to glycolytic metabolism dysfunction.32 This physiological feedback system, controlling progesterone and estrogen production, may be impaired by the increased production of ROS, compromising the pregnancy.33 However, all the ROS action on normal and healthy gestational status as well in a pathological state still needs to be better understood. The success of a placental implant is due to trophoblastic invasion, remodeling of the spiral artery, nutrient supply, and oxygen diffusion, including the oxidative stress balance. A change in one of these processes, likely in cancer evolution, interferes with the cellular signaling pathways, such as the mechanistic target of rapamycin (mTOR) and the O-linked N-acetylglucosamine transferase (OGT) system, impairing the placental and fetal activities.6, 8 As mentioned above, the production of ROS is usual in a pregnancy because of the high cellular synthesis activity. However, especially in the third trimester of pregnancy, the increase in ROS levels in response to higher mitochondrial activity is related to some of the diseases that may happen during pregnancy, including cancer. At this time, the trophoblastic invagination allows the relative pressure of oxygen (pO2) to increase as a result of the opening of the spiral artery. In order to manage a sudden increase of pO2, there are some important cellular changes such as increased expression of PGC1 mRNA to synthesize a higher number of antioxidants agents, including some important enzymes such as catalase, glutathione peroxidase, and superoxide dismutase. These points are completely disturbed during pregnancy with cancer, showing an imbalance between ROS production and the activation of antioxidant products in the placenta and fetal tissues in tumor-bearing rats.6, 32, 34 Some studies have shown that trophoblast proliferation was directly managed by mTOR signaling linked to nutrient availability,33 which was changed during cancer evolution. In our previous studies, the placental metabolomic tissue profile was changed showing decreases in glucose, succinate, and ATP content. This was associated with an increase in lactate levels as well as impacts on the tricarboxylic cycle and mitochondrial electron transport chain pathways directly related to placental energy production.6, 32, 34 In another point of view, tumor evolution causes an increased inflammatory process associated with enhanced global tissue oxidative stress. The reduction of antioxidant enzyme activities in the placenta leads to an increase of ROS levels and lipid peroxidation, which may contribute to irreversible placental damage and compromise fetal development.6, 32, 34 In addition,

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a vicious cycle occurs when placental damage and irregular blood pressure can also promote a state of hypoxia, leading to organ and fetus oxidative stress. The placental tissue responds to the hypoxia process by OGT signaling in HIF-1a-induced angiogenesis. This could also compromise the vascularization, reducing the placenta growth and activity and further restricting fetal growth. In this case, the reduced growth, resorption, and even death of the fetus that occurred in an experimental model likely happened as a secondary process not only by maternal nutritional deficiency, as mentioned above, but also by deleterious effects induced by tumor factors. These could include the proteolysis-induced factor, which directly impairs mTOR and OGT signaling pathways, affecting the placental and fetal function.6, 34, 35 Considering the cancer evolution during pregnancy, the mother and the fetus could also suffer from cachexia syndrome, increasing the damage of oxidative stress established in skeletal muscle cells and inducing skeletal muscle spoliation. Adding to this, our previous studies have shown that fetal skeletal muscle was intensively wasted in association with higher oxidative stress, where the tumor growth affected the antioxidative enzyme activities and increased lipid peroxidation (malondialdehyde), affecting the fetal gastrocnemius muscle activity.5 The cachexia state changed the metabolomic profile in the host plasma composition and even in the fetal serum, which conducted to impacted metabolic pathways related to the nitrogen metabolism and mitochondrial energy deficiency.19 In addition, there was a differentiation in the molecular expressions of some compounds such as angiotensin II, TGF-b, myostatin, glucocorticoids, TNF- alpha, and interleukin 1 and 6, which contribute even more to the hypercatabolic state. Beyond these characteristics, the host and fetal muscle cells have many repair mechanisms to control the oxidative level, although there is a lack of a mechanism that increases the oxidative status associated with disrupted mitochondrial organelles.

Nutritional supplementation and positive effects in pregnancy and oxidant and antioxidant responses Meanwhile, an antioxidant diet protects cells against damage caused by ROS. The nutritional deficiency of proteins or micronutrients, such as vitamins and minerals, can negatively affect antioxidant capacity because there is a lack of amino acids to synthesize the antioxidant enzyme; faults of many micronutrients that constitute the active site of the antioxidant enzyme; or the absence of micronutrients that are cofactors for antioxidant enzyme regulation. Some micronutrients such as selenium, copper, and zinc are important as they have antioxidant stress activity, cooperating with embryonic and placental development. Besides, the placenta is provided with some defenses to oxidative stress such as seleniumdependent glutathione peroxidases that help artery remodeling as well as other enzymes.36 Beyond those nutrients and enzymes, some hormones such as estrogen and progesterone play important roles as antioxidants. Because NADPH has an oxidative subunit related to ROS production, estrogen can control its production in the vascular smooth muscle cell, minimizing the oxidative stress. However, the mechanism of how estrogen interferes in the production of NADPH and O2 is still not completely understood. Progesterone has important involvement in breast cancer modulation, inducing apoptotic and angiogenic pathways as well as modulating the action and effects of ROS production, showing an antitumor effect. It is well known that in cancer, ROS and antioxidants are increased, helping cancer cells acquire drug resistance. In contrast, progesterone can induce the activation of the caspase and p53 pathways, promoting apoptosis and leading to a decrease in the number of tumor cells. Certain specific amino acids such as leucine can also initiate cell signaling pathways and stimulate PI3K/Akt/mTOR. Leucine is one of the branched-chain amino acids (BCAAs) that plays an essential role in the skeletal muscle metabolism. This appears to be unique to this amino acid, which leads to improved protein synthesis and slows down proteolysis.8, 19, 37–39 Dietary and nutrient supplementation, especially leucine, can minimize the damage caused by a tumor over fetal growth, such as minimizing the higher mobilization of host proteins, which is diverted to tumor growth instead of fetal development.6, 19 Leucine increases the protein synthesis signaling through the activation of mTOR, and also improves the placenta tissue as the mTOR stimulates trophoblast proliferation.6, 40 Experimental studies in pregnant tumor-bearing rats showed that nutritional supplementation with leucine, solely or in conjunction with isoleucine and valine, promotes the recovery of plasma insulin as well as the ability to increase protein synthesis, minimizing the changes induced by cancer.24, 25 Besides, the leucine-rich diet led to a reduction of oxidative stress, improving the antioxidant responses in the fetal gastrocnemius muscle as well in the placenta tissue.5, 6, 8 As previously reported, leucine oxidation is increased in tumor-bearing animals, and as verified in our experiments, a leucine-rich diet leads to a metabolic shift that provides more energy to the host tissues.8 This finding supports the high demand of both skeletal muscle and tumor tissue, and also by the placenta and fetus, which appears to contribute to the enhanced amino acid turnover. Indeed, our findings show that changes involved in oxidative stress by placental dysfunction may lead to fetal programming.41, 42 Furthermore, the placenta malfunction can lead to fetal intrauterine growth restriction. In this way, there is strong evidence that these alterations, even in the placenta or the fetus, would bring some epigenetic alterations that can

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lead to post adult diseases such as metabolic, heart, or even cancer disease. Summing up, improving the maternal responses can minimize the tumor-induced damage in the placenta and fetal tissues.

Maternal diet influence in cancer evolution and host responses Maternal antioxidant diet could affect the defenses of the adult offspring tumor-bearing hosts The most severe syndrome that happens in some types of cancer evolution is the cachexia state, which still remains irreversible and could be responsible for approximately 30% of all cancer-related deaths.21, 43 Therefore, in this case, any interventions that can minimize or prevent this state as well as the tumor effects are valuable. Considering that 30%–50% of cancer cases could be prevented, the nutritional scheme and lifestyle are some important factors that can diminish the cancer risk.44 Therefore, a balanced diet associated with a specific nutritional supplementation might be a preventive approach against cancer. In this case, as during gestation and lactation, the metabolic programming of the offspring is modulated by the maternal diet composition, where the nutrient intake during this time is the most critical environmental factor that can affect gene expression related to the metabolic pathways in the offspring. As this modulation can be preserved throughout life, the effects of maternal nutrition over adult offspring health have been extensively studied,45, 46 including as a preventive strategy against diseases such as cancer and also the cachexia state.21, 41, 42, 47 Antioxidant nutrients such as flavonoids, phenolic compounds, vitamins, amino acids, and polyunsaturated fatty acids (PUFA) are often studied and used to combat cancer; recently, their preventive capacity has also been explored. Quercetin consumption was found to be inversely related to lung cancer risk, or, in animal models, as a preventive compound against colon cancer.48, 49 Gingerol has chemopreventive properties as well, acting through the inhibition of COX-2 expression and the suppression of NF-kB DNA binding activity in mouse skin.50, 51 Also, vitamin C has been found to reduce the risk of gastrointestinal cancers.52 Similarly, these preventive properties of antioxidants are also of great interest during the maternal period. In a study with female offspring from dams fed an n-3 PUFA-rich diet during pregnancy and lactation, the breast cancer risk was lower than in those fed an n-6 PUFA-rich diet.53 In another study with a maternal diet enriched with blueberries, enhanced mammary differentiation status in the prepubescent female offspring rats was observed, which could also attenuate breast cancer risk.54 The mechanisms responsible for this preventive action are studied by nutriepigenomics. That is the study of nutrients and their effects on health status through epigenetic modifications,55 which can be due to DNA methylation, histone modifications, and RNA interference. In cancer, global hypomethylation, the downregulation of miRNAs, the hypermethylation of some specific promoters, histone deacetylation, and the upregulation of epigenetic machines are frequent in neoplastic tissue and also cause damage to other host tissues.56 Adding to this, which can be related to the mechanistic process of chemoprevention, the epigenetic modifications are associated with gene silencing, including the detoxifying enzymes due to acetylation or methylation of proteins such as NF-kB and HSP90 chaperone.57 Moreover, it has been observed that epigenetic modifications led by the maternal diet providing fetal nutrition are associated with cancer predisposition and development.58 In this way, the branched-chain amino acid leucine acts as a cell signaling , stimulating the protein synthesis, and also providing the fuel molecule—acetyl-CoA—related to the production of ATP, by tricarboxylic acid cycle. In cancer cachexia, nutritional supplementation can be an essential therapeutic approach, acting as a coadjutant to the clinical treatment as leucine is an important molecule to evaluate its effects on the impaired muscle protein turnover. In order to modify the composition of the maternal diet by altering the metabolic responses of offspring to the nutrients, it is necessary to consider the capacity of these nutrients to cross the placental barrier and alter the breast milk composition. The maternal leucine supplementation can provide some benefit because this amino acid can cross the placental barrier actively, altering its serum concentration in the fetus.4 Also, this amino acid can alter breast milk composition and influence the offspring through breastfeeding.59 Another common nutrient supplemented in the maternal diet is PUFA omega-3, which can influence the triglyceride and essential fatty acid availability to the fetus and neonates rats. It depends on the placental selectivity, which is related to placental membrane receptors, to deliver to fetal circulation. Also, the eicosapentaenoic and docosahexaenoic acid content in maternal milk is also correlated to the mother’s nutritional lifestyle.60 Studies indicate that the high consumption of omega-3 in relation to omega-6 throughout life can reduce tumor growth, attenuating the cachexia state and increasing the survival rate of tumor-bearing rats.21, 41, 42 Considering their antioxidant properties and also their role in epigenetic markers (through DNA methylation due to their action in the one-carbon metabolism), it is interesting to associate omega-3 supplementation in the maternal diet for the prevention of diseases in adult offspring. A recent study showed positive effects of maternal nutrition, which was supplemented with leucine and/or fish oil, a rich source of omega-3, over offspring responses to cancer cachexia in adulthood. The maternal-supplemented tumor-bearing offspring showed improved survival, especially in a combination of both nutrients administrated during gestation and

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lactation. Also, an important finding was the capacity of the metabolic programming provided by these nutrients in diminishing the liver oxidative stress, and also improving the skeletal muscle mTOR signaling, probably leading to an improvement in the host responses against the disease.41, 42 Considering the importance of cancer treatment and prevention, the modification of the impaired oxidative stress responses in this condition, especially in the cachexia syndrome, needs to be carefully studied to better understand the mechanisms and interventions to counteract this condition. The maternal diet composition is essential to the mother’s response and health, and pregnancy evolution is also a key factor in the offspring’s metabolic programming, modulating the offspring responses until adulthood. The maternal administration of some nutrients to modify the offspring response against cancer needs more study to show the epigenetic mechanisms that could modify the fetal gene expression and the real efficacy and security of these compounds to the mother and child. Studies with animal models are being conducted to clarify these points, mainly focusing on specific genes and pathways involved in cancer development and progression. Expanded knowledge in this area could provide new therapies and interventions against the damage and oxidative stress impairment in cancer in the future.

Conclusion In summary, in Fig. 1, we show a schematic view of nutritional supplementation minimizing the cancer-induced damage in maternal, placenta, and fetal tissues. In addition, the maternal nutritional supplementation could also minimize the tumorinduced damage in the adult offspring host. The evidence of the leucine role in improving skeletal muscle protein synthesis and minimizing muscle degradation, associated with some metabolomic findings, is also related to the positive effects over the placenta and fetal tissues. As found in our studies and reported by other research groups, leucine is a suitable coadjuvant treatment in an experimental model of cancer during pregnancy. However, the selection of the best choice of therapy remains mostly an empirical approach because our knowledge of the biology of cancer during pregnancy is still growing.

Cancer during pregnancy Leucine - rich diet Tumour

O H3C

Leucine nutritional suplementation

OH

Positive effects in host mother carcass

CH3 NH2

Improve MTOR signaling ↑ Protein synthesis

↑ Body fat sorces

Normal foetal intra uterine growth Improved placental function decreased foetal reasorption Normal oxidative stress Antioxidant/oxidative stress balance in both placenta and foetus

Improve liver function

Maintain cardiac function

Cancer in adult offspring Tumour

Cardiac function

Maternal diet influence • Leucine and or Ω-3 Improve MTOR signaling

Lower cachexia index Increase survival rate Reduced liver oxidative stress

Improve body fat

• Ω-3/Ω-6

Maternal influence

↓ Breast cancer risk

Nutritional suplementation (Pregnancy + lactation) Adult-offspring tumor-bearing

Liver function

FIG. 1 Summary of nutritional supplementation (and maternal diet) modulating the cancer-induced damages in maternal, placenta, and fetal tissues and also in adult offspring tumor-bearing hosts.

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Summary points l l l l

l

This chapter focuses on cancer during pregnancy and nutritional supplementation. Oxidative stress usually happens in pregnancy, but is intensified by the cancer evolution. Tumor-induced damage increases the reactive oxygen species in the placenta and fetus. Nutritional supplementation with leucine and/or omega-3 minimizes the oxidative stress in the maternal host, placenta, and fetal tissues. The influence of maternal nutritional supplementation modulates the tumor-induced damage in adult offspring, minimizing the cachexia state.

Acknowledgments The authors are thankful for the financial support of CAPES (Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior), CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnolo´gico #302524/2016-9; #301771/2019-7), and FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo #2014/13334-7; #2015/21890-0; #2017/02739-4; #2017/10809-2; #2019/13937-7). The authors gratefully thank R.W. Santos for technical support and Designer A.C.Gomes Marcondes for the graphical art.

References 1. Weisz B, Schiff E, Lishner M. Cancer in pregnancy: maternal and fetal implications. Hum Reprod Update 2001;7:384–93. 2. Hepner A, Negrini D, Azeka E, et al. Cancer during pregnancy : the oncologist overview. World J Oncol 2019;10:28–34. 3. Zagouri F, Dimitrakakis C, Marinopoulos S, Tsigginou A, Dimopoulos MA. Cancer in pregnancy: disentangling treatment modalities. ESMO Open 2016;1:1–6. 4. Viana LR, Gomes-Marcondes MCC. Leucine-rich diet improves the serum amino acid profile and body composition of fetuses from tumor-bearing pregnant mice. Biol Reprod 2013;88:121. 5. Cruz B, Gomes-Marcondes MCC. Leucine-rich diet supplementation modulates foetal muscle protein metabolism impaired by Walker-256 tumour. Reprod Biol Endocrinol 2014;12:2. 6. Cruz BLG, da Silva PC, Tomasin R, et al. Dietary leucine supplementation minimises tumour-induced damage in placental tissues of pregnant, tumour-bearing rats. BMC Cancer 2016;16:58. 7. Su Y, Wu J, He J, et al. High insulin impaired ovarian function in early pregnant mice and the role of autophagy in this process. Endocr J 2017;64:613–21. 8. Viana LRLR, Gomes-Marcondes MCC. A leucine-rich diet modulates the tumor-induced down-regulation of the MAPK/ERK and PI3K/Akt/mTOR signaling pathways and maintains the expression of the ubiquitin-proteasome pathway in the placental tissue of NMRI mice. Biol Reprod 2015;92:49. 9. Gomes-Marcondes MCC, Curi R. Consequences of Walker 256 tumour growth for the placental/foetal development in rats. Cancer Res Ther Control 1998;5:277–83. 10. Johansson ALV, Andersson TML, Hsieh CC, et al. Tumor characteristics and prognosis in women with pregnancy-associated breast cancer. Int J Cancer 2018;142:1343–54. 11. Gomes-Marcondes MCC, Cury L, Curi R. Consequences of Walker 256 tumor growth for the placental/fetal development in rats. Cancer Res Ther Control 1998;5. 12. Amant F, Vandenbroucke T, Verheecke M, et al. Pediatric outcome after maternal cancer diagnosed during pregnancy. N Engl J Med 2015;373:1824–34. 13. Ventrucci G, de Mello MA, Gomes-Marcondes MC. Effects of leucine supplemented diet on intestinal absorption in tumor bearing pregnant rats. BMC Cancer 2002;2:7. 14. Ventrucci G, Mello MAR, Gomes-Marcondes MCC. Effect of a leucine-supplemented diet on body composition changes in pregnant rats bearing Walker 256 tumor. Braz J Med Biol Res 2001;34:333–8. 15. Valcarce C, Cuezva J, Medina J. Increased gluconeogenesis in the rat at term gestation. Life Sci 1985;37:553–60. 16. Bispham J, Gopalakrishnan GS, Dandrea J, et al. Maternal endocrine adaptation throughout pregnancy to nutritional manipulation: consequences for maternal plasma leptin and cortisol and the programming of fetal adipose tissue development. Endocrinology 2003;144:3575–85. 17. Meguid MM, Pichard C. Cytokines: the mother of catabolic mediators! Curr Opin Clin Nutr Metab Care 2003;6:383–6. 18. Prentice AM, Goldberg GR. Energy adaptations in human pregnancy: limits and long-term consequences. Am J Clin Nutr 2000;71:49–51. 19. Viana LR, Canevarolo R, Luiz ACP, et al. Leucine-rich diet alters the 1H-NMR based metabolomic profile without changing the Walker-256 tumour mass in rats. BMC Cancer 2016;16:764. 20. Sandri M. Protein breakdown in cancer cachexia. Semin Cell Dev Biol 2015. https://doi.org/10.1016/j.semcdb.2015.11.002. 21. Argiles JM, Stemmler B, Lo´pez-Soriano FJ, Busquets S. Inter-tissue communication in cancer cachexia. Nat Rev Endocrinol 2018;15:9–20. 22. Viana LR, Gomes-Marcondes MCC. A leucine-rich diet modulates the tumor-induced down-regulation of the MAPK/ERK and PI3K/Akt/mTOR signaling pathways and maintains the expression of the ubiquitin-proteasome pathway in the placental tissue of NMRI Mice1. Biol Reprod 2015;92:49. 23. Viana LR, Gomes-Marcondes MCC. Leucine-rich diet improves the serum amino acid profile and body composition of fetuses from tumor-bearing pregnant Mice1. Biol Reprod 2013;88:1–8.

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24. Ventrucci G, Mello MAR, Gomes-Marcondes MCC. Proteasome activity is altered in skeletal muscle tissue of tumour-bearing rats a leucine-rich diet. Endocr Relat Cancer 2004;11:887–95. 25. Ventrucci G, Mello MAR, Gomes-Marcondes MCC. Leucine-rich diet alters the eukaryotic translation initiation factors expression in skeletal muscle of tumour-bearing rats. BMC Cancer 2007;7:42. 26. Al-Gubory KH, Fowler PA, Garrel C. The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. Int J Biochem Cell Biol 2010;42:1634–50. 27. Agarwal A, Gupta S, Sekhon L, Shah R. Redox considerations in female reproductive function and assisted reproduction: from molecular mechanisms to health implications. Antioxid Redox Signal 2008;10:1375–404. 28. Hu XQ, Song R, Zhang L. Effect of oxidative stress on the estrogen-NOS-NO-K Ca channel pathway in uteroplacental dysfunction: its implication in pregnancy complications. Oxidative Med Cell Longev 2019;2019. https://doi.org/10.1155/2019/9194269. 29. Schoots MH, Gordijn SJ, Scherjon SA, van Goor H, Hillebrands JL. Oxidative stress in placental pathology. Placenta 2018;69:153–61. 30. Gupta S, Agarwal A, Banerjee J, Alvarez JG. The role of oxidative stress in spontaneous abortion and recurrent pregnancy loss: a systematic review. Obstet Gynecol Surv 2007;62:335–47. 31. Aris A, Benali S, Ouellet A, Moutquin JM, Leblanc S. Potential biomarkers of preeclampsia: inverse correlation between hydrogen peroxide and nitric oxide early in maternal circulation and at term in placenta of women with preeclampsia. Placenta 2009;30:342–7. 32. Toledo MT, Ventrucci G, Marcondes MCCG. Cancer during pregnancy alters the activity of rat placenta and enhances the expression of cleaved PARP, cytochrome-c and caspase 3. BMC Cancer 2006;6:168. 33. Rizzo A, Roscino MT, Binetti F, Sciorsci RL. Roles of reactive oxygen species in female reproduction. Reprod Domest Anim 2012;47:344–52. 34. Toledo MT, Ventrucci G, Gomes-Marcondes MC. Increased oxidative stress in the placenta tissue and cell culture of tumour-bearing pregnant rats. Placenta 2011;32:859–64. 35. Yano CL, Ventrucci G, Field WN, Tisdale MJ, Gomes-Marcondes MCC. Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes. BMC Cancer 2008;8:24. 36. Khera A, Vanderlelie JJ, Perkins AV. Selenium supplementation protects trophoblast cells from mitochondrial oxidative stress. Placenta 2013;34:594–8. 37. Cruz B, Oliveira A, Gomes-Marcondes MCC. L-leucine dietary supplementation modulates muscle protein degradation and increases proinflammatory cytokines in tumour-bearing rats. Cytokine 2017;96:253–60. 38. Cruz B, Oliveira A, Ventrucci G, Gomes-Marcondes MCC. A leucine-rich diet modulates the mTOR cell signalling pathway in the gastrocnemius muscle under different Walker-256 tumour growth conditions. BMC Cancer 2019;19:349. 39. Nie C, He T, Zhang W, Zhang G, Ma X. Branched chain amino acids: beyond nutrition metabolism. Int J Mol Sci 2018;19. https://doi.org/10.3390/ ijms19040954. 40. Zhang QX, Ws QN. Altered expression of mtor and autophagy in term normal human placentas. Romanian J Morphol Embryol 2017;58:517–26. 41. da Miyaguti Silva NA, de Oliveira SCP, MCC G-M. Maternal nutritional supplementation with fish oil and/or leucine improves hepatic function and antioxidant defenses, and minimizes cachexia indexes in Walker-256 tumor-bearing rats offspring. Nutr Res 2018;51:29–39. 42. da Miyaguti Silva NA, de Oliveira SCP, Gomes-Marcondes MCC. Maternal leucine-rich diet minimises muscle mass loss in tumour-bearing adult rat offspring by improving the balance of muscle protein synthesis and degradation. Biomolecules 2019;9. https://doi.org/10.3390/biom9060229. 43. Palesty JA, Dudrick SJ. What we have learned about cachexia in gastrointestinal cancer. Dig Dis 2003;21:198–213. 44. Mayne ST, Playdon MC, Rock CL. Diet, nutrition, and cancer: past, present and future. Nat Rev Clin Oncol 2016;13:504–15. 45. Remely M, Stefanska B, Lovrecic L, Magnet U, Haslberger AG. Nutriepigenomics. Curr Opin Clin Nutr Metab Care 2015;18:328–33. 46. Lee HS. Impact of maternal diet on the epigenome during in utero life and the developmental programming of diseases in childhood and adulthood. Nutrients 2015;7:9492–507. 47. Mabasa L, Cho K, Walters MW, Bae S, Park CS. Maternal dietary canola oil suppresses growth of mammary carcinogenesis in female rat offspring. Nutr Cancer 2013;65:695–701. 48. Camargo CA, da Silva MEF, da Silva RA, Justo GZ, Gomes-Marcondes MCC, Aoyama H. Inhibition of tumor growth by quercetin with increase of survival and prevention of cachexia in Walker 256 tumor-bearing rats. Biochem Biophys Res Commun 2011;406:638–42. 49. Camargo CA, Gomes-Marcondes MCC, Wutzki NC, Aoyama H. Naringin inhibits tumor growth and reduces interleukin-6 and tumor necrosis factor alpha levels in rats with Walker 256 carcinosarcoma. Anticancer Res 2012;32:129–33. 50. Shukla Y, Singh M. Cancer preventive properties of ginger: a brief review. Food Chem Toxicol 2007;45:683–90. 51. Lee JK, Lee MK, Yun YP, et al. Acemannan purified from aloe vera induces phenotypic and functional maturation of immature dendritic cells. Int Immunopharmacol 2001;1:1275–84. 52. Lee KW, Lee HJ, Surh Y-J, Lee CY. Vitamin C and cancer chemoprevention: reappraisal. Am J Clin Nutr 2018;78:1074–8. 53. Su H-M, Hsieh P-H, Chen H-F. A maternal high n-6 fat diet with fish oil supplementation during pregnancy and lactation in rats decreases breast cancer risk in the female offspring. J Nutr Biochem 2010;21:1033–7. 54. Wu X, Rahal O, Kang J, Till SR, Prior RL, Simmen RCM. In utero and lactational exposure to blueberry via maternal diet promotes mammary epithelial differentiation in prepubescent female rats. Nutr Res 2009;29:802–11. 55. Tammen SA, Friso S, Choi SW. Epigenetics: the link between nature and nurture. Mol Asp Med 2013;34:753–64. 56. Mathias PCF, Elmhiri G, De Oliveira JC, et al. Maternal diet, bioactive molecules, and exercising as reprogramming tools of metabolic programming. Eur J Nutr 2014;53:711–22.

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57. Gerhauser C. Cancer chemoprevention and nutri-epigenetics: state of the art and future challenges. In: Pezzuto MJ, Suh N, editors. Natural products in cancer prevention and therapy. Berlin, Heidelberg: Springer; 2012. p. 73–132. 58. Vanden BW. Epigenetic impact of dietary polyphenols in cancer chemoprevention: lifelong remodeling of our epigenomes. Pharmacol Res 2012;65:565–76. 59. Melnik BC. Milk—a nutrient system of mammalian evolution promoting mTORC1-dependent translation. Int J Mol Sci 2015;16:17048–87. 60. Mennitti LV, Oliveira JL, Morais CA, et al. Type of fatty acids in maternal diets during pregnancy and/or lactation and metabolic consequences of the offspring. J Nutr Biochem 2015;26:99–111.

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Chapter 13

Inflammation and oxidatively induced DNA damage: A synergy leading to cancer development Ioanna Tremia, Somaira Nowsheenb, Khaled Azizb, Shankar Sivac,d, Jessica Venturae, Vasiliki I. Hatzif, Olga A. Martind, and Alexandros G. Georgakilasg a

DNA Damage Laboratory, Physics Department, School of Applied Mathematics and Physical Sciences, National Technical University of Athens (NTUA),

Athens, Greece, b Medical Scientist Training Program, Mayo Graduate School, Mayo Clinic, College of Medicine, Rochester, MN, United States, c

Department of Radiation Oncology, Peter MacCallum Cancer Centre, The University of Melbourne, Melbourne, VIC, Australia, d Sir Peter MacCallum

Department of Oncology, The University of Melbourne, Melbourne, VIC, Australia, e Department of Obstetrics & Gynaecology, The University of Melbourne and Royal Women’s Hospital, Melbourne, VIC, Australia, f Laboratory of Health Physics & Environmental Health, Institute of Nuclear Technology & Radiation Protection, National Center for Scientific Research “Demokritos”, Athens, Greece, g DNA Damage Laboratory, Physics Department, School of Applied Mathematics and Physical Sciences, National Technical University of Athens (NTUA), Athens, Greece

List of abbreviations AG AID BER COX2 c-PTIO CRT CSFS DAMPs DC DDR DMSO DSB EGFR HIF HMGB1 IBD ICAM IFN-g IL IR JNK L-NNA MDSC MHC MIF 1 NADP NER NFkB NO NSCLC

aminoguanidine activation-induced cytidine-deaminase base excision repair cyclooxygenase 2 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide calreticulin colony-stimulating factors damage associated molecular patterns dendritic cell DNA damage response dimethyl sulfoxide double strand break epidermal growth factor receptor hypoxia-inducible factor high-mobility group-protein B1 inflammatory bowel diseases intercellular adhesion molecule interferon gamma interleukin ionizing radiation c-Jun N-terminal kinase N(omega)-nitro-L-arginine myeloid-derived suppressor cells major histocompatibility complex macrophage migration inhibitory factor nicotinamide adenine dinucleotide phosphate nucleotide excision repair nuclear factor kappa beta nitric oxide nonsmall cell lung cancer

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00013-4 © 2021 Elsevier Inc. All rights reserved.

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OCDLs PTC PTEN RNS ROS SABR SASP STAT TAM TGFb TLR4 TNF VHL

A Oxidative stress and cancer

oxidatively induced clustered DNA lesions papillary thyroid carcinoma phosphatase and tensin homologue reactive nitrogen species reactive oxygen species stereotactic ablative body radiotherapy senescence-associated secretory phenotype signal transducer and activator of transcription tumor associated macrophages transforming growth factor-b toll-like receptor 4 tumor necrosis factor von Hippel-Lindau

Introduction Cancer is a very real threat to people of all ages and despite decades of research we have failed to conquer the disease. Oncogenic transformation is due to the accumulation of various mutations, whether acquired or inherited and caused by endogenous and/or exogenous agents. These bestow pro-survival capacities to the transformed cells and often allow them to evade and modify the immune response. Multiple genes in the human body precisely control cell growth. Errors in these genes lead to further alterations or mutations. Accumulation of many mutations over time usually leads to a malignant state manifested by high chromosomal instability. The human body is under continuous attack from both external and internal insults which results in numerous DNA lesions per cell per day (10,000
100,000).1 These lesions can block DNA replication and transcription leading to mutations and possibly transformation and carcinogenesis. Just one unrepaired double-strand break (DSB) can be lethal to the cell or highly mutagenic. Failure to repair any DNA damage leads to apoptotic or necrotic cell death. The DNA damage response (DDR) network detects DNA lesions, signals their presence, and promotes DNA repair. Defects in this pathway are often seen in cancer. DNA damage can be induced by oxidation and this may eventually progress to carcinogenesis. In addition, cancer is considered a pro-inflammatory disease and a number of current therapies target this pro-inflammatory state within the tumor microenvironment. Thus, in this chapter, we discuss the role(s) of oxidatively induced DNA damage and inflammation in cancer. Overall, a better understanding of the synergy between oxidative DNA damage, inflammation, and cancer, that is, a “lethal triptych” will provide the center for future therapies.

Oxidative DNA damage Oxidative DNA damage is an inevitable consequence of endogenous and exogenous events, such as cellular metabolism and toxic insults such as exposure to chemicals or IR. Oxidative stress has been associated with various serious diseases including cancer, Alzheimer’s, arteriosclerosis diabetes, gastrointestinal disorders, and aging. Oxidative damage occurs when the body is exposed to excessive amounts of electrically charged, aggressive oxygen and nitrogen compounds: reactive oxygen and nitrogen species (ROS, RNS). Whether endogenous or exogenous, these compounds can modify major cellular components such as DNA but also proteins, lipids, and carbohydrates. The effects of free radicals on lipids and lipid components have been thoroughly studied until recently.2, 3 Focusing on the DNA, purine and pyrimidine bases and sugar moieties can be affected by oxidation. Oxidatively induced DNA lesions, multiple DNA lesions in close proximity (clusters or OCDLs), play a critical role in carcinogenesis mainly due to their repair resistance.4 DNA-protein crosslinks can also result from oxidation. Though oxidative modifications occur in proteins, lipids, and DNA, since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is the modifications to the DNA, which can cause mutations and lead to genomic instability (Fig. 1).

Mechanisms of induction Oxidation is a critical component of energy production by mitochondria, the inflammatory response and, in general, by the cellular defense system. Acute inflammatory response recruits activated leukocytes that can cause extensive DNA damage by secreting various chemical mediators. Some of the oxygen-derived products include hydroxyl radical and superoxide

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FIG. 1 Persistent oxidative stress leads to neoplastic transformation. The key steps are the production of reactive oxygen and nitrogen species (ROS/ RNS) which can lead to the accumulation of mutations and therefore preneoplastic states. In this first initiation step, components of the immune system seem to play a pivotal role through their recruitment in these damage/mutation sites. The occurrence of a tissue malignancy seems always to coincide with genomic and chromosomal instability.

radical. The hydroxyl radical reacts with biological molecules such as DNA, causing damage to the heterocyclic DNA bases and the sugar moiety by a variety of mechanisms. Hydroxyl radical reacts with purines and pyrimidines of DNA by addition to double bonds and by the abstraction of an H from the methyl group of thymine and from each of the CdH bonds of 20 -deoxyribose leading to modifications.5 This oxidative stress can also lead to DSBs. In order to cope with the oxidation damage cells, use several defense compounds such as antioxidants, antioxidant enzymes, and DNA repair mechanisms.

Pathways of repair Oxidative DNA damage is repaired by multiple, overlapping DNA repair pathways. Two major mechanisms exist to repair oxidatively induced DNA lesions: base-excision repair (BER) and nucleotide-excision repair (NER). In BER-mediated repair, DNA glycosylase usually detects the damaged base and mediates base removal prior to nuclease, polymerase, and ligase proteins bridging the gap and completing the repair process. On the other hand, NER-mediated repair recognizes base lesions that distort the helical structure. The damaged base is excised as a 22–30 base oligonucleotide resulting in single-stranded DNA that is repaired by proteins such as DNA polymerase before proceeding to ligation. There are two pathways that differ in the mechanism of helix recognition: transcription-coupled NER specifically targets lesions that transcription while global-genome NER covers the other lesions. Other repair pathways include mismatch repair, nonhomologous end joining, and homologous recombination all of which repair DSBs.6

Role of inflammation in the induction of oxidative stress and DNA damage leading to cancer Inflammation is a key component of the tumor microenvironment and a recognized hallmark of cancer.7, 8 The causal linkage between inflammation and cancer was initially suggested in the 19th century following the observation that tumors often developed in settings of chronic inflammation and that pro-inflammatory cells were present in biopsied tumor specimens.9 Accumulating evidence shows that chronic inflammation is, in fact, associated with an increased risk of cancer development. Moreover, chronic inflammation is linked to between 15% and 20% of worldwide cancer deaths.8 The interlink between inflammation and cancer involves two major pathways which are interconnected: an extrinsic mechanism, where a constant inflammatory state (chronic inflammation) contributes to increased cancer risk; and an intrinsic mechanism, where genetic events (e.g., oncogenes) induce neoplastic transformation triggering the inflammatory cascade.8 The relationship between cancer and inflammation is discussed in detail below and is summarized in Figs. 1–4.

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Extrinsic pathway of carcinogenesis Inflammatory or infectious conditions can increase cancer risk via the extrinsic pathway. Leukocytes producing inflammatory mediators are primarily responsible for triggering inflammation. Chronic inflammation can be induced by, among other sources, chronic infections, exposure to noxious agents that trigger inflammation (e.g., gastric acid reflux, tobacco, asbestos, and other chemicals) and autoimmune conditions.8 Due to the presence of ROS and RNS different mutagenic lesions may occur such as 8-oxodG and 8-nitroguanine, with the second being most common between various types of inflammation-related carcinogenesis.10 Pathogenic infections such as those due to Hepatitis B and C viruses, human papillomavirus (HPV) or Helicobacter pylori results in chronic inflammation that favors initiation and progression of tumors.11 The formation of 8-oxodG at cancer sites of patients with these diseases has been strongly indicated.12 In all cases, the production of DNA damage and the accumulation of mutations and epigenetic changes is considered critical. Autoimmune diseases such as inflammatory bowel disease for colon cancer and prostatitis for prostate cancer, mechanical, radiation, and chemical insults can also induce inflammation associated with human malignancy (Fig. 4). The role of the tumor microenvironment (stroma) is being increasingly appreciated as being a critical part of carcinogenesis. Inflammatory cells are an important component of the stroma and milieu fosters proliferation, survival, and migration.13 Figs. 2, 4, and 5 illustrate how chronic inflammation may contribute to carcinogenesis. Chronic inflammation promotes the development of blood vessels and the remodeling of the extracellular matrix fostering the perfect environment in which a mutation bearing normal cell can turn potentially malignant. In addition, immune cells like neutrophils and macrophages produce ROS via a plasma membrane-bound nicotinamide adenine dinucleotide phosphate (NADP). Based on in vitro and in vivo data, ROS and RNS that play a vital role in normal cellular metabolism

FIG. 2 Inflammatory pathways lead to cancer. As explained in the text the major events and sources contributing to persistent inflammation can be of extrinsic or intrinsic nature. The two pathways can be identified as major contributors to the inflammatory milieu: the intrinsic pathway where genetic events (e.g., mutations in oncogenes) induce neoplastic transformation triggering the inflammatory cascade and the extrinsic pathway where chronic inflammation (e.g., infections and low doses of IR) significantly increases the risk for different types of cancer. The two pathways converge, resulting in the activation of transcription factors (e.g., NFkB, STAT3, HIF) that coordinate the production of inflammatory mediators and the activation of various leukocytes generating an inflammatory microenvironment that nurtures cancer progression. The resulting activation of several transcription factors, inflammatory cells, and chemical mediators like cytokines has been closely bonded to the creation of uncontrolled cell proliferation, mitigation of apoptosis, abnormal angiogenesis, and other molecular changes leading to cancer. The production of DNA damage is considered a critical step. In addition, a high inflammatory response has been also related to tumor cell migration and metastatic ability.

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FIG. 3 Signaling of the inflammatory pathways leading to tissue abnormal changes. Aberrant signaling can lead to increased angiogenesis, cell proliferation, and invasion which, in turn, can lead to abnormal growth premalignant and finally malignant states.

FIG. 4 Overview of pathways leading to cancer. As described in the text, the association of inflammation with the generation of ROS/RNS and DNA damage induces several relating pathways of DNA damage response (DDR) like signaling and induction of DNA repair proteins, cell cycle arrest, and proliferation changes. In every step, there are two major safeguarding mechanisms and these are DNA repair and apoptosis assuming they work properly. A premalignant state will be characterized by loss of all these control mechanisms due to the accumulation of mutations, epigenetic changes, and finally genomic and chromosomal instability.

are inflammation-generated mediators of DNA damage. An increase in oxidative stress leads to a spike in ROS/RNS formation.14 These highly reactive species can easily bind to proteins, lipids, and DNA. Since proteins and lipids are usually turned over, damage to these macromolecules is usually not detrimental to the cell. However, damage to the DNA can lead to cancer.4 Interestingly, tumor promoters are able to recruit inflammatory cells and stimulate them to generate ROS/RNS which, in turn, generate DNA lesions and lead to mutations. Numerous reports suggest that tumor growth in vivo, inflammation, and OCDLs are interconnected. Moreover, tumors can induce their DNA damaging effects in distant tissues and organs.15 Since clustered DNA lesions (both DSBs and OCDLs) are highly mutagenic, these results are biologically relevant.16 Reports also suggest that OCDL scan is induced by a cytokine CCL2-based mechanism.17 Researchers are actively pursuing these avenues of inflammation-induced carcinogenesis.

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Proliferation Migration Invasion

Chronic inflammation/ irritation

Bacteria

Macrophages Lymphocytes

DNA

Tumor

Neutrophils

ROS/RNS

Adhesion Cell death Growth suppressors Immune destruction FIG. 5 An overview of the suggested interplay between inflammation and associated oxidative stress. This interplay can affect key aspects of tumorigenesis, angiogenesis, and metastasis. In this model, the generation of oxidative stress and potentially DNA damage through the inflammatory responses involving macrophages and different cytokines like the MCP-1/IL-6 are considered critical.13

Several recent reports support the involvement of ROS in cancer-related processes. For instance, ROS production has been demonstrated to be required for mediating K-ras-induced lung cancer in mice.18 Moreover, ROS released by damaged cells can induce inflammation and trigger the production of pro-inflammatory cytokines by functioning as signaling molecules. New molecular pathways involving mitochondrial damage and ROS production are being actively investigated. These not only play a significant role in DNA damage and activation of oncogenes but also in different aspects of inflammation. This suggests that ROS play an important role in the promotion of inflammation and tumorigenesis by modulating cancer-related signaling pathways. Clinical data indicate that chronic inflammation promotes carcinogenesis. For instance, patients with inflammatory bowel diseases (IBD, ulcerative colitis, and Crohn’s disease) have five- to sevenfold increased risk of developing colorectal cancer. Alarmingly, 43% of patients with ulcerative colitis develop colorectal cancer after 25–35 years.19 Chronic airway inflammatory conditions such as asbestosis, silicosis, exposure to airborne particulate matter, idiopathic pulmonary fibrosis, and tuberculosis have been reported to trigger nonsmoking related cancer development.20 Another form of lung disease is mesothelioma which is caused by exposure to asbestos and asbestos-induced chronic inflammation, and subsequent production of ROS and DNA damage. Chronic inflammation also leads to gastric cancer. Aberrant expression of activation-induced cytidine-deaminase (AID), a member of the cytidine-deaminase family that acts as a DNA- and RNA-editing enzyme, is induced by H. pylori and is observed in this malignancy.21H. pylori-mediated upregulation of AID results in accumulation of nucleotide alterations in gastric cells which ultimately leads to the development of gastric cancer.21 Moreover, tumor necrosis factor (TNF) stimulation in human bile-duct cells induces ectopic AID production, which results in chronic biliary inflammation and the development of cholangiocarcinoma. Therefore, AID may be the link between chronic inflammation and DNA damage in these tumors. Oxidative DNA damage and inflammation is also implicated in schistosomiasis,22 lung, liver, and breast cancers. Elevated levels of DNA damage is seen upon urine analysis of patients with schistosomiasis.22 The cells are more prone to DNA damage induced by the ROS/RNS produced by activated inflammatory cells. This, in turn, leads to an increased risk for bladder cancer in adults.22 Chronic infection with hepatitis B or C viruses or ingestion of aflatoxin that causes ROS and subsequent DNA damage production leads to hepatocellular carcinoma and is considered as a significant cause of cancerassociated mortality in Asia and Africa.23 Oxidative DNA damage may be involved in the development of breast cancer as well. Increased steady-state levels of DNA damage and ROS have been reported in invasive ductal carcinoma.23 Whether the changes are due to decreased DNA repair and/or increased oxidative DNA damage remains to be confirmed.

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Intrinsic pathway The intrinsic pathway is induced by genetic events such as activation of various types of oncogenes by mutation, chromosomal rearrangement or amplification, and the inactivation of tumor-suppressor genes. These cells produce inflammatory mediators which creates an inflammatory microenvironment in tumors without prior underlying inflammatory condition.24 Results reported in the literature show that various oncogenic mechanisms are involved in cancer-related inflammation pathways8 (Figs. 2 and 3). For example, inhuman papillary thyroid carcinoma (PTC), a tumor characterized by the presence of chemokine-guided macrophage and dendritic cell infiltration, rearrangements in the protein tyrosine kinase RET play a key role in the pathogenesis.25 One of the signaling molecules in PTC colony-stimulating factors (CSFS) promotes leukocyte recruitment and survival. Interleukin 1b (IL-1b) is also secreted and is one of the main inflammatory cytokines. Cyclooxygenase 2 (COX2) is frequently expressed in cancer and is involved in the synthesis of prostaglandin E(2) which can promote tumor growth by binding its receptors and activating signaling pathways which regulate cell proliferation, migration, apoptosis, and angiogenesis. Chemokines attract monocytes and dendritic cells leading to secretion of CCL2 and CCL20 and, as expected, these molecules have been reported to be pro-tumorigenic. Angiogenic chemokines such as CXCL8 coordinate induction and inhibition of matrix-degrading enzymes which promote tumor progression and survival. Upregulation of L-selectin and expression of the chemokine receptor CXCR4 that promote metastasis are also observed in these cells.8, 25 Thus, an early, causative and sufficient genetic event promotes an inflammatory microenvironment which, in turn, leads to tumor formation. The inflammatory cascade and tumor progression can be triggered by the activation of oncogenes or inactivation of tumor suppressors. For example, in the ras family oncogenes activation induces expression and production of inflammatory mediators. Expression of ras in a cervical carcinoma cell line induces the production of CXCL8 which promotes angiogenesis and tumor progression.8 Moreover, mild chronic pancreatitis and K-ras mutation induce pancreatic intraepithelial neoplasia and invasive ductal carcinoma.26 Similarly, Braf, which is frequently activated in malignant melanoma, induces cytokines which create a pro-tumorigenic microenvironment.27 The myc oncogene encodes a transcription factor that is overexpressed in many human tumors. Deregulation of myc is important in the initiation and maintenance of key aspects of the tumor phenotype. In association with inflammatory cells and mediators, myc promotes cell proliferation and remodeling of the extracellular milieu. Myc-mediated alterations include secretion of chemokines which recruit mast cells and help sustain the formation of new vessels and tumor growth.8 The epidermal growth factor receptor (EGFR) family plays an important role in cancer. EGFR activation in glioma induces COX2 expression which is involved in the synthesis of prostaglandin E(2) which can promote tumor growth by binding its receptors and activating signaling pathways which regulate cell proliferation, migration, apoptosis, and angiogenesis. COX2 expression is an independent prognostic factor in glioma. Production of inflammatory mediators can also be regulated by tumor suppressor proteins such as von Hippel-Lindau/ hypoxia-inducible factor (VHL/HIF), transforming growth factor-b (TGF-b) and phosphatase and tensin homologue (PTEN). The chemokine receptor CXCR4 is frequently expressed on malignant cells and has been implicated in cell survival and metastasis. CXCR4 and TNF-a lie downstream of the VHL/HIF axis in human renal-cell carcinoma.8 Mutation of PTEN in nonsmall cell lung cancer (NSCLC) results in upregulation of HIF-1 activity and subsequent HIF-1-dependent transcription of the CXCR4 gene. CXCR4 regulates migration of lung cells through activation of Rac1 and matrix metalloproteinases. CXCR4 also modulates the action of ERK, IKK, NFkB, and integrins which promote metastasis of the lung cancer.28 Data from breast carcinoma suggests that inactivation of the gene encoding the type II TGF-b receptor stimulates the production of CXCL5 and CXCL12, which draws myeloid-derived suppressor cells (MDSC). CXCL5 induces Raf/ MEK/ERK activation, Elk-1 phosphorylation, and Snail upregulation. Activation of Elk-1 facilitates recruitment of phosphorylated mitogen- and stress-activated protein kinase 1, which in turn enhances histone H3 acetylation and phosphorylation of Snail promoter, resulting in Snail enhancement and E-cadherin downregulation. This facilitates metastasis of breast cancer.8, 29 Thus, oncogenes and tumor suppressor genes can induce inflammation.

The link between extrinsic and intrinsic pathways Some of the molecules involved in both the intrinsic and extrinsic pathways include transcription factors, such as nuclear factor-kB (NF-kB), signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor 1a (HIF1a).30 These transcription factors modulate the inflammatory response and promote tumorigenesis via soluble mediators including cytokines for example, IL-1, IL-6, and IL-23, chemokines CCL2 and CXCL8, and other cellular components, for example, tumor-associated macrophages.8 These factors recruit and activate various leukocytes, mainly of

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myelomonocytic lineage. The cytokines also activate key transcription factors in various cell types such as inflammatory, stromal, and tumor cells. This results in a cascade where even more inflammatory mediators are generated and a cancerrelated inflammatory milieu is created.24 NFkB is a key transcription factor that potentially is a link between tumor cells and inflammatory cells. NFkB has a variety of roles including facilitating proliferation and survival of malignant cells by activating genes that regulate cell cycle progression (e.g., cyclin D and c-myc) and apoptosis (e.g., cIAPs, A1/BFL1, Bcl2, c-Flip), promoting angiogenesis and metastasis, disrupting adaptive immunity, and altering responses to hormones and chemotherapeutic agents.8 In this respect, NFkB induces the expression of inflammatory cytokines such as key enzymes in the prostaglandin synthase pathway (COX2), adhesion molecules, nitric oxide (NO) synthase, and angiogenic factors which promote inflammation as well as tumorigenesis. Hepatocarcinogenesis is substantially reliant on NFkB activation in both parenchymal (hepatocytes) and nonparenchymal cells of the liver.8 STAT3 is also implicated in both extrinsic and intrinsic pathway.30 Constitutively activated STAT3 increases tumor cell proliferation, survival and invasion, and subdues antitumor immunity. Persistent activation of STAT3 leads to inflammation which promotes tumor formation. This dual role of STAT3 in tumor inflammation and immunity involves upregulation of pro-oncogenic inflammatory pathways, including NFkB and IL-6-gp130-JAK pathways, and downregulation of STAT1 and NFkB-mediated Th1 antitumor immune responses.

Soluble mediators and cellular components Inflammation is sustained by molecules such as TNF-a. Tumor-derived TNF-a supports the growth and development of skin, pancreatic, liver, and bowel tumors.31 Constitutively produced TNF-a is associated with increased release of chemokines such as CCL2, CXCL12, CXCL8, CXCL1, CXCL13, CCL5, CCL17, and CCL22, IL-1, IL-6, VEGF, and macrophage migration inhibitory factor (MIF-1).8 Tumor-associated macrophages (TAM) represent the major inflammatory component of the stroma of many tumors and affect different aspects of the tumor.8 TAM accumulation has been reported to promote angiogenesis via production VEGF and platelet-derived endothelial cell growth factor.32 Moreover, myeloid cells in the tumor milieu also play a role as an angiogenic switch at different levels. Mast cells, eosinophils, neutrophils, and effectors of the adaptive immune response are capable of tolerating the inflammatory reactions that lead to cancer.

Tissue injury There are significant overlaps between key features of wound healing and tumor development. These include stem cell and myofibroblast activation, enhanced cell proliferation, inflammation, and neoangiogenesis. Chronic injury results in an aberrant healing and regenerative response that ultimately stimulates the growth and development of initiated cells. Indeed, in the initial phase, the body interprets tumors as wounds and similar to healing tissues, activated platelets are present in tumors. This phase of tumor growth is governed by the actions of the stroma which is similar to physiologic tissue repair.4 However, during late tumor growth, the tumor becomes independent of stromal signaling for progression and survival. So far we have focused on the interaction of extrinsic and intrinsic mechanisms of inflammation and their role in the induction of carcinogenesis. It should be noted though that the inflammatory response is also critical in other aspects of tumor progression as well such as tissue invasion and metastasis. Angiogenesis significantly augments vascular invasion of migrating cells. Matrix metalloproteases and their inhibitors are essential for angiogenesis and remodeling of the extracellular matrix. Similar to cancer, cell proliferation is enhanced in a wound which results in tissue regeneration. However, unlike cancer, cell proliferation and inflammation subside after the foreign particle is removed or the repair is complete.4 Chronic tissue damage and inflammation can indeed promote the growth and progression of cancer. For instance, v-Src oncogene cannot induce cancer unless supplemented by tissue injury and ensuing tissue renewal.33 Similarly, pancreatic insult is required to unravel the oncogenic potential of activated K-Ras. Finally, tissue injury inflicted by tobacco smoke influences lung cancer development, and suppression of cell death-related pathways (c-Jun N-terminal kinase and JNK) or death-induced pro-inflammatory cytokines (TNF and IL-6) reduces tumor development. Taken together, these results support the notion that a substantial fraction of all cancer cases is likely to be initiated and promoted by chronic tissue injury. Given that persistent inflammation promotes genetic instability,8 targeting cancer-related inflammation is a possible treatment strategy that can minimize normal tissue injury. The global inflammation that prevails in cancer can be targeted to restore normal tissue homeostasis and, perhaps, can be used in cancer prevention.

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Nontargeted effects, inflammation, oxidative stress, and DNA damage Bystander and abscopal effects The term “bystander” effect was first used in radiation biology to explain the results obtained in cell cultures irradiated with a-particles (energetic helium nuclei with the short range of absorption which can be produced by cyclotrons or synchrotrons). Although only a few cells were traversed by a-particles, many more exhibited sister chromatid exchanges, indicating that nontargeted cells also sustained damage.34 Subsequently, the term has been used in various scenarios to describe the ability of cells affected by an agent to convey manifestations of damage to other cells not directly targeted by the agent or necessarily susceptible to it themselves.35 These indirectly affected, bystander cells exhibited various types of genomic destabilization such as altered clonogenic survival, changed the frequency of gene mutations, induction of apoptosis and micronuclei, altered expression of stress-related genes, elevated frequencies of malignant transformation of mammalian cells in vitro, altered DNA damage and repair and senescence arrest, and various epigenetic changes36 (reviewed in Refs. 35, 37). All these indirect (systemic) consequences in bystander cells are delayed effects, and though similar to the direct effects of radiation, can follow different kinetics. For instance, elevated levels of phosphorylated histone H2AX (g-H2AX) have been found in both irradiated and bystander cultures indicating the presence of DNA DSBs.38, 39 In contrast to direct effects of radiation, bystander effects show no true radiation dose response40 and are detectable at doses as low as 10 mGy.41 The nonlinear dose-response was first demonstrated in vitro34, 42 and recently in an in vivo study involving various synchrotron radiation settings including different doses and irradiation field sizes.43 The results of this study indicated that radiation settings did not substantially influence the persistent biological effects observed in out-offield tissues following synchrotron irradiation of the right hind leg of mice. Not all cell types and not every cell in a bystander culture are equally responsive.44 For example, rapidly proliferating cancer cells in culture and, generally, proliferating cells in S-phase appear to be highly susceptible.45 However, other data suggest that this is not the only factor determining bystander vulnerability.46 In such cells, ROS generated directly or indirectly as a result of cell damage interact with bystander cell DNA, producing lesions ranging from base or sugar modifications to abasic sites and single-stranded breaks. ROS-induced DNA damage can interfere with both replication and transcription in proliferating cells and transcription in nonproliferating cells, leading to DSB formation. Bystander effects have been noted in response to a number of cellular stresses including UV exposure and nonradiation sources of cellular damage such as media from tumor and aging cells.46 Therefore, bystander effects can be generalized as an overall cell population response to the presence of cells undergoing stresses of various types.47 Of particular interest in regards to human health are the nontargeted or systemic effects that have been reported by radiation oncologists for decades—reactions in normal unirradiated tissues after radiation therapy of a particular part of the body. These out-of-field or abscopal effects have been described as an action at a distance from the irradiated volume but within the same organism.48 Abscopal effects have been also shown to be a general phenomenon; they result from a number of other localized stimuli, for example, surgery, hyperthermia, and laser immunotherapy.37 The discovery of the radiation-induced bystander effect has prompted the description of abscopal effects as distant in vivo bystander effects, and their further investigation. Several studies reported in vivo radiation-induced bystander effects in animal models. Using strategies that involve partial head or body 1 Gy X-ray irradiation, profound genetic and epigenetic changes were identified in shielded organs, such as skin and spleen.49, 50 The results of the in vivo RIBE can be transmitted to future generations. The radiation-induced bystander effect is a potential contributor to the well-documented clinical phenomenon of secondary cancers,51 a major concern in cancer RT, affecting more than 1% of patients.37 The frequency of secondary malignancies that arise as a function of distance from the irradiated area,52 and irradiation volume and doses53 have been investigated. Typically at distances closer to the irradiated area (at least 5 cm) 22% of tumors arise,52 while primary tumors that receive doses of less than 2.5 Gy were shown to be associated with the development of 31% secondary malignancy outside the irradiated area.53 A recent review compiled a list of inhibitors that have been shown to minimize or completely abrogate bystander effects, which could ultimately curb the manifestation of radiation-associated secondary cancers.54 These inhibitors interrupt well-known mechanisms that drive bystander effects and can be categorized into three main groups, (1) inhibitors of intercellular gap junction communication, (2) detoxifiers of reactive species, and (3) agents with antiinflammatory properties.54

Bystander signaling in vitro Two major mechanisms have been identified as playing a role in the transmission of bystander responses in cellular models, physical contact, via gap junction mediation, and soluble factors, such as ROS, NO, and cytokines often referred as Damage Associated Molecular Patterns (DAMPs) (reviewed in Ref. 37). DAMPs, also known as alarmins, are molecules released by

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stressed (damaged) cells undergoing apoptosis or necrosis that act as endogenous danger signals to promote and intensify the inflammatory response. Some of the most well-known DAMPs are high mobility group box-1 (HMGB1), S100A8 (MRP8 and calgranulin A) and S100A9 (MRP14 and calgranulin B), and serum amyloid A (SAA). High serum levels of these DAMPs have been associated with many inflammatory diseases, including sepsis, arthritis, atherosclerosis, lupus, Crohn’s disease, and cancer. Therapeutic strategies are being developed to modulate the expression of these DAMPs for the treatment of these diseases. For the bystander signaling, in the absence of gap junctions (e.g., in the medium transfer experiments), several studies found suppression of bystander responses when various inhibitors and ROS and NO scavengers were added to the media of donor cells and recipient cells. The use of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) as a NO-specific scavenger, the incidence of bystander micronuclei yields reduced, which indicated that NO contributes to the bystander effect.55 Various inflammation-related cytokines that have been found at elevated levels in medium conditioned by irradiated cells. Notably, stress events other than IR, such as UVC, UVA, and unirradiated cancer cells release similar to irradiated cultures cytokines in the culture medium.46 They can target bystander cells directly; cytokine TGF-b, when added to cell cultures, induces elevated levels of DSBs similar to those induced by the conditioned medium, and addition of the blocking anti-TGF-b antibody reversed the effect.46, 56 Indirectly, through activation of cytokine receptor-mediated pathways, bystander cells also start expression and production of IL-8, IL-6, IL-33, RGE2, and other factors.52 When gene expression profile was compared between unirradiated and bystander normal human fibroblasts, the transcription level of COX-2 was found consistently upregulated in bystander cells by more than threefold. Addition of COX-2 inhibitor NS-398 suppressed COX-2 activity and decreased bystander mutagenesis.57 Recently several publications addressed the role of the stimulatory neurotransmitter 5-hydroxytryptamine (serotonin) which is a serum component in a cell culture medium, on the radiationinduced bystander effect. Some publications report a trend for increasing bystander response with increasing serotonin concentration, while others found no effect.58–60

Role of cytokines for bystander signaling The cell-cell communication in vivo is mediated by the immune system and is more complex. Recently, the abscopal DNA damage response in normal tissues has been described which was influenced by early-stage tumors growing in mice.61 The presence of a tumor has been shown to induce inflammatory and DNA damage responses in the immediate tumor microenvironment, possibly due to the production of ROS and cytokines, similar to the radiation-induced bystander effect signaling. Results obtained in cell culture indicate that tumors could influence normal cell cultures; normal cells sustain elevated levels of DNA damage when incubated with medium previously conditioned on tumor cells, and similar cytokines released into the medium of unirradiated tumor and irradiated normal cells.46 Syngeneic tumors (B16 melanoma, reticulum cell sarcoma, and colon adenocarcinoma) were implanted subcutaneously into mice, and 2 weeks later, the levels of two types of DNA damage in different tissues were measured. Elevated levels of DSBs, as marked by g-H2AX foci, and OCDLs, were present in several distant tissues, such as duodenum, colon, stomach, rectum, and skin.15 Both DSBs and OCDLs are potentially serious lesions and lead to genome instability if not fully repaired.16 Ovary and lung did not exhibit elevated g-H2AX foci, but had elevated OCDL levels. This wider incidence of elevated OCDLs versus g-H2AX foci may be attributable to the mechanisms of lesion formation. g-H2AX foci form in tissues with larger fractions of proliferating cells, such as those in the gastrointestinal tract, in which replication forks may participate in DSB formation, while OCDLs form equally well in every tissue. Out of 59 cytokines measured in mouse serum, CCL2/MCP-1, CCL7/MCP-3, and CXCL1/IP-10, were over threefold elevated in tumor-bearing mice. Elevated numbers of activated macrophages were found in gastrointestinal tract organs and skin. This suggested that macrophages in these distant tissues secrete ROS that induces DNA damage in the cells of the host organs. This also substantiated an association between inflammation and bystander DNA damage responses in these tumor-bearing mice. Interestingly, this systemic oxidative DNA damage in normal tissues both neighboring to and distant from injected tumors can be abrogated by feeding mice with the antioxidant tempol-rich food suggesting that the endogenous antioxidant systems could be efficiently boosted by a well-designed antioxidant therapy to suppress the oxidative load in the organism.62 The role of CCL2 in cancer development has been controversial, with evidence of pro- and antitumorigenic effects. Recent studies suggest that CCL2 contributes to cancer growth.63, 64 There is mounting evidence linking inflammation and cancer. ROS are secreted from activated immune cells and stressed epithelial cells, resulting in DNA damage and genomic instability that may contribute to carcinogenesis.65–67 In a study of patients treated with radiation therapy for nonsmall cell lung cancer, accumulation of unrepaired DNA damage in out-of-fields normal tissues was associated with changes in CCL2 plasma levels,68, 69 which also were associated with high-grade lung toxicity.69 To examine whether the association between CCL2 and tumor-induced bystander DNA damage was causal, the tumors were implanted into CCL2 knockout mice.

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Strikingly, there was no measurable increase in distant DNA damage in the tumor-bearing CCL2 KO mice, suggesting that CCL2 is essential in the tumor-induced genotoxic response in vivo.15 The proposed model states that the bystander DNA damage in the tumor-bearing mice is due to the presence of activated macrophages in the distant tissues.61 These activated macrophages at the irradiation site of injured tissue secrete CCL2 and TGFb into the extracellular environment, which then bind to their respective receptors in out-of-field tissues, including CCL receptor type-2 (CCR2)70 and TFGb receptor 1 (TGFbR1).71 These receptors can interact with various cellular pathways consequently leading to TGFb upregulation and increased COX-2 expression. Increased expression of COX-2 is linked to biochemical failure, distant metastasis and radiation toxicity in RT patients with prostate cancer.72 Therefore, CCL2, TGFb, and COX-2 are critical factors in bystander signaling and carcinogenesis,37 and are attractive targets to manipulate abscopal effects. In support of this notion, therapies targeting these factors have already been considered in the clinical setting to block TGFb signaling in RT breast cancer patients73 and inhibit metastatic cascade in glioblastoma74 and nonsmall cell lung cancer patients75 (by using a CCL2neutralizing antibody or small molecule to inhibit CCR2 reviewed in Refs. 14, 70, 76). Another good example of systemic cytokine-mediated intercellular communication is the relationship between senescent cells and surrounding normal tissues in an organism. Cellular senescence is a part of normal aging, as well as a preventive strategy to stop the proliferation of cells undergoing malignant transformation.77 This antiproliferative response can be driven by oncogene activation or loss of tumor suppressor signaling. A direct connection between cellular senescence and inflammation was established recently indicating a crucial role in oncogene-induced cellular senescence the senescence-associated secretory phenotype (SASP), a cross talk between senescent cells and their environment by secretion of numerous cytokines, chemokines, growth factors, and proteases. For example, IL-6 and IL-8, two well-known proinflammatory cytokines, seem to play a central role in premature cellular senescence induction. CCL2 appears as the most upregulated factor and a critical component in the SASP from melanoma cells.78 Moreover, the SASP from senescent melanoma cells or recombinant CCL2 induces DNA damage in naı¨ve melanoma cells, another indication that CCL2 triggers bystander effects.78 On the detrimental side of the SASP effects, the chronic presence of senescent cells secreting numerous proteins has been predicted to significantly alter normal tissue structure and functions, not only in the local milieu but in the whole organism. In the absence of a tumor, synchrotron X-ray irradiation is capable of inducing persistent abscopal effects to normal outof-field tissues in mice. A recent report demonstrated that a short pulse of synchrotron X-ray irradiation on the right hind leg (200 and 810 ms for 10 and 40 Gy) was sufficient to induce significant and persistent DNA damage (DSBs and OCDLs), apoptosis, and local and systemic immune responses in out-of-field tissues.43 Direct irradiation of skin tissue induced an innate immune response (due to increases in macrophages/DC and neutrophils) while in out-of-field duodenum both the innate and adaptive immune response (macrophages/DC, neutrophils, and T-cells) was activated.43 In addition to these persistent immune responses in out-of-field duodenum, increases in oxidative stress, inflammation and senescent cells, and decrease in proliferation were observed in the same tissues. This report also showed significant alterations in a range of plasma cytokines including CSF1R, IL-10, TIMP1, VEGF, TGFb1, and TGFb2, representing a misbalance in the cellular microenvironment in the irradiated area, which likely triggered activation of other factors responsible for the propagation of the systemic effects observed in this study. A mechanism to explain the widespread and persistent abscopal effect observed in out-of-field tissues was proposed.43 At the irradiated site, macrophages and neutrophils become activated via phagocytosis of radiation-induced apoptotic cells79 and secrete cytokines.80 Either directly or by triggering activation of other factors, cytokines, in turn, activate distant tissue-associated macrophages (and other immune cells) that generate free radicals and lead to persistent oxidative stress,37, 80 resulting in OCDL formation in out-of-field tissues. In highly proliferative tissues such as intestine, oxidative DNA lesions can develop into DSBs, which can lead to apoptotic cell death. To identify which components of the immune response drive abscopal effects a recent study used synchrotron-X-rayirradiated immune-deficient mice with a range of immune system abnormalities to tease out which immune system components were essential abscopal effect propagators.81 Contrary to healthy wild type mice, little or no change in DNA damage and apoptosis was observed in out-of-field tissues of immune-deficient mice, indicating that the abscopal effect relies on a functional immune response for its propagation to occur. Since no change in DNA damage and apoptosis was observed in CCL2 knockout mice, which lack the ability to recruit monocytes, macrophages/DC, and memory T-cells to sites of inflammation in damaged tissues,70, 82 NSG (NOD SCID gamma) mice with severe immune deficiencies,83, 84 and C57BL6/J mice treated with anti-CSF1R neutralizing antibody which renders mice macrophage-depleted,83 it indicates that macrophages and CCL2 play key roles in initiating and propagating abscopal effects in out-of-field tissues following localized synchrotron radiation.81 Therefore, targeting the innate immunity via CSF1R in macrophages and/or blocking TGFb/CCL2 can potentially simultaneously protect out-of-field tissues, inhibit metastasis and primary tumor growth.85, 86 Tumor suppression can potentially be further enhanced by using a CSF1R inhibitor which can reduce the secretion of radiation-induced CSF1R from tumors, subsequently decreasing the level of immunosuppressive myeloid cells.87

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Radiation-induced inflammation The concept that IR (IR) as a stress factor interferes with both targeted and nontargeted tissues is supported by multiple sources evidence of systemic response to radiation. As we already mentioned free radicals play an important role in cellular metabolism and cell signaling. However, after exposure to IR a redundant amount of ROS and NO is formed which can damage cellular components and genome. Chronic inflammation is strongly connected with oxidative damage after exposure to IR. After IR an increased number of immune system cells such as macrophages and T-cells may occur which can lead to the accumulation of several inflammatory mediators (NF-kB and SMAD2/3, cytokines, TNF-a, TGF-b, and IFN-g). An increased number of these mediators are connected to ROS and NO.88 Radiation induces cellular oxidative stress that results in damage of not only nuclear DNA but also mitochondrial DNA leading to a decrease in respiratory chain activity and loss of mitochondrial function. The outcome is persistent metabolic oxidative stress that could continue to cause further oxidative damage to critical biological structures after long radiation exposure.89 This radiation-induced damage to mitochondrial DNA in directly targeted or bystander tissues could become heritable and contribute to radiationinduced genomic instability. Genomic instability in nonirradiated normal tissues has been reported to be mediated by late cytokine response, as in case of long-lived COX-2 pathway cytokine-dependent DNA damage and apoptosis response in nonirradiated mouse bone marrow cells after bone marrow was retrospectively irradiated. Such mechanistic studies provide insight into the nature of signaling molecules participating in targeted and nontargeted effects that potentially can be manipulated to increase therapeutic gain in radiotherapy. Exposure to IR has long been known to modulate the immune capacity of irradiated subjects, with a recognized dose/ effect relationship.90 Radiation exposure directly damages hematopoietic stem cells and alters the capacity of bone marrow stromal elements to support and maintain hematopoiesis. Data from the atomic bomb victims suggest a threshold dose to the acute radiation hematological syndrome characterized by severe immune-compromise and subsequent death. In solid tumors these forms of unscheduled cell death can lead to a pro-inflammatory environment and an increase in cell-to-cell signaling. In this scenario, the innate immune system is important in mediating the antitumor effects of localized IR. For example, a preclinical murine study in as early as 1979 demonstrated that in vivo tumor control probability to radiation was profoundly influenced by the host immune-competence in a transplanted murine fibro-sarcoma model.91 However, even in the presence of a competent immune system an established tumor system is usually adapted to avoid immune recognition in the absence of additional antitumor stimulus. This section focuses on the complex induced immune response of the tumor and host secondary to radiotherapy.

Local tumor environment and radiation Immune cells associated with the complex tumor microenvironment can function to promote or suppress the adaptive immune response. Tumor-associated macrophages which are consistently colocated within the tumor microenvironment are pro-angiogenic and can assist in tumor growth. In established and advanced neoplasia, when persistent tumor cells have escaped the immune attack, M2-polarized macrophages predominate the tumor microenvironment and suppress adaptive immunity. The response to IR can trigger inflammation; however, the interpretation of this process by the innate immune system appears to be dependent on a variety of factors. In tumor cells, doses of 5% over past 6 months, or



BMI (Body Mass Index) 2%, or



Sarcopenia (defined as “low lean body mass (mostly muscle); fatigue is common, strength may be lessened, and physical function limited”) and wl>2%

TABLE 3 Probable functions of omega-3 in cancer cachexia. 

Production of anti-inflammatory agents



Decrease proinflammatory factors



Increase tumor cell susceptibility to apoptosis



Reduce the neoangiogenesis and metastasis



Improving of insulin signaling and sensitivity, and increase appetite

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Role of omega-3 PUFAs in chemoresistant cancers Chemotherapy efficacy is limited by intrinsic or acquired drug resistance of cancer cells.38 Several studies demonstrate that patients benefit from the combination of n-3 long-chain polyunsaturated fatty acids (o-3 LCPUFAs) and chemotherapy39, 40 and that the o-3 LCPUFAs (EPA and DHA) are considered as chemosensitizing agents and revertants of multidrug resistance throw alteration in gene expression, modulation of cellular proliferation and differentiation, induction of apoptosis, generation of reactive oxygen species, and lipid peroxidation.41 Corsetto et al. indicate that a key mechanism in the control of cell drug uptake and efflux is related to o-3 LCPUFA influence on membrane lipid composition. The incorporation of docosahexaenoic acid in the lipid rafts produces significant changes in their physical–chemical properties affecting content and functions of transmembrane proteins, such as growth factors, receptors, and ATP-binding cassette transporter that are crucial for tumor cell growth. In particular, o-3 LCPUFAs often alter the lipid compositions more in chemoresistant cells (that are rich of cholesterol and lipid rafts) than in chemosensitive cells, suggesting a potential adjuvant role in the treatment of drug-resistant cancers.41 The effects of o-3 PUFAs on multidrug resistance (MDR) colon cancer cells are well explained in the literature42; in this, cancer a high rate of cholesterol synthesis is responsible of the phenotype unresponsive to different drugs, unrelated for chemical structure and mechanism of action.43, 44 One of the main mechanisms of MDR is the overexpression of membrane ATP-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp/ABCB1) that limits the intracellular accumulation and toxicity of several anticancer agents and is directly related to the amount of cholesterol in the plasma membrane.45, 46 As a result of these events, o-3 PUFAs overcome drug resistance and allow MDR cells to response to chemotherapy.42 The effects of o-3 PUFAs are not limited to colon cancer cells, because the increased demand of cholesterol is a typical feature of several types of chemoresistant tumors43, 44; in fact a considerable number of in vivo studies have shown a good chemosensitizing effect of this fatty acids in tumors resistant to anthracyclines and taxanes,25, 47–49 drugs used in hematological and solid malignancies. This feature and safety enlarges the potential number of oncological patients who may benefit from o-3 LCPUFA supplementation.

Application to other cancers and conditions The reason of using EPA and DHA in patients with breast cancer was due to the results of cross-selectionated studies, which showed that higher intake of these fatty acid was associated with decreased risk of cancer-related mortality.50 Other studies have shown that omega 3 fatty acid could decrease the risk of breast cancer51 and adverse events related to it. Their supplementation has shown: reduced malignant epithelial cells proliferation markers (Ki67) in prostate cancer, reversed cachexia in advanced pancreatic cancer, improved liver and pancreas function in postoperative patients with abdominal cancer, and increased chemotherapeutical effects and survival in patients with lung cancer.52, 53 One study54 investigated the impact of GST polymorphisms on the relationship between omega-3 fatty acid and breast cancer risk in postmenopausal Chinese women in Singapore. An increased protective effect of dietary intake of marine-based omega-3 fatty acid was identified in women with genetic polymorphisms for reduced GST activity. The high consumption of marine sources of omega-3 fatty acid with reduced GST activity polymorphisms (GSTT1-null genotype) had at least a 64% reduction of risk compared to the low consumer counterparts, with some polymorphisms experiencing an even greater protection. In this prospective cohort study of US men and women,55 we found that greater consumption of the long-chain omega3 PUFAs EPA and DHA was associated with an 18% reduced risk of mortality from all causes and a 23% decrease in mortality from cancer. There was a 13% nonsignificant decrease in mortality from CVD, which contributed to the total mortality risk reduction.

Conclusions Since the 1970s, omega-3 have been the focus of several investigations, due to their capacity to suppress inflammatory mechanisms, especially in cancer patients, and in the last years it has also become feasible to find potential molecular targets, with the opportunity to use them as adjuvant therapy in several types of cancer, especially since the receptors of free fatty acids have been identified more accurately. However, the specific processes involved in their effect have not yet been identified, even if, for example, numerous hypotheses have been made, such as an alteration in the uptake and metabolism of lipids, a decrease in generating proinflammatory factors, a decrease in angiogenesis, and an increase in the sensitivity of cells to apoptosis.18

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The first systematic review concluded that there was insufficient data to establish whether omega-3 had an incisive effect at the oxidative level, and more generally beneficial in patients with neoplastic cachexia. The most recent reviews of the literature have instead highlighted a substantial beneficial and anti-inflammatory role of omega-3 fatty acids when used in cancer patients undergoing different treatment regimens such as radiotherapy, chemotherapy, or combination of the two treatments.56 However, there is still a general incoherence in clinical data, but despite this the guidelines issued by the American Society for Parenteral and Enteral Nutrition (ASPEN), and the guidelines issued by the European Society for Parenteral and Enteral Nutrition (ESPEN) recommend supplementation with omega-3 fatty acids as an option in patients with cancer on progressive weight loss and malnutrition. If one can agree with these general indications for the benefit of the use of omega 3 in cancer patients, future randomized controlled trials should be directed mainly and specifically to determine the pharmacodynamics and pharmacokinetics of omega 3, their specific targets of action, the effects on the individual type of cancer, and especially the most effective dosage.57 This would allow not only to identify which patients can benefit from this intervention, at what stage of disease, and with which category of treatment, but also to discover new approaches that could involve these molecules in combination with antitumor agents in tumor-specific therapy and not only through an antioxidant and anti-cachectic action.

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Oxidative stress plays a fundamental role in the pathogenesis of inflammatory, autoimmune, neurodegenerative, and cancer diseases. Omega-3 fatty acids have shown over the years an activity of prevention on some types of cancer and more recently have aroused interest in a potential therapeutic role against neoplastic cachexia. They also show an improvement on oxidative stress in patients undergoing chemotherapy or radiotherapy. Tumor cachexia is closely linked to oxidative stress and chronic inflammation, worsening the catabolic state of the subject. The administration of omega-3, if carried out at the beginning of therapy, can prevent the development of an unrecoverable catabolic phase. The supplementation with omega-3 fatty acids in cancer patients has a proven efficacy on cancer cells that develop resistance to chemotherapy and that’s the reason why they can be considered chemosensitizing agents. The ESPEN and ASPEN guidelines suggest the use of omega-3 fatty acids in cancer patients to maintain or increase appetite, food intake, and also to avoid progressive weight loss and lean mass reduction. Despite the incoherence of clinical data and the need of further randomized trials, the use of omega-3 in cancer patients appears to have significant benefits as anti-inflammatory agents, mainly during radiotherapy and chemotherapy.

Acknowledgments All authors have read and approved the final manuscript. None of the authors had any conflict of interests.

References 1. Chiurchiu` V, Maccarone M. Chronic inflammatory disorder and their redox control: from molecular mechanisms to therapeutic opportunities. Antioxid Redox Signal 2011;15(9):2605–41. 2. Norling LV, Ly L, Dalli J. Resolving inflammation by using nutrition therapy: roles for specialized pro-resolving mediators. Curr Opin Clin Nutr Metab Care 2017;20(2):145–52. 3. Calder PC. Marine o-3 fatty acids and inflammatory processes: effects, mechanisms and clinical relevance. Biochim Biophys Acta 1851;2015:469–84. 4. Murray M, Hraiki A, Bebawy M, Pazderka C, Rawling T. Anti-tumor activities of lipids and lipid analogues and their development as potential anticancer drugs. Pharmacol Ther 2015;150:109–28. 5. Serini S, Fasano E, Piccioni E, Cittadini AR, Calviello G. Dietary n-3 polyunsaturated fatty acids and the paradox of their health benefits and potential harmful effects. Chem Res Toxicol 2011;24:2093–105. 6. Manson JE, Cook NR, Lee I-M, Christen W, Bassuk SS, Mora S, et al. Marine n 3 fatty acids and prevention of cardiovascular disease and cancer. N Engl J Med 2019;380:23–32. 7. Merendino N, Costantini L, Manzi L, Molinari R, D’Eliseo D, Velotti F. Dietary o 3 polyunsaturated fatty acid DHA: a potential adjuvant in the treatment of cancer. Biomed Res Int 2013;2013:310186. 8. Bruera E, Strasser F, Palmer JL, Willey J, Calder K, Amyotte G, et al. Effect of fish oil on appetite and other symptoms in patients with advanced cancer and anorexia/cachexia: a double-blind, placebo-controlled study. J Clin Oncol 2003;21:129–34.

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9. Clarke JT, Cullen-Dean G, Regelink E, Chan L, Rose V. Increased incidence of epistaxis in adolescents with familial hypercholesterolemia treated with fish oil. J Pediatr 1990;116:139–41. 10. EFSA Panel on Dietetic Products. Nutrition and Allergy (NDA). Scientific opinion on the tolerable upper intake level of eicosapentaenoic acid (EPA), docosahexaenoic acid (DH) and docosapentaenoic acid (DPA). EFSA J 2012;10(7):2815. 11. Pardini RS. Nutritional intervention with omega-3 fatty acids enhances tumor response to anti-neoplastic agents. Chem Biol Interact 2006;162:89– 105. 12. Hardman WE. N-3 fatty acids and cancer therapy. J Nutr 2004;134:3427S–30S. 13. Haqq J, Howells LM, Garcea G, Dennison AR. Targeting pancreatic cancer using a combination of gemcitabine with the omega-3 polyunsaturated fatty acid emulsion. Lipidem™. Mol Nutr Food Res 2015;60:1437–47. 14. Michael-Titus AT, Priestley JV. Omega-3 fatty acids and traumatic neurological injury: from neuroprotection to neuroplasticity? Trends Neurosci 2014;37:30–8. 15. Elbarbary NS, Ismail EA, Farahat RK, El-Hamamsy M. u-3 fatty acids as an adjuvant therapy ameliorates methotrexate-induced hepatotoxicity in children and adolescents with acute lymphoblastic leukemia: a randomized placebo-controlled study. Nutrition 2016;32:41–7. 16. Fearon KC, Von Meyenfeldt MF, Moses AG, Van Geenen R, Roy A, Gouma DJ, et al. Effect of a protein and energy dense N-3 fatty acid enriched oral supplement on loss of weight and lean tissue in cancer cachexia: a randomised double blind trial. Gut 2003;52(10):1479–86. 17. Zhang YF, Gao HF, Hou AJ, Zhou YH. Effect of omega-3 fatty acid supplementation on cancer incidence, non-vascular death, and total mortality: a meta-analysis of randomized controlled trials. BMC Public Health 2014;14:204. 18. Freitas RDS, Campos MM. Protective effects of omega-3 fatty acids in cancer-related complications. Nutrients 2019;11:945. ´ lvarez M, Luengo-Perez LM, Grande E, A ´ lvarez-Herna´ndez J, Sendro´s-Madron˜o MJ, et al. Nutritional support and parenteral 19. Virizuela JA, Camblor-A nutrition in cancer patients: an expert consensus report. Clin Transl Oncol 2018;20:619–29. 20. Ryan AM, Power DG, Daly L, Cushen SJ, Nı´ E. Cancer-associated malnutrition, cachexia and sarcopenia: the skeleton in the hospital closet 40 years later. Proc Nutr Soc 2016;75:199–211. 21. Mcmillan DC. The systemic inflammation-based Glasgow prognostic score: a decade of experience in patients with cancer. Cancer Treat Rev 2013;39:534–40. 22. Diakos CI, Charles KA, Mcmillan DC, Clarke SJ. Cancer-related inflammation and treatment effectiveness. Lancet Oncol 2014;15:493–503. 23. Martins E, Oliveira ACDM, Pizato N, Muniz-junqueira MI, Magalha˜es KG, Nakano EY. The effects of EPA and DHA enriched fish oil on nutritional and immunological markers of treatment naı¨ve breast cancer patients: a randomized double-blind controlled trial. Nutr J 2017;16:71. 24. Chagas TR, Borges DS, PFD dO, Mocellin MC, Barbosa AM, Camargo CQ. Oral fish oil positively influences nutritional-inflammatory risk in patients with haematological malignancies during chemotherapy with an impact on long-term survival: a randomised clinical trial. J Hum Nutr Diet 2017;30:681–92. 25. Bougnoux P, Hajjaji N, Ferrasson MN, Giraudeau B, Couet C, Floch OL. Improving outcome of chemotherapy of metastatic breast cancer by docosahexaenoic acid: a phase II trial. Br J Cancer 2009;101:1978–85. 26. Tan BH, Fearon KC. Cachexia: prevalence and impact in medicine. Curr Opin Clin Nutr Metab Care 2008;11:400–7. 27. Fearon KC, Voss AC, Hustead DS, Cancer Cachexia Study Group. Definition of cancer cachexia: effect of weight loss, reduced food intake, and systemic inflammation on functional status and prognosis. Am J Clin Nutr 2006;83:1345–50. 28. Churm D, Andrew IM, Holden K, Hildreth AJ, Hawkins C. A questionnaire study of the approach to the anorexia-cachexia syndrome in patients with cancer by staff in a district general hospital. Support Care Cancer 2009;17:503–7. 29. Argiles JM, Busquets S, Stemmler B, Lo´pez-Soriano FJ. Cachexia and sarcopenia: mechanisms and potential targets for intervention. Curr Opin Pharmacol 2015;22:100–6. 30. Dewey A, Baughan C, Dean T, Higgins B, Johnson I. Eicosapentaenoic acid (EPA, an omega-3 fatty acid from fish oils) for the treatment of cancer cachexia. Cochrane Database Syst Rev 2007;2007:CD004597. 31. Ries A, Trottenberg P, Elsner F, Stiel S, Haugen D, Kaasa S, et al. A systematic review on the role of fish oil for the treatment of cachexia in advanced cancer: an EPCRC cachexia guidelines project. Palliat Med 2011;26:294–304. 32. Colomer R, Moreno-Nogueira JM, Garcı´a-Luna PP, Garcı´a-Peris P, Garcı´a-deLorenzo A, Zarazaga A, et al. N-3 fatty acids, cancer and cachexia: a systematic review of the literature. Br J Nutr 2007;97:823–31. 33. Camargo Cde Q, Mocellin MC, de Pastore Silva JA, Fabre ME, Nunes EA, Trindade EB. Fish oil supplementation during chemotherapy increases posterior time to tumor progression in colorectal cancer. Nutr Cancer 2016;68(1):70–6. 34. Sanchez-Lara K, Turcott JG, Juarez-Hernandez E, Nunez-Valencia C, Villanueva G, Guevara P, et al. Effects of an oral nutritional supplement containing eicosapentaenoic acid on nutritional and clinical outcomes in patients with advanced non-small cell lung cancer: randomised trial. Clin Nutr 2014;33(6):1017–23. 35. Van der Meij BS, Langius JA, Spreeuwenberg MD, Slootmaker SM, Paul MA, Smit EF, et al. Oral nutritional supplements containing n-3 polyunsaturated fatty acids affect quality of life and functional status in lung cancer patients during multimodality treatment: an RCT. Eur J Clin Nutr 2012;66(3):399–404. 36. Murphy RA, Yeung E, Mazurak VC, Mourtzakis M. Influence of eicosapentaenoic acid supplementation on lean body mass in cancer cachexia. Br J Cancer 2011;105:1469–73. 37. Gorjao R, Santos CMMD, Serdan TDA, Diniz VLS, Alba-Loureiro TC, Cury-Boaventua MF, et al. New insights on the regulation of cancer cachexia by N-3 polyunsaturated fatty acids. Pharmacol Ther 2019;196:117–34. 38. Zheng HC. The molecular mechanisms of chemoresistance in cancers. Oncotarget 2017;8:59950–64.

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39. De Aguiar Pastore Silva J, Emilia de Souza Fabre M, Waizberg DL. Omega-3 supplements for patients in chemotherapy and/or radiotherapy: a systematic review. Clin Nutr 2015;34:359–66. 40. Laviano A, Rianda S, Molfino A, Rossi FF. Omega-3 fatty acids in cancer. Curr Opin Clin Nutr Metab Care 2013;16:156–61. 41. Corsetto PA, Colombo I, Kopecka J, Rizzo AM, Riganti C. Review w-3 long chain polyunsaturated fatty acids as sensitizing agents and multidrug resistance revertants in cancer therapy. Int J Mol Sci 2017;18:2770. 42. Gelsomino G, Corsetto PA, Campia I, Montorfano G, Kopecka J, Catella B, et al. Omega 3 fatty acids chemosensitize multidrug resistant colon cancer cells by down-regulating cholesterol synthesis and altering detergent resistant membranes composition. Mol Cancer 2013;12:137. 43. Kopecka J, Campia I, Olivero P, Pescarmona G, Ghigo D, Bosia A, et al. A LDL-masked liposomal-doxorubicin reverses drug resistance in human cancer cells. J Control Release 2011;149:196–205. 44. Riganti C, Castella B, Lopecka J, Campia I, Coscia M, Pescarmona G, et al. Zoledronic acid restores doxorubicin chemosensitivity and immunogenic cell death in multidrug-resistant human cancer cells. PLoS One 2013;8:e60975. 45. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2002;2:48–258. 46. Eckford PD, Sharon FJ. Interaction of the P-glycoprotein multidrug efflux pump with cholesterol: effects on ATPase activity, drug binding and transport. Biochemistry 2008;47:13686–98. 47. Hardman WE, Avula CP, Fernandes G, Cameron IL. Three percent dietary fish oil concentrate increased efficacy of doxorubicin against MDA-MB 231 breast cancer xenografts. Clin Cancer Res 2001;7:2041–9. 48. Kornfeld S, Goupille C, Vibet S, Chevalier S, Pinet A, Lebeau J, et al. Reducing endothelial NOS activation and interstitial fluid pressure with n-3 PUFA offset tumor chemoresistance. Carcinogenesis 2012;33:260–7. 49. Colas S, Mahe`o K, Denis F, Goupille C, Hoinard C, Champeroux P, et al. Sensitization by dietary docosahexaenoic acid of rat mammary carcinoma to anthracycline: a role for tumor vascularization. Clin Cancer Res 2006;12:5879–86. 50. Prakash M, Mrunal K, Rashmi D, Amol C, Kavita S, Ruchikh K. Improved antioxidant status by omega-3 fatty acids supplementation in breast cancer patients undergoing chemotherapy: a case series. J Med Case Rep 2015;9:148. 51. Khodarahmim M, Azadbakht L. The association between different kinds of fat intake and breast cancer risk in women. Int J Prev Med 2014;5(1):6–15. 52. Minich DM, Brown BI. A review of dietary (phyto) nutrients for glutathione support. Nutrients 2019;11:2073. 53. Manson JE, Bassuk SS, Lee IM. The vitamin D and omega-3 trial (VITAL): rationale and design of a large randomized and controlled trial of vitamin D and marine omega-3 fatty acids supplements for the primary prevention of cancer and cardiovascular diseases. Contemp Clin Trials 2012;33(1):159–71. 54. Gago-Dominiguez M, Castelao JE, Sun C-L, Van Den Berg D, Koh W-P, Lee HP, et al. Marine n-3 fatty acid intake, glutathione S-transferase polymorphisms and breast cancer risk in post-menopausal Chinese women Singapore. Carcinogenesis 2014;25:2143–7. 55. Bell GA, Cantor ED, Lampe JW, Kristal AR, Heckbert SR, White E. Intake of long chain n-3 fatty acid from diet and supplement in relation mortality. Am J Epidemiol 2014;179(6):710–20. 56. Gorjao R, Dos Santos CMM, Serdan TDA, Diniz VLS, Alba-Loureiro TC, Cury-Boaventura MF, et al. New insights on the regulation of cancer cachexia by N-3 polyunsaturated fatty acids. Pharmacol Ther 2019 Apr;196:117–34. 57. Lavriv DS, Neves PM, Ravasco P. Should omega-3 fatty acids be used for adjuvant treatment of cancer cachexia. Clin Nutri ESPEN 2018;25:18–25.

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Chapter 23

Statins, cancer, and oxidative stress Tahoora Shomalia and Mahboobeh Ashrafib a

Division of Pharmacology and Toxicology, Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran, b Division of

Biochemistry, Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

List of abbreviations CAFs Cav-1 COX-2 HIF 1a LDL MDA mTOR NFkB PGE2 PI3K ROS SOD TAC TNFa

cancer associated fibroblasts caveolin-1 cyclooxygenase 2 hypoxia inducible factor 1a low-density lipoprotein malondialdehyde mammalian target of rapamycin nuclear factor kB prostaglandin E2 phosphatidylinositol 3 kinase reactive oxygen species superoxide dismutase total antioxidant capacity tumor necrosis factor a

Oxidative stress is defined as an imbalance between oxidants including free radicals, reactive species, reactive metabolites, and antioxidants. Reactive species are comprised of reactive oxygen species (ROS), reactive nitrogen species, reactive sulfur species, and reactive chlorine species, which are derived from oxidative metabolism.1 They are ions, radicals, or molecules with an unpaired electron in their most outer layer of electrons. ROS are the most abundant of active species and include anion superoxide, hydroxyl radical, hydrogen peroxide, singlet oxygen, and ozone.2 Physiological concentration of ROS is required for normal signaling pathways,3 activation of enzymes,4 control of certain gene expression,5 formation of disulfide bounds in protein folding process,6 and induction of apoptosis,7 but excessive production can damage macromolecules such as nucleic acids, proteins, and lipids. In living cells, endogenous ROS is produced in mitochondria as a by-product of oxidative metabolism, peroxisomes by oxidases, plasma membrane by cyclooxygenase, lipoxygenase and NADPH oxidase, endoplasmic reticulum by cytochrome P450, cytochrome b5, and cytoplasm by xanthine oxidase.8 Oxidative stress is associated with a long list of human diseases including cardiovascular diseases,8 complications of diabetes,9 liver diseases,10 neurodegenerative diseases,11 and cancers.12 Several studies have reported that excessive production of ROS is oncogenic and oxidative stress is implicated in different stages of carcinogenesis such as initiation, promotion, progression, and metastasis.13 Among different ROS, hydrogen peroxide, superoxide, and hydroxyl radicals are the best studied in cancer cells.14 Major sources of ROS production in cancer cells include increased activity of mitochondria or peroxisomes,15 oncogenic signaling [tumor necrosis factor a (TNFa), platelet-derived growth factor, transforming growth factor b, epidermal growth factor, and insulin],16–20 NADPH oxidase, and infiltrating macrophages.21 In the initiation step of carcinogenesis, overproduction of ROS can lead to DNA oxidative damage and mutations such as base modification, single- or double-strand breaks of DNA, base damage, and gene amplifications, which in turn activate the oncogenes. The changes result in genomic instability and carcinogenesis.22 Moreover, overproduction of ROS can damage cell membranes via lipid peroxidation. Malondialdehyde (MDA) as one of the end products of this process has mutagenic and carcinogenic effects.23 High levels of ROS in cancer cells can activate phosphatidylinositol 3 kinase (PI3K)/AKT (protein kinase B)/mammalian target of rapamycin (mTOR) signaling as a survival pathway.24 mTOR signaling, that is, hyper-activated in many Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00023-7 © 2021 Elsevier Inc. All rights reserved.

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cancers, contributes to cell proliferation, growth, survival, and metabolism.25 In addition, in tumor cells, ROS inactivates PTEN (phosphatase and tensin homolog) pathway that initiates apoptosis.24 PTEN as a tumor suppressor gene codes a lipid phosphatase that dephosphorylates PI3P (phosphatidylinositol 3, 4, 5 triphosphate) and prevents the activation of PI3K/ AKT/mTOR pathway.26 Recently, there is a direct evidence that indicate the role of oxidative stress-induced epigenetic changes in transformation of cells into malignant phenotype.27 In addition, ROS can induce overexpression of cyclooxygenase 2 (COX-2) as a mediator in inflammation, that is, related to promotion step of carcinogenesis or accumulation of preneoplastic cells.28, 29 Prostaglandin E2 (PGE2) production by COX-2 can promote tumor growth via its effect on cell proliferation and apoptosis.30 Progression step of carcinogenesis is concomitant with genetic and phenotypic changes of cells, so that the growth of neoplastic cells with further mutations is fast. In 2011, Goh et al. reported that overproduction of ROS and mitochondrial oxidative stress can promote tumor progression and metastasis in breast cancer.31 Recent studies have shown that cancer cells can induce oxidative stress in tumor stroma such as adjacent fibroblast cells as cancer-associated fibroblasts (CAFs) that are present in cancerous tissues, because ROS can be released in the extracellular matrix through exosomes or aquaporins.32 Then, ROS overproduction triggers onset of authophagy, mithophagy, glycolysis and pyruvate, lactate, and ketone body production in these cells. Caveolin-1 (Cav-1) is a major component of caveolae and is highly expressed in fibroblasts, adipocytes, and endothelial cells. Down expression of Cav-1 due to Ras mutation, c-Myc overexpression, and loss of p53 is shown in CAFs.33 Cav-1 inhibits the activity of NO synthase.34 Physiological concentrations of NO are necessary for vascular function, but NO overproduction exerts oxidative stress and activates aerobic glycolysis in CAFs with loss of Cav-1. High-energy metabolites are secreted from CAFs and their entrance into adjacent cancer cells can promote growth, proliferation, and protection against apoptosis.35 This model was identified as “the autophagic tumor stroma model of cancer metabolism”.35 Also, oxidative stress in tumor stroma can promote tumor spreading in a way that local or systemic administration of antioxidants blocks tumor metastasis in cancer models.36, 37 In solid tumors, there is an area in the center of tumors with hypoxia hallmark. Hypoxia inducible factor 1a (HIF 1a) is a transcription factor, that is, implicated in hypoxia response of tumors. Target genes of HIF1a are angiogenic genes such as vascular endothelial growth factor,38 calcitonin receptor-like receptor as a G-protein-coupled receptor,38 semaphorin 4D (Sema4D) as one member of 20 semaphorins,38 stem cell factor and angiopoietin 2 (ANGPT2),38 metastatic genes including adhesion molecules,39 matrix metalloproteinases (MMP2 and MMP9), transcription factor TWIST, CXC chemokine receptor-4, c-Met and CC chemokine receptor 7 (CCR7), anaerobic metabolism genes such as glucose transporters 1 and 3, 6-phosphofructo-2-kinase, phosphoglycerate kinase 1, pyruvate kinase M2 and monocarboxylate transporter MCT4, and genes with negative effect on differentiation such as Notch downstream genes.38 Hypoxia and overproduction of ROS can activate HIF1a resulting to expression of its target genes and tumor progression.40 Also, several studies have shown that overproduction of ROS and hypoxia in cancer cells can activate nuclear factor kB (NFkB) as a transcription factor that plays crucial roles in initiation and progression steps of carcinogenesis by effect on cell survival, proliferation, and inflammation.41 In cancer, chronic inflammation is accompanied with moderately elevation in NFkB activity that promotes cell survival with upregulation of antiapoptotic genes and adhesion molecules (in leucocytes).42, 43 Anoikis is a type of programmed cell death, that is, induced by detachment of cells from extracellular matrix. It is reported that ROS production in cancer cells can induce anoikis resistance that results in cell survival and progression of cancer.44 Metastasis is spreading of tumor cells from primary site to other sites through blood flow or lymph system. The expression of adhesion molecules and matrix metalloproteinases is important in the migration of tumor cells and metastasis and ROS production in cancer cells can upregulate the expression of these genes.45 In addition to high production of ROS, reduction of antioxidants in cancer states can result in the development of oxidative stress. In the same way, reduction in the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase in cell lines or tumor cells was reported in many studies.46, 47 Also, TNFa as a cytokine that can be detected in the blood of cancer patients reduces the expression of catalase in liver tissue.48 On the other hand, lethal concentrations of ROS in cancer cells due to radiation and other anticancer agents can induce cell death.49 Statins, with their names originates from the word “stasis,” are natural products (and their analogs) initially isolated from a Penicillium culture in a microbiological study originally conducted to find new antibacterial agents and led to the discovery of drugs, which are now considered as most commonly prescribed agents for treating hypercholesterolemia. These drugs bring blood cholesterol into stasis by competitive inhibition of the crucial enzyme hydroxymethyl glutarylCoA reductase, majorly due to the resemblance of their structure (lactone ring) to mevalonate as the enzyme’s product. The resultant reduction in cholesterol biosynthesis decreases hepatic cholesterol content. This encourages liver to avidly scavenge low-density lipoprotein (LDL) cholesterol in circulating blood, where the activity and expression of LDL receptors in hepatocytes’ membrane increase in response to these agents. Reduced production accompanied by increased

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catabolism of apo B100 also happens after statin administration that not only affects blood cholesterol concentration but also can reduce triglycerides levels; although the magnitude and importance of each event (decreased production vs increased catabolism of apo B100) is affected by type of hypercholesterolemia as well as dose of statin used among others.50 While mevastatin, lovastatin, and simvastatin’s lactone rings need to be metabolically opened in order to be active, second and third generations of statins have an already open lactone ring and, therefore, are not prodrugs.50 Pravastatin and rosuvastatin have hydrophilic nature with slow passive diffusion across cell membranes, where organic anion transporter1B1 is a major factor for the uptake of pravastatin into hepatocytes.51 Other statins including lovastatin, simvastatin, cerivastatin, fluvastatin, pitavastatin, and atorvastatin are considered as lipophilic statins. Simvastatin, lovastatin, cerivastatin, and atorvastatin are metabolized primarily by liver cytochrome P450-3A4 enzymes, which increase the possibility of drug interactions.52 Statins that lack a significant hepatic metabolism (pravastatin), metabolized by more than one cytochrome P450 isoenzyme (fluvastatin), or those with their metabolism could be mediated through alternative cytochrome P450 isoenzymes (cerivastatin) are the least prone to drug interactions.53 Lovastatin, simvastatin, and fluvastatin have short half-lives and are recommended to be administered in the evening due to the fact that cholesterol biosynthesis peaks during night. Atorvastatin, rosuvastatin, and extended release fluvastatin have longer half-lives. Longer-acting statins can be used according to the patient compliance.54 Apart from plasma cholesterol reduction, statins have pleiotropic effects that are not only important in protection from cardiovascular diseases but also play a role in amelioration of other conditions, including osteoporosis, neurodegenerative diseases, and cancer.55 Statins usually show a good safety profile as class, but like any other drug they are not void of adverse effects. The most important side effects include myopathy and rhabdomyolysis. Occasional hepatic injury which can be reversible and some adverse renal effects, e.g., acute renal failure may also be encountered.56 While antioxidant effects of statins are among their pleiotropic properties, which can be due to interference with the synthesis of isoprenoid intermediates and is especially important in cardiovascular diseases,57 oxidative stress due to metabolism of statins is involved in various levels of their adverse effects on skeletal muscles, liver, and kidney. In fact, it has been suggested that the effects of statins on oxidative status are site dependent.58 Formation of ROS in skeletal muscle, liver, and kidney plays a critical role in statin-induced oxidative stress and related toxicities. Statin-induced ROS generation results in oxidative stress and can put antioxidant defense system under extensive pressure. Damage to different cellular macromolecules including lipids, DNA, and proteins is logically anticipated. MDA and thiobarbituric acid reactive substances levels as markers of lipid peroxidation have shown to be increased due to statin administration in both in vitro and in vivo studies for demonstrating the mechanism for statin-induced liver damages. It must be emphasized that lipid peroxidation due to statins can be dose or time dependent. In contrast to lipid peroxidation, protein oxidation is rarely reported in studies on statin toxicity. In vivo models have shown diminished activity of antioxidant enzymes and decreased glutathione concentration as a mechanism of liver injury,59 which can be dose dependent.60 Change in oxidative status of muscles by statins can be related to muscle type. Collectively statins can affect antioxidant enzymes activity, antioxidant signaling pathways, and expression of related genes in noncancerous tissues.58 Currently, statins are finding their niche in cancer prevention and/or therapy.61 The tumor type and class of statins (hydrophilic vs lipophilic agents) can majorly influence the effect of statins on cancer incidence.62 Different mechanisms are proposed for the beneficial effects of statins on cancer and their effects on oxidative status are a decisive factor in this regard. In fact, like the case of noncancerous cells, statins show a dual effect on oxidative status in cancer cells. In vitro studies have shown that lipophilic statins (among them simvastatin is mostly studied) can cause cytotoxicity and cell death in cancer cells as well as inhibition of cellular proliferation by a prooxidant action. These effects are observed in HepG2 cells,63 k-ras-transformed thyroid cells,64 MCF-7 breast cancer cells,65 human papilloma virus positive and negative cervical cancer cells,66 murine CT26 colon carcinoma cells,67 lymphoma cells,68 and A549 lung cancer cells.69 Others have reported that simvastatin rises ROS levels and induces senescence in human melanoma cells accompanied by elevated expression of catalase and peroxiredoxin-1.70 Moreover, lovastatin at the pharmacological relevant concentration has induced DNA damage, oxidative stress, and autophagy in cancer cells but not in normal mesothelial cells.71 In contrast, reports are available that suggest the involvement of antioxidant mechanisms in anticancer effects of statins. In a study by, pitavastatin suppressed intestinal polyp development and oxidative stress represented by 8-nitroguanosine in the small intestinal epithelial cells of Min mice.72 Atorvastatin has shown antioxidant effects in rats with nitrosamineinduced bladder cancer.73 Lovastatin has inhibited malignant B cell proliferation by reducing intracellular ROS.74 In B16
F10 murine melanoma cells bearing mice, anticancer effect of liposome-encapsulated simvastatin has been associated with antioxidant effects.75 Licarete et al. showed an antioxidant effect of simvastatin in B16
F10 murine melanoma cells.76 Moreover, it has been demonstrated that simvastatin induces dose-dependent upregulation of Nrf2 (a transcription factor to activate cellular antioxidant response) expression in colon cancer cells and induces selected antioxidant enzymes.77 Atorvastatin enhanced the cell-killing effect of irradiation by reducing endogenous ROS levels and prolonging the life span of

258 SECTION

B Antioxidants and cancer

radiation-induced ROS via a decrease in the level of NADPH oxidases and SOD activity in PC-3 prostate cancer cells to enhance the cell-killing effect.78 In A-2058 human melanoma malignum cells, simvastatin has shown antioxidant properties.79 In a study by Karimi et al., simvastatin usage increased total antioxidant capacity (TAC) level, paraoxonase 1 activity in serum and decreased total oxidant status and MDA levels in tumors similar to tamoxifen in mice with 7, 12-dimethylbenzathracene-induced breast cancer.80 It is interesting to mention that although there are few in vivo reports on the effect of statins on oxidative status in cancer, all of them have shown antioxidant properties of these agents. The outcomes of different studies on the effects of statins on oxidative stress in cancer are summarized in Table 1. Taken together, it seems that the dual effects of statins on oxidative stress may be important in their anticancer properties. TABLE 1 effects of statins on oxidative status in different studies on cancer. Type of study Tumor/cancer cell parameters

Effect on oxidative status

Oxidative status parameters

Reference

In vitro

In vivo

Agent used

HepG2 cells

–

Simvastatin

Cell death and ATP synthesis

Prooxidant

DNA oxidative damage

[64]

k-rastransformed thyroid cells

Athymic mice injected with FRTL5-K-Ras cells

Lovastatin

Apoptosis (in vitro study), tumor volume (in vivo study)

Prooxidant (in vitro study)

ROS levels, Mn-SOD expression

[65]

MCF-7 breast cancer cells

–

Atorvastatin, fluvastatin, and simvastatin

Proliferation, DNA synthesis, viability, cell cycle, cell death, cell membrane integrity, and mitochondrial membrane potential

Prooxidant

ROS generation

[66]

CaSki, HeLa (HPV+) and ViBo (HPV ) cervical cancer cells

–

Atorvastatin, fluvastatin and simvastatin

Proliferation, cell cycle and cell death

Prooxidant

ROS production

[67]

Murine CT26 colon carcinoma cells and B16BL6 melanoma cells

–

Simvastatin

Cell viability and lactate dehydrogenase release assay, apoptosis, caspase activity

Prooxidant

GSH/GSSG ratio, ROS level, Cu/Zn SOD, Mn-SOD, CAT, GPx1, heme oxygenase 1, and SESN 3 expression

[68]

–

Min mice

Pitavastatin

Number of intestinal polyps, mRNA expression levels of cyclooxygenase-2, IL-6, etc.

Antioxidant

8-Nitroguanosine level in intestinal epithelium

[73]

–

Rats with nitrosamineinduced bladder cancer

Atorvastatin

Macroscopic, histological and immunohistochemical (p53, ki67, CD31) evaluation, Incidence, number and volume of tumors, inflammation parameters, etc.

Antioxidant

Serum MDA and total anti-oxidant status

[74]

Human mesothelioma cell line ZL55

–

Lovastatin

Proliferation, DNA damage, cell cycle and survival, autophagy

Prooxidant

Heme oxygenase-1 expression

[72]

Statins, cancer, and oxidative stress Chapter

23

259

TABLE 1 effects of statins on oxidative status in different studies on cancer—cont’d Type of study

Effect on oxidative status

In vitro

In vivo

Agent used

Tumor/cancer cell parameters

WM9 human metastatic melanoma cells

–

Simvastatin

Senescence

Prooxidant

ROS levels, expression of catalase and peroxiredoxin-1

[71]

A20 and EL4 lymphoma cells

–

Atorvastatin, fluvastatin, and simvastatin

DNA fragmentation, activation of proapoptotic members and anti-apoptotic molecule Bcl-2, mitochondrial membrane potential, apoptosis, etc.

Prooxidant

ROS level

[69]

Simvastatin, lovastatin, and pravastatin

Proliferation and viability, proapoptotic effects, tumor cell production HIF 1a

Prooxidant?

SOD activity, total nonenzymatic antioxidant status

[81]

B16.F10 melanoma cells

Oxidative status parameters

Reference

A549 lung cancer cells

–

Simvastatin

Proliferation

Prooxidant

ROS levels, MDA level, total SOD and Mn-SOD activity, Mn-SOD expression

[70]

Human B lymphoma Daudi cells

–

Lovastatin

Proliferation

Antioxidant

ROS level

[75]

–

B16.F10 murine melanomabearing mice

Liposomeencapsulated simvastatin

Tumor growth, proteins associated with tumor angiogenesis, and inflammation

Antioxidant

Tumor MDA and NO levels and CAT activity

[76]

HT-29 and HCT 116 human colon cancer cell lines

–

Simvastatin

Viability

Antioxidant?

Nrf2 expression and nuclear translocation of Nrf2, Expression of heme oxygenase-1, NAD(P)H: quinine oxidoreductase 1, g-glutamate-cysteine ligase catalytic subunit

[78]

B16.F10 murine melanoma cells

–

Simvastatin

Proliferation, viability, inflammatory/angiogenic protein levels, HIF 1a expression

Antioxidant

MDA levels

[77]

PC-3 prostate cancer cells

–

Atorvastatin

Clonogenic assay and a cell survival curve, apoptosis

Antioxidant before radiation, prooxidant after radiation

ROS level, NADPH oxidases protein level, SOD protein level and activity

[79]

A-2058 human melanoma malignum cells

–

Simvastatin

Proliferation

Antioxidant

Free radical concentrations

[80]

–

Mice with DMBAinduced breast cancer

Simvastatin

Tumor volume and PGE2 levels, serum carcinoma antigen 15–3 (CA15–3)

Antioxidant

Serum total antioxidant capacity and paraoxonase 1 activity, tumor total oxidant status, and MDA level

[81]

260 SECTION

B Antioxidants and cancer

Applications to other cancers or conditions Statins are well known for their pleiotropic effects, which include their ability to influence the oxidative status of cells. These agents are not only extensively prescribed in cardiovascular diseases but are also under investigation to find a niche in the treatment of other disease states such as osteoporosis, neurodegenerative diseases, and cancer. The tumor type and class of statins (hydrophilic vs lipophilic agents) can majorly influence the effect of statins on cancer incidence. Different mechanisms are proposed for the beneficial effects of statins on cancer and their effects on oxidative status are a decisive factor in this regard. In fact, like the case of noncancerous cells, statins show a dual effect on oxidative status in cancer cells. Their dual effects on oxidative status is shown in a variety of cancer cell lines including breast, cervix, thyroid, and colon cancer cells among others and has been associated with promising results. Studies on laboratory animal models of cancer such as breast and bladder cancer and melanoma-bearing rodents have also shown interesting facts about anticancer effects of statins. Simvastatin is the most routinely addressed statin for its effects on cancer in in vitro and in vivo experiments although its effects needs to be more clarified in future studies.

Summary points l

l l

l

l l

l

Oxidative stress is implicated in different stages of carcinogenesis such as initiation, promotion, progression, and metastasis. Lethal concentrations of reactive ROS in cancer cells due to radiation and other anticancer agents can induce cell death. Statins have pleiotropic effects that are not only important in protection from cardiovascular diseases but also play a role in confronting other conditions, including osteoporosis, neurodegenerative diseases, and cancer. Different mechanisms are proposed for the beneficial effects of statins on cancer with their effects on oxidative status as a decisive factor in this regard. Like the case of noncancerous cells, statins show a dual effect on oxidative status in cancer cells. Lipophilic statins (among them simvastatin is mostly studied) can cause cytotoxicity and cell death in cancer cells as well as inhibition of cellular proliferation by a prooxidant action. Although there are few in vivo reports on the effect of statins on oxidative status in cancer, all of them have shown antioxidant properties of these agents.

References 1. Apak R, Capanoglu E, Shahidi F. Biomarkers of oxidative stress and cellular-based assays of indirect antioxidant measurement. In: Measurement of antioxidant activity and capacity: recent trends and applications. Wiley; 2018. p. 166. 2. Simic MG, Bergtold DS, Karam LR. Generation of oxy radicals in biosystems. Mutat Res-Fund Mol M 1989;214(1):3–12. 3. Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 2000;279(6):L1005–28. 4. Giannoni E, Buricchi F, Raugei G, Ramponi G, Chiarugi P. Intracellular reactive oxygen species activate Src tyrosine kinase during cell adhesion and anchorage-dependent cell growth. Mol Cell Biol 2005;25(15):6391–403. 5. Turpaev KT. Reactive oxygen species and regulation of gene expression. Biochemistry (Mosc) 2002;67(3):281–92. 6. Shimizu Y, Hendershot LM. Oxidative folding: cellular strategies for dealing with the resultant equimolar production of reactive oxygen species. Antioxid Redox Signal 2009;11(9):2317–31. 7. Hoidal JR. Reactive oxygen species and cell signaling. Am J Respir Cell Mol Biol 2001;25(6):661–3. 8. Cervantes Gracia K, Llanas-Cornejo D, Husi H. CVD and oxidative stress. J Clin Med 2017;6(2):22. pii: E22. 9. Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ Res 2010;107(9):1058–70. 10. Cichoz-Lach H, Michalak A. Oxidative stress as a crucial factor in liver diseases. World J Gastroenterol 2014;20(25):8082–91. 11. Uttara B, Singh AV, Zamboni P, Mahajan RT. Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Curr Neuropharmacol 2009;7(1):65–74. 12. Sosa V, Molin
e T, Somoza R, Paciucci R, Kondoh H, Leonart ME. Oxidative stress and cancer: an overview. Ageing Res Rev 2013;12(1):376–90. 13. Assi M. The differential role of reactive oxygen species in early and late stages of cancer. Am J Physiol Regul Integr Comp Physiol 2017;313(6): R646–53. 14. Liou GY, Storz P. Reactive oxygen species in cancer. Free Radic Res 2010;44(5):479–96. 15. Dahabieh MS, Di Pietro E, Jangal M, Goncalves C, Witcher M, Braverman NE, et al. Peroxisomes and cancer: the role of a metabolic specialist in a disease of aberrant metabolism. Biochim Biophys Acta Rev Cancer 2018;1870(1):103–21. 16. Young CN, Koepke JI, Terlecky LJ, Borkin MS, Boyd Savoy L, Terlecky SR. Reactive oxygen species in tumor necrosis factor-a-activated primary human keratinocytes: implications for psoriasis and inflammatory skin disease. J Invest Dermatol 2008;128(11):2606–14. 

Statins, cancer, and oxidative stress Chapter

23

261

17. Bae YS, Sung JY, Kim OS, Kim YJ, Hur KC, Kazlauskas A, et al. Platelet-derived growth factor-induced H2O2 production requires the activation of phosphatidylinositol 3-kinase. J Biol Chem 2000;275(14):10527–31. 18. Krsti
c J, Trivanovi
c D, Mojsilovi
c S, Santibanez JF. Transforming growth factor-beta and oxidative stress interplay: implications in tumorigenesis and cancer progression. Oxid Med Cell Longev 2015;2015:654594. 19. Bae YS, Kang SW, Seo MS, Baines IC, Tekle E, Chock PB, et al. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation. J Biol Chem 1997;272(1):217–21. 20. Goldstein BJ, Mahadev K, Wu X, Zhu L, Motoshima H. Role of insulin-induced reactive oxygen species in the insulin signaling pathway. Antioxid Redox Signal 2005;7(7–8):1021–31. 21. Skonieczna M, Hejmo T, Poterala-Hejmo A, Cieslar-Pobuda A, Buldak RJ. NADPH oxidases: insights into selected functions and mechanisms of action in cancer and stem cells. Oxid Med Cell Longev 2017;2017:9420539. 22. Levine AS, Sun L, Tan R, Gao Y, Yang L, Chen H, et al. The oxidative DNA damage response: a review of research undertaken with Tsinghua and Xiangya students at the University of Pittsburgh. Sci China Life Sci 2017;60:1077–80. 23. Yonei S, Furui H. Lethal and mutagenic effects of malondialdehyde, a decomposition product of peroxidized lipids, on Escherichia coli with different DNA-repair capacities. Mutat Res 1981;88(1):23–32. 24. Kumari S, Badana AK, Murali Mohan G, Shailender G, RamaRao M. Reactive oxygen species: a key constituent in cancer survival. Biomark Insights 2018;6:13. 25. Yu JS, Cui W. Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination. Development 2016;143(17):3050–60. 26. Lu XX, Cao LY, Chen X, Xiao J, Zou Y, Chen Q. PTEN inhibits cell proliferation, promotes cell apoptosis, and induces cell cycle arrest via downregulating the PI3K/AKT/hTERT pathway in lung adenocarcinoma A549. Cells Bio Med Res Int 2016;2016. https://doi.org/10.1155/2016/2476842. Article ID 2476842, 8 pages. 27. Mahalingaiah PK, Ponnusamy L, Singh KP. Oxidative stress-induced epigenetic changes associated with malignant transformation of human kidney epithelial cells. Oncotarget 2017;8(7):11127–43. https://doi.org/10.18632/oncotarget.12091. 28. Onodera Y, Teramura T, Takehara T, Shigi K, Fukuda K. Reactive oxygen species induce Cox-2 expression via TAK1 activation in synovial fibroblast cells. FEBS Open Bio 2015;5:492–501. https://doi.org/10.1016/j.fob.2015.06.001. 29. Wilson KT, Fu S, Ramanujam KS, Meltzer SJ. Increased expression of inducible nitric oxide synthase and cyclooxygenase-2 in Barrett’s esophagus and associated adenocarcinomas. Cancer Res 1998;58:2929–34. 30. Wang D, DuBois RN. Prostaglandins and cancer. Gut 2006;55(1):115–22. https://doi.org/10.1136/gut.2004.047100. 31. Goh J, Enns L, Fatemie S, Hopkins H, Morton J, Pettan-Brewer C, et al. Mitochondrial targeted catalase suppresses invasive breast cancer in mice. BMC Cancer 2011;11:191. https://doi.org/10.1186/1471-2407-11-191. 32. Tafani M, Sansone L, Limana F, Arcangeli T, De Santis E, Polese M, et al. The interplay of reactive oxygen species, hypoxia, inflammation, and sirtuins in cancer initiation and progression. Oxid Med Cell Longev 2016;2016:3907147. https://doi.org/10.1155/2016/3907147. 33. Koleske AJ, Baltimore D, Lisanti MP. Reduction of caveolin and caveolae in oncogenically transformed cells. Proc Natl Acad Sci U S A 1995;92:1381–5. 34. Vakkala M, Kahlos K, Lakari E, Paakko P, Kinnula V, Soini Y. Inducible nitric oxide synthase expression, apoptosis and angiogenesis in in situ and invasive breast carcinomas. Clin Cancer Res 2000;6:2408–16. 35. Pavlides S, Tsirigos A, Migneco G, Whitaker-Menezes D, Chiavarina B, Flomenberg N, et al. The autophagic tumor stroma model of cancer: role of oxidative stress and ketone production in fueling tumor cell metabolism. Cell Cycle 2010;9:3485–505. 36. Nishikawa M, Hashida M, Takakura Y. Catalase delivery for inhibiting ROS mediated tissue injury and tumor metastasis. Adv Drug Deliv Rev 2009;61:319–26. 37. Hyoudou K, Nishikawa M, Ikemura M, Kobayashi Y, Mendelsohn A, Miyazaki N, et al. Prevention of pulmonary metastasis from subcutaneous tumors by binary system-based sustained delivery of catalase. J Control Release 2009;137:110–5. 38. Liu W, Shen SM, Zhao XY, Chen GQ. Targeted genes and interacting proteins of hypoxia inducible factor-1. Int J Biochem Mol Biol 2012; 3(2):165–78. 39. Lee SH, Lee YJ, Han HJ. Role of hypoxia-induced fibronectin-integrin beta1 expression in embryonic stem cell proliferation and migration: involvement of PI3K/Akt and FAK. J Cell Physiol 2011;226:484–93. 40. Jung SN, Yang WK, Kim J, Kim HS, Kim EJ, Yun H, et al. Reactive oxygen species stabilize hypoxia-inducible factor-1 alpha protein and stimulate transcriptional activity via AMP-activated protein kinase in DU145 human prostate cancer cells. Carcinogenesis 2008; 29(4):713–21. 41. Hoesel B, Schmid JA. The complexity of NF-kB signaling in inflammation and cancer. Mol Cancer 2013;12:86. 42. Chen F, Castranova V, Shi X. New insights into the role of nuclear factor-kB in cell growth regulation. Am J Pathol 2001;159(2):387–97. 43. Hang CH, Shi JX, Li JS, Li WQ, Yin HX. Up-regulation of intestinal nuclear factor kappa B and intercellular adhesion molecule-1 following traumatic brain injury in rats. World J Gastroenterol 2005;11(8):1149–54. 44. Giannoni E, Fiaschi T, Ramponi G, Chiarugi P. Redox regulation of anoikis resistance of metastatic prostate cancer cells: key role for Src and EGFRmediated pro-survival signals. Oncogene 2009;28:2074–86. 45. Svineng G, Ravuri C, Rikardsen O, Huseby NE, Winberg JO. The role of reactive oxygen species in integrin and matrix metalloproteinase expression and function. Connect Tissue Res 2008;49(3):197–202.

262 SECTION

B Antioxidants and cancer

46. Laurent A, Nicco C, Ch
ereau C, Goulvestre C, Alexandre J, Alves A, et al. Controlling tumor growth by modulating endogenous production of reactive oxygen species. Cancer Res 2005;65(3):948–56. 47. Oberley TD, Oberley LW. Antioxidant enzyme levels in cancer. Histol Histopathol 1997;2:525–35. 48. Beier K, V€ olkl A, Fahimi HD. NF-alpha downregulates the peroxisome proliferator activated receptor-alpha and the mRNAs encoding peroxisomal proteins in rat liver. FEBS Lett 1997;412(2):385–7. 49. Yang H, Villani RM, Wang H, Simpson MJ, Roberts MS, Tang M, et al. The role of cellular reactive oxygen species in cancer chemotherapy. J Exp Clin Cancer Res 2018;37:266. https://doi.org/10.1186/s13046-018-0909-x. 50. Sirtori CR. The pharmacology of statins. Pharmacol Res 2014;88:3–11. 51. Nakai D, Nakagomi R, Furuta Y, Tokui T, Abe T, Ikeda T, et al. Human liver-specific organic anion transporter, LST-1, mediates uptake of pravastatin by human hepatocytes. J Pharmacol Exp Ther 2001;297(3):861–7. 52. Corsini A, Bellosta S, Baetta R, Fumagalli R, Paoletti R, Bernini F. New insights into the pharmacodynamic and pharmacokinetic properties of statins. Pharmacol Ther 1999;84(3):413–28. 53. Igel M, Sudhop T, von Bergmann K. Metabolism and drug interactions of 3-hydroxy-3-methylglutaryl coenzyme A-reductase inhibitors (statins). Eur J Clin Pharmacol 2001;57(5):357–64. 54. Awad K, Serban MC, Penson P, Mikhailidis DP, Toth PP, Jones SR, et al. Effects of morning vs evening statin administration on lipid profile: A systematic review and meta-analysis. J Clin Lipidol 2017;11(4):972–85. 55. Allen SC, Mamotte CDS. Pleiotropic and adverse effects of statins-do epigenetics play a role? J Pharmacol Exp Ther 2017;362(2):319–26. 56. Sˇimi
c I, Reiner Zˇ. Adverse effects of statins—myths and reality. Curr Pharm Des 2015;21(9):1220–6. 57. Costa S, Reina-Couto M, Albino-Teixeira A, Sousa T. Statins and oxidative stress in chronic heart failure. Rev Port Cardiol 2016;35(1):41–57. 58. Liu A, Wu Q, Guo J, Ares I, Rodrı´guez JL, Martı´nez-Larran˜aga MR, et al. Statins: adverse reactions, oxidative stress and metabolic interactions. Pharmacol Ther 2019;195:54–84. 59. Motawi TK, Teleb ZA, El-Boghdady NA, Ibrahim SA. Effect of simvastatin and naringenin coadministration on rat liver DNA fragmentation and cytochrome P450 activity: an in vivo and in vitro study. J Physiol Biochem 2014;70(1):225–37. 60. Farag MM, Mohamed MB, Youssef EA. Assessment of hepatic function, oxidant/antioxidant status, and histopathological changes in rats treated with atorvastatin: effect of dose and acute intoxication with acetaminophen. Hum Exp Toxicol 2015;34(8):828–37. 61. Iannelli F, Lombardi R, Milone MR, Pucci B, De Rienzo S, Budillon A, et al. Targeting mevalonate pathway in cancer treatment: repurposing of statins. Recent Pat Anticancer Drug Discov 2018;13(2):184–200. 62. Santos CR, Schulze A. Lipid metabolism in cancer. FEBS J 2012;279(15):2610–23. 63. Tavintharan S, Ong CN, Jeyaseelan K, Sivakumar M, Lim SC, Sum CF. Simvastatin: a possible role in statin-induced hepatotoxicity? J Mol Med (Berl) 2008;86(12):1341–51. 64. Laezza C, Fiorentino L, Pisanti S, Gazzerro P, Caraglia M, Portella G, et al. Reduced mitochondrial coenzyme Q10 levels in HepG2 cells treated with high-dose Lovastatin induces apoptosis of k-ras-transformed thyroid cells via inhibition of ras farnesylation and by modulating redox state. Toxicol Appl Pharmacol 2007;223(2):173–9. 65. Sa´nchez CA, Rodrı´guez E, Varela E, Zapata E, Pa´ez A, Masso´ FA, et al. Statin-induced inhibition of MCF-7 breast cancer cell proliferation is related to cell cycle arrest and apoptotic and necrotic cell death mediated by an enhanced oxidative stress. Cancer Invest 2008;26(7):698–707. 66. Crescencio ME, Rodrı´guez E, Pa´ez A, Masso FA, Montan˜o LF, Lo´pez-Marure R. Statins inhibit the proliferation and induce cell death of human papilloma virus positive and negative cervical cancer cells. Int J Biomed Sci 2009;5(4):411–20. 67. Qi XF, Kim DH, Yoon YS, Kim SK, Cai DQ, Teng YC, et al. Involvement of oxidative stress in simvastatin-induced apoptosis of murine CT26 colon carcinoma cells. Toxicol Lett 2010;199(3):277–87. 68. Qi XF, Zheng L, Lee KJ, Kim DH, Kim CS, Cai DQ, et al. HMG-CoA reductase inhibitors induce apoptosis of lymphoma cells by promoting ROS generation and regulating Akt, Erk and p38 signals via suppression of mevalonate pathway. Cell Death Dis 2013;4:e518. 69. Li Y, Fu J, Yuan X, Hu C. Simvastatin inhibits the proliferation of A549 lung cancer cells through oxidative stress and up-regulation of SOD2. Pharmazie 2014 Aug;69(8):610–4. 70. Guterres FA, Martinez GR, Rocha ME, Winnischofer SM. Simvastatin rises reactive oxygen species levels and induces senescence in human melanoma cells by activation of p53/p21 pathway. Exp Cell Res 2013;319(19):2977–88. 71. Shi Y, Felley-Bosco E, Marti TM, Stahel RA. Differential effects of lovastatin on cisplatin responses in normal human mesothelial cells versus cancer cells: Implication for therapy. PLoS One 2012;7(9):e45354. 72. Teraoka N, Mutoh M, Takasu S, Ueno T, Yamamoto M, Sugimura T, et al. Inhibition of intestinal polyp formation by pitavastatin, a HMG-CoA reductase inhibitor. Cancer Prev Res (Phila) 2011;4(3):445–53. 73. Parada B, Reis F, Pinto A, Sereno J, Xavier-Cunha M, Neto P, et al. Chemopreventive efficacy of atorvastatin against nitrosamine-induced rat bladder cancer: antioxidant, anti-proliferative and anti-inflammatory properties. Int J Mol Sci 2012;13(7):8482–99. 74. Song X, Liu BC, Lu XY, Yang LL, Zhai YJ, Eaton AF, et al. Lovastatin inhibits human B lymphoma cell proliferation by reducing intracellular ROS and TRPC6 expression. Biochim Biophys Acta 2014;1843(5):894–901. 75. Alupei MC, Licarete E, Patras L, Banciu M. Liposomal simvastatin inhibits tumor growth via targeting tumor-associated macrophages-mediated oxidative stress. Cancer Lett 2015;356(2 Pt B):946–52. 76. Licarete E, Sesarman A, Rauca VF, Luput L, Patras L, Banciu M. HIF-1a acts as a molecular target for simvastatin cytotoxicity in B16.F10 melanoma cells cultured under chemically induced hypoxia. Oncol Lett 2017;13(5):3942–50.

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77. Jang HJ, Hong EM, Kim M, et al. Simvastatin induces heme oxygenase-1 via NF-E2-related factor 2 (Nrf2) activation through ERK and PI3K/Akt pathway in colon cancer. Oncotarget 2016;7(29):46219–29. 78. Yu H, Sun SQ, Gu XB, Wang W, Gao XS. Atorvastatin prolongs the lifespan of radiation-induced reactive oxygen species in PC-3 prostate cancer cells to enhance the cell killing effect. Oncol Rep 2017;37(4):2049–56. 79. Zdybel M, Chodurek E, Pilawa B. Effect of simvastatin in different concentrations on free radicals in A-2058 human melanoma malignum cells-EPR studies. J Cell Biochem 2018;11. 80. Karimi B, Ashrafi M, Shomali T, Yektaseresht A. Therapeutic effect of simvastatin on DMBA-induced breast cancer in mice. Fundam Clin Pharmacol 2019;33(1):84–93. 81. Alupei MC, Licarete E, Cristian FB, Banciu M. Cytotoxicity of lipophilic statins depends on their combined actions on HIF-1a expression and redox status in B16.F10 melanoma cells. Anticancer Drugs 2014;25(4):393–405.

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Chapter 24

Role of anthocyanins in oxidative stress and the prevention of cancer in the digestive system Elvira Gonzalez de Mejiaa, Miguel Rebollo-Hernanzb, Yolanda Aguilerab, and Maria A. Martı´n-Cabrejasb a

Department of Food Science and Human Nutrition, Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Champaign, IL,

United States, b Institute of Food Science Research, CIAL (UAM-CSIC), Department of Agricultural Chemistry and Food Science, Universidad Auto´noma de Madrid, Madrid, Spain

List of abbreviations ANC ANCD AQP3 CagA CI DPPH FRAP GIC GPx HAT HIF-1a HR MDA OR ORAC ROS RR SET SIRT SOD TEAC VEGF

anthocyanins anthocyanidin carcinogenesis-related protein aquaporin cytotoxin-associated gene A confidence interval 2,2-diphenyl-1-picrylhydrazyl ferric reducing antioxidant potential gastrointestinal cancer glutathione peroxidase hydrogen atom transfer-based hypoxia-related transcription factor hypoxia-inducible factor 1a hazard ratio malondialdehyde odds ratio oxygen radical absorbance capacity reactive oxygen species relative risk scavenging electron transfer-based tumor suppressor sirtuins superoxide dismutase Trolox equivalent antioxidant capacity vascular endothelial growth factor

Introduction Cancer is the main public health issue worldwide, being the second leading cause of death in the United States (US).1 Cancer is one of the diseases that has the most significant impact on society. Although cancer incidence has augmented over the years, its mortality has diminished due to the advances in treatment. Nevertheless, cancer prevention still needs to improve.2 Gastrointestinal cancer (GIC) is a comprehensive term for numerous types of cancers affecting the digestive system, which includes the esophagus, stomach, small intestine, colon, rectum, anus, liver, gallbladder, and pancreas.3 Table 1 summarizes the estimated statistics of new cases of GIC in the United States in 2019 by sex and cancer type. In total, approximately 328,030 new GIC cases will be identified, representing 18.6% of cancer diagnosed. From all the GICs, most cases are associated with colorectal, pancreas, and liver cancer. Similarly, most deaths are associated with colorectal and pancreatic cancer. Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00024-9 © 2021 Elsevier Inc. All rights reserved.

265

266 SECTION

B Antioxidants and cancer

TABLE 1 Projected cancer statistics in the digestive system (2019). Estimated new cases

Estimated deaths

Male

Female

Both sexes

Male

Female

Both sexes

Digestive system

186,080 (21.4%)

141,950 (15.9%)

328,030 (18.6%)

97,110 (30.2%)

68,350 (24.0%)

165,460 (27.3%)

Esophagus

7.4

2.7

5.4

13.4

4.5

9.7

Stomach

9.3

7.2

8.4

7.0

6.3

6.7

Small intestine

3.0

3.5

3.2

0.9

1.0

1.0

Colon

27.8

35.0

30.9

28.5

34.2

30.8

Rectum

14.4

12.2

13.5

–

–

–

Anus, anal canal, and anorectum

1.5

3.9

2.5

0.5

1.1

0.8

Liver and intrahepatic bile duct

15.8

8.8

12.8

22.2

14.9

19.2

Gallbladder and other biliary

3.1

4.6

3.8

1.7

3.4

2.4

a

Pancreas

16.1

18.9

17.3

24.5

32.1

27.7

Other digestive organs

1.6

3.0

2.2

1.3

2.4

1.7

Projected cancer statistics for 2019 classified based on the digestive system, by sex, including estimated new cases and estimated deaths. Data are expressed as a percentage of the total cases of gastrointestinal cancer. a Deaths for colon and rectal cancers are combined because many deaths from rectal cancer are misclassified as colon cancer. Adapted from Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69:7–34.

Diet can be considered as one of the lifestyle factors that most influences GIC cancer incidence and mortality. Numerous reports have stated that vegetables and fruits-based diets are strongly associated with a significant reduction in GIC cancer risk.4, 5 Phytochemicals present in plant foods, associated with potential health benefits related to the prevention of cancer, are flavonoids, phenolic acids, stilbenes, lignans, and carotenoids.6 Flavonoids are a large group of phenolic compounds and are usually involved in the protection of plants against external hazards. They are gaining interest due to their potent antioxidant activity against oxidative stress and potentially related disorders.7 Both in vitro and in vivo studies have confirmed that flavonoids possess anticarcinogenic potential. Likewise, numerous population-based studies have described the relationship between the intake of flavonoids and reduced cancer risk.8 Anthocyanins (ANC) are recognized as one of the main classes of flavonoids, water-soluble plant pigments, responsible for the deep purple/red/blue colors observed in plantbased foods.9 Epidemiological studies have proposed that the consumption of ANC may lower the risk of cancer due, to some extent, to their antioxidant and antiinflammatory properties.10 Hence, this chapter aimed to review the literature regarding the potential of consuming ANC and food rich in these phytochemicals in the prevention of oxidative stress and cancer in the digestive system.

Applications to other cancers In this chapter, we reviewed the role of anthocyanins in the prevention of cancer of the digestive system. We clearly showed that ANC from different sources are efficient scavengers of free radicals related to GIC protection. We also showed that ANC could reduce oxidative stress via direct scavenging of different radicals and the regulation of the antioxidant cellular defense response. Anthocyanidins and ANC also possess the ability to prevent GIC through mechanisms different from oxidative stress regulation. ANC inhibit cell proliferation modulating the cell cycle. ANC can selectively arrest the proliferation of cancer cells without changing the proliferation of nontumor cells. ANC can elicit cancer cell apoptosis via the internal mitochondrial pathway (inducing the release of cytochrome c and the modulation of proapoptotic proteins), and the external death receptor pathway (enhancing the expression of FAS and its ligand, FASL). ANC can also suppress angiogenesis by reducing the expression of VEGF and VEGF receptors. ANC also decrease the expression of matrix

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metalloproteinase and urokinase plasminogen activator, thus inhibiting cancer cell invasion. The literature reports other potential health benefits of anthocyanins in the prevention of diabetes, obesity, and other cancers apart from GIC.11 There is a clear need for interventional studies that consider an appropriate characterization and quantification of ANC in the foods of interest. Also, there is a need to better comprehend the mechanisms of action of ANC from foods on gastrointestinal cancer prevention.

Oxidative stress and gastrointestinal cancer Oxidative stress Oxidative metabolism is indispensable for the survival of the cells. The production of energy connected with the catabolism of nutrients, the detoxification of many xenobiotics, and the immune response is linked to oxidative processes.12 Besides these processes, oxygen is implied in the production of highly reactive mediators, known as reactive oxygen species (ROS). Most ROS are free radicals, molecules possessing unpaired electrons at a higher energy level, and high reactivity.13 Environmental, dietary, and physiological factors can produce a redox imbalance, prompting oxidation, which may originate what is known as oxidative stress. If ROS are overproduced, endogenous or exogenous systems may not control the excess; then, oxidation of lipidic membranes, low-density lipoproteins, cellular macromolecules, such as proteins and DNA, can occur. Antioxidant defense systems act on the substrates susceptible to oxidation under tight control to preserve the physiological redox equilibrium. The defense activity of some enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, might be initiated under redox imbalances.14 Oxidative stress has a primary causative role in the development of many chronic diseases and aging. Thus, ascertaining the role antioxidants play in delaying oxidation, results in either the prevention or the retardation of the oxidative stress; this attracts research efforts and resources.15 Chronic ROS conduce to several events associated with inflammation and carcinogenesis, including DNA damage, gene expression changes, and tissue remodeling. Moreover, lipid peroxidation generates malondialdehyde (MDA), which induces angiogenesis and the activation of carcinogenic signaling pathways (Wnt/b-catenin and PI3K/Akt).16 Along with these cellular carcinogenic activities, oxidative stress produced by cytotoxic anticancer agents can damage and destroy cancer cells. Therefore, these opposing facts generate confusion in regard to the regulation of oxidative stress in cancer management.

Oxidative stress in gastric cancer During the development of gastric cancer, Helicobacter pylori stimulates ROS generation in chronic gastritis patients. The presence of CagA (cytotoxin-associated gene A) protein, which is localized in the mitochondria, has been proposed as a cause of ROS production (Fig. 1A). CagA protein may induce inflammation and carcinogenesis through oxidative stress, not only causing DNA damage but also preventing the adequate functioning of DNA reparation mechanisms. CagA protein also upregulates Wnt/b-catenin signaling, increasing cellular proliferation, which results in carcinogenesis.17 CagA protein-derived ROS induce increased hypoxia-inducible factor 1a (HIF-1a) expression, through the suppression of the tumor suppressor sirtuins (SIRT) 3.18 Aquaporin 3 (AQP3) promoter, a carcinogenesis-related protein, may be regulated by HIF-1a. The ROS-HIF-1a-AQP3-ROS loop may thus be an essential factor leading to the development of gastric cancer.19 In the cancer states, the elicitation of oxidative stress is included in anticancer responses.20 Cancer cells are resistant to treatments owing to their enhanced protection against ROS. Cancer stem cells are characterized for escaping from ROS, which denotes the essential role of oxidative stress in the control of cancer progression and the necessity for managing it appropriately.21

Oxidative stress in liver cancer During liver cancer development, hepatitis B virus, hepatitis C virus, or lipids (in the liver of nonalcoholic fatty liver disease) prompt ROS production, which results in chronic inflammation, liver fibrosis, and hepatocarcinogenesis (Fig. 1B).22, 23 Once hepatocarcinogenesis has been developed, the role of oxidative stress changes. An appropriate level of oxidative stress ought to be sustained in order to control the progression of liver carcinogenesis. The management of chronic hepatitis and the subsequent hepatocarcinogenesis without impairing the physiological roles of oxidative stress is a challenging requirement when looking for antioxidant therapeutic compounds.

268 SECTION

B Antioxidants and cancer

H. pylori

HBV HCV NAFLD

Inflammation Fibrosis

Fatty acids CagA

ROS

ROS

Cellular damage

Cellular damage AQP3 HIF-1α

Carcinogenesis

(A)

AMPK

Wnt/β-catenin

Carcinogenesis

Gastric cells

Hepatic cells

(B) Sporadic colon cancer Alcohol Obesity Lifestyle

Cellular damage

Colitic colon cancer Intestinal bowel disease-derived inflammation

ROS

NF-κB

Carcinogenesis

(C)

Colon cells

FIG. 1 Different stressors generate oxidative stress in the digestive system; not being controlled, ROS lead to carcinogenesis. Molecular mechanism of oxidative stress in the development of gastric (A), hepatic (B), and colon (C) cancers.

Oxidative stress in colorectal cancer ROS also has a crucial role in the initiation of colon cancer. In sporadic-type colorectal cancer, stressors such as certain foods and alcohol consumption, or obesity have been described to promote colon carcinogenesis (Fig. 1C). The gastrointestinal tract is a source of oxidative stress being continuously exposed to ingested materials and pathogens, including bacteria.24 Cancer cells present elevated oxidative stress levels compared to healthy cells, which is associated with their enhanced metabolic activity.20 Plasmatic oxidative stress levels are correspondingly increased.25 In colitic cancer, inflammatory bowel disease-related inflammation can induce ROS, which promotes mucosal lesions.26 Even if oxidative stress is connected with the development of colorectal cancer derived from colitis, the molecular mechanism of carcinogenesis in inflammatory bowel disease remains unclear.27 Noncancerous cells produce exacerbated levels of antioxidant enzymes, possibly as a defense mechanism against cancer-producing oxidative stress. The expression of antioxidant-related enzymes is activated in colorectal tumor

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samples and adjacent nontumor tissues, indicating that adjacent noncancerous cells assist in reducing the ROS produced cancer cells.28 Given that oxidative stress conduces preferably to the death of cancer cells, the use of oxidants is an approach for the treatment of colorectal cancer. These opposing effects need consideration during anticancer therapies aiming at the modulating of oxidative stress. Consequently, future research will need to elucidate the molecular mechanisms whereby antioxidants act in colorectal cancer.

Oxidative stress in pancreatic cancer Pancreatic cancer exhibits poor prognoses despite new therapies developed. Pancreatic cancer is associated with obesity, other cancers, and oxidative stress conditions.29, 30 The maintenance of redox homeostasis in pancreatic cancer patients is essential as they tend to loss of skeletal muscle mass after chemotherapy and surgery treatments.31 Oxidative stress plays a key role in the cancer microenvironment, but the control of oxidative stress in other pancreas-related cancer, such as pancreaticobiliary tract cancers, remains controversial.32 The use of antioxidants in pancreatic cancer must contemplate the oxidative stress-related environment. The management of carcinogenesis without altering the physiological functions of oxidative stress can be problematic.

Anthocyanins: Properties and dietary sources Chemistry of anthocyanins ANC are a class of phenolic compounds responsible for the bright red, blue, and purple colors of fruits and vegetables. ANC are water-soluble glycosides, and acyl-glycosides of anthocyanidins.33 The most common sugar moieties attached to the ANC aglycone are glucose, galactose, and rhamnose, but aglycones might be found combined with other sugars.34 ANC aglycones can be classified into six common types, namely pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin, based on the different substituents on flavylium B-ring (Fig. 2). Furthermore, sugars bound to the ANC aglycone can be similarly attached to other saccharides through glycosidic bonds or acylated with organic aromatic or aliphatic acids.35

Food sources ANC can be found in various types of frequently consumed plant foods, viz., fruits, vegetables, cereals, nuts, and legumes. Likewise, fruit-based beverages obtained from wines, and juices, may also present high concentration of ANC.36 Fruits have considerable content of ANC in comparison to the other plant foods. Specifically, berries as ac¸ai, blackberry, blueberry, chokeberry, cranberry, elderberry, raspberry, and strawberry are good sources of ANC. Also, red and black currants exhibit a high concentration of ANC. Among vegetables, red cabbage, eggplant, radicchio, radishes, and purple sweet potatoes exhibit a high content of ANC. Even though nuts and seeds cannot be considered as sources of ANC, if compared to fruits and vegetables, they contain some ANC, cyanidin is present primarily in almonds, hazelnuts, pecans, pistachios, and walnuts. Among cereals, purple wheat, purple and black rice, and purple and red corn are the most highlighted sources. Finally, legumes such as black and red kidney beans, and black cowpeas contain a very high concentration of ANC.

Bioavailability and metabolism Once ANC are consumed, only a small fraction can be absorbed after suffering stomach and small intestine digestion (up to 35%).37 Nonetheless, ANC might exert biological activity along their transit through the gastrointestinal tract and are transformed by colonic microbiota reaching the large intestine.38 Fig. 2C depicts the main structures found during ANC absorption and metabolism. After food consumption, ANC are partially degraded by saliva. Then they enter in the stomach, where they are rapidly absorbed, being identified in blood and urine as ANC, anthocyanidin, methylated, glucuronidated, and sulfonated.39 In the small intestine, the stability of ANC is reduced due to the neutral or mildly alkaline conditions. Absorption occurs mostly in the jejunum, in the form of anthocyanidin due to the hydrolytic activity of enzymes present there. Nonetheless, a small amount of ANC is absorbed in the duodenum, whereas there is no absorption in the ileum and the colon.40 ANC can be hydrolyzed to anthocyanidins mainly by lactase phlorizin hydrolase on the brush border of the epithelial cells or by b-glucosidase inside epithelial cells.41 ANC can be absorbed utilizing a glucose transporter while the hydrophobic anthocyanidins are more likely to enter the enterocyte through passive diffusion.42 After reaching circulation, ANC are transported to the liver where they are further metabolized. Along the gastrointestinal tract, ANC are substrates for several enzymes located in the small intestine, the colon, and then the liver, including hydrolyzing and conjugating

270 SECTION

B Antioxidants and cancer

FIG. 2 Representative ANC in different fruits and their metabolic transformations throughout the GI tract. Chemical structure of anthocyanidins (A), examples of food sources indicating an average concentration in each fruit (B), and the chemical species formed in different parts of the body (C). Adapted from Tian L, Tan Y, Chen G, Wang G, Sun J, Ou S, Chen W, Bai W. Metabolism of anthocyanins and consequent effects on the gut microbiota. Crit Rev Food Sci Nutr 2019;59:982–91.

enzymes, phase I and phase II, respectively.38 Finally, unabsorbed ANC are metabolized by the intestinal microbiota producing diverse compounds, including phenolics such as protocatechuic and gallic acids. The permeability of ANC across the gastrointestinal mucosa is considered quite high. ANC are found in high concentrations in intestinal tissues compared to their reduced circulating concentration. ANC in gastrointestinal tissues may achieve mM concentrations. Thus, ANC could reach biologically active concentrations in gastrointestinal tissues and exhibiting their protective effects before reaching circulation.43 The most recent results emphasize that not only the presence of ANC in plasma imposes their health effects but also their metabolites. Metabolites produced during digestion, mainly in the colon, by intestinal microbiota, are also absorbed and present in the gastrointestinal tract. Hence, the presence of ANC and their metabolites in the gastrointestinal tract, and then in liver and blood, suggest that ANC might have an impact on GIC rather than in other cancers, due to their presence in higher concentrations. Nevertheless, the elucidation of the compounds (parent ANC or metabolites) responsible for the exerted effects warrants further research.

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Role of anthocyanins in the prevention of oxidative stress ROS are generated in cells during physiological processes and play a role in body functions in cell signaling, the immune system, among others. Nonetheless, they can promote cellular damage, which conduces to degenerative diseases, including cancer, if ROS are excessively produced.44 Oxidative stress is developed when redox homeostasis is off-balance. Oxidative stress is a deregulated state in which the overproduction of ROS/reactive nitrogen species (RNS) overcome endogenous antioxidant capacity, conducing to the oxidation of proteins, DNA, and lipids. Along with aging, this balance is inclined in favor of the oxidants.45 The intake of dietary antioxidants, ANC, among others, is essential to control an equilibrium between oxidants and antioxidants.

Direct chemical mechanisms The effectiveness of ANC as potent antioxidants has been evaluated both in vitro and in vivo. ANC can scavenge free radicals and block the chain reaction causing the beginning of oxidative damage (Fig. 3A).46 The antioxidant activity of ANC is linked to their phenolic structure. ANC are able to quench ROS, such as superoxide ðO2 ∙
Þ, singlet oxygen (1O2), hydroxyl radical (OH∙), peroxide (ROO∙), and hydrogen peroxide (H2O2).47 The antioxidant capacity of ANC present in diverse fruits has been proved with a wide variety of assays following different chemical mechanisms, among them, oxygen-radical absorbance capacity (ORAC), hydrogen transfer-based (HAT), ferric-reducing antioxidant potential (FRAP), trolox equivalent antioxidant capacity (TEAC), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging activity. The assays are centered on electron transfer-based (SET); scavenging activity toward superoxide;

(A)

(B) FIG. 3 Anthocyanins are multimechanistic antioxidants, acting via direct and indirect pathways. In healthy cells, the control of oxidative stress lead to cancer prevention; however, in cancer cells, anthocyanins antioxidant effects might be counterproductive, diminishing the efficacy of the therapy by increasing cells defenses. Antioxidant mechanisms of anthocyanins (A) and molecular implications in oxidative stress in healthy and cancer cells (B).

272 SECTION

B Antioxidants and cancer

peroxynitrite (ONOO
) scavenging activity; inhibition of human low-density lipoprotein oxidation); inhibition of lipid peroxidation; and ability to chelate heavy metals such as iron, zinc, and copper. ANC and anthocyanidins are highly reactive toward ROS because of their electron deficiency. Nevertheless, ANC antioxidant activity depends on their chemical substitutions, having different activities for scavenging diverse ROS and RNS according to the different functional groups on their flavylium B-ring.48 The antioxidant capacity of ANC is associated to their structural orientation; hydroxyl groups efficiency to donate an electron from its hydrogen atom to a free radical and stabilize the unpaired electron depends on the ring orientation.49 Generally, there is a relationship between the number of free hydroxyls around the pyrone ring and the ANC antioxidant capacity; the higher number of hydroxyls, the higher antioxidant capacity. Likewise, ANC antioxidant capacity is linked to glycosylation. ANC-radical scavenger capacity is lower than that of the corresponding anthocyanidin, due to the reduced ability of the ANC radical to delocalize electrons.50 The contribution of the ANC flavylium ring functional groups to the efficiency of antioxidant activity is –OH > –OCH3 ≫ –H. Hence, anthocyanidin’s antioxidant capacity diverges in the order: delphinidin > petunidin > malvidin 5 cyanidin > peonidin > pelargonidin.51

Indirect molecular mechanisms ANC can also stimulate endogenous antioxidant defenses in the cell via different mechanisms (Fig. 3A). ANC enhance or restore the activity of the antioxidant enzymes SOD and GPx, therefore increasing glutathione content. Moreover, ANC reduce the formation of oxidative adducts in DNA and endogenous ROS by inhibiting NADPH oxidase and xanthine oxidase, or by modifying mitochondrial respiration.52 Indirect ANC antioxidant effects derived from the activation of the NF-E2-related factor 2 (Nfr2)-antioxidant response element (ARE) pathway result in the transcription of genes that code for these enzymes (Fig. 3B). The boosting effect of ANC on ARE-regulated phase II enzyme expression is essential for the defense of cells against oxidative stress. Even if oxidative stress is enough for activating Nrf2-ARE signaling pathway and therefore induce the transcription of Nrf2 target genes, ANC can promote oxidative stress-mediated activation due to their specific redox cycling properties.53 Nrf2 activation by redox-sensitive cofactors such as ANC might be considered as an attractive cancer prevention strategy.

Role of anthocyanins in the prevention of gastrointestinal cancer Fruit and vegetable sources of ANC have been proposed as preventive agents for GIC, due to their chemopreventive or chemotherapeutic properties. Research on the anticancer properties of the ANC comprise evidence from in vitro cell culture and in vivo animal models, along with results from human epidemiological and clinical studies. ANC, preventing ROS overproduction and activating antioxidant defenses (Nfr2-ARE pathway), could prevent cancer initiation and then repress carcinogenesis (Fig. 3B).54 Regardless of the ANC crucial role in cancer prevention by ROS scavenging or the regulation of defense response, in cancerous cells and tissues, a completely opposite situation is found. ROS induce cell death, and Nrf2ARE induction can provide growth advantage to cancer cells, thereby triggering resistance to chemotherapies. Despite the positive results observed using cell culture and animal models,55 epidemiological studies have presented limited evidence on the efficacy of ANC in the prevention of GIC. Table 2 summarizes the most recent studies on the association of ANC consumption, among other flavonoids, and the development of esophageal, gastric, colorectal, hepatic, or pancreatic cancer. From the three studies found on esophageal cancer, only one suggested that ANC could significantly reduce the risk of esophageal cancer. Only one out of seven studies proposed a negative association between ANC intake and reduced gastric cancer risk. Nonetheless, regarding colorectal cancer, positive effects associated with ANC intake can be abstracted from Table 2. Five out of the six studies projected a reduced risk of colorectal cancer associated with the consumption of ANC. Only one study suggested a reduced risk of hepatic cancer and none studies for pancreatic cancer. From those results, the effectiveness of ANC on GIC cancer needs further clarification. Literature only suggests that the consumption of ANC could be beneficial in the prevention of colorectal cancer. Hence, more studies will be needed to confirm these effects. Limited clinical research can be found on the impact of consuming ANC and ANC-rich foods on GIC prevention/alleviation. Most studies have been conducted on colorectal cancer patients, as observed in Table 3. Only eight clinical studies were found on the effects of ANC-rich foods on GIC cancer. Two of those studies were carried out on patients of esophageal cancer. The consumption of black raspberries and strawberries was associated with a reduction of oxidative stress and inflammatory marker, respectively. On the other hand, six studies investigated ANC effects on colorectal cancer. Four out of those six studies evaluated the effects of black raspberries intake. In general, the consumption of ANC-rich berries reduced the proliferation and increased

TABLE 2 Epidemiological studies on the effects of ANC on esophageal, gastric, colorectal, hepatic, and pancreatic cancers. Methods

Foods/ Compounds

Results

References

Esophageal cancer Flavonoids

Data suggested that total flavonoid intake was not associated with a reduced risk of esophageal cancer

Adenocarcinoma HR: 1.03, 95% CI: 0.82–1.29 Squamous cell carcinoma HR: 0.86, 95% CI: 0.58–1.27

56

ANC

Data suggested that total anthocyanin intake was not associated with a reduced risk of esophageal cancer

Adenocarcinoma HR: 0.89, 95% CI: 0.70–1.11 Squamous cell carcinoma HR: 0.68, 95% CI: 0.46–1.00

56

Flavonoids

Data suggested that total flavonoid intake was not associated with a reduced risk of esophageal cancer

Adenocarcinoma OR: 0.92, 95% CI: 0.63–1.37 Squamous cell carcinoma OR: 0.87, 95% CI: 0.53–1.41

57

ANC

Data suggested that total anthocyanin intake was associated with a reduced risk of esophageal cancer

Adenocarcinoma HR: 0.43, 95% CI: 0.29–0.66 Squamous cell carcinoma HR: 0.43, 95% CI: 0.26–0.70

57

Flavonoids

Total flavonoid intake was associated with a reduced risk of esophageal cancer

HR: 0.70, 95% CI: 0.50–0.99 HRlog2: 0.87, 95% CI: 0.78–0.98

58

ANC

Data suggested that total anthocyanin intake was not associated with a reduced risk of esophageal cancer

HR: 0.82, 95% CI: 0.57–1.19 HRlog2: 0.92, 95% CI: 0.83–1.02

58

Case-control study 329 cases and 2700 controls aged 20–85 years, from Spain Food frequency questionnaires Phenol Explorer Database Analyzed in quartiles of flavonoid subclasses

Flavonoids

Total flavonoid intake was associated with a reduced risk of gastric cancer

OR: 0.60, 95% CI: 0.40–0.89 ORlog2: 0.76, 95% CI: 0.65–0.89

59

ANC

Data suggested that total anthocyanin intake was associated with a reduced risk of gastric cancer when using the log2 transformation of the flavonoid intake

OR: 0.68, 95% CI: 0.46–1.01 ORlog2: 088, 95% CI: 0.80–0.96

59

Prospective cohort study 469,008 participants 12 years follow up Food frequency questionnaires Analyzed in quintiles of flavonoid subclasses

Flavonoids

Data suggested that total flavonoid intake was not associated with a reduced risk of gastric cancer

Cardia HR: 1.02, 95% CI: 0.78–1.34 Noncardia HR: 1.11, 95% CI: 0.86–1.44

56

ANC

Data suggested that total anthocyanin intake was not associated with a reduced risk of gastric cancer

Cardia HR: 1.05, 95% CI: 0.80–1.39 Noncardia HR: 0.94, 95% CI: 0.72–1.23

56

Prospective cohort study 469,008 participants 12 years follow-up Food frequency questionnaires Analyzed in quintiles of flavonoid subclasses

Case-control study 274 cases and 662 controls aged 30–79 years, from USA Food frequency questionnaires Phenol Explorer Database Analyzed in quartiles of flavonoid subclasses Prospective cohort study 521,448 participants 11 years follow-up Food frequency questionnaires Analyzed in quartiles of flavonoid subclasses Gastric cancer

Continued

TABLE 2 Epidemiological studies on the effects of ANC on esophageal, gastric, colorectal, hepatic, and pancreatic cancers—cont’d Methods

Foods/ Compounds

Results

References

Flavonoids

Data suggested that total flavonoid intake was not associated with a reduced risk of gastric cancer

Cardia OR: 1.32, 95% CI: 0.87–2.00 Noncardia OR: 0.94, 95% CI: 0.72–1.23

57

ANC

Data suggested that total anthocyanin intake was not associated with a reduced risk of gastric cancer

Cardia OR: 0.71, 95% CI: 0.46–1.10 Noncardia OR: 0.70, 95% CI: 0.47–1.03

57

Case-control study 334 cases and 334 controls aged 35–75 years, from Korea Food frequency questionnaires USDA database Analyzed in tertiles of flavonoid subclasses

Flavonoids

Total flavonoid intake was associated with a reduced risk of gastric cancer (in general and in women but not in men)

OR: 0.49, 95% CI: 0.31–0.76

60

ANC

Total anthocyanin intake was not associated with a reduced risk of gastric cancer

OR: 0.73, 95% CI: 0.46–1.15

60

Observational study 477,312 subjects aged 35– 70 years, from 10 European countries Follow-up of 11 years Food frequency questionnaires USDA and Phenol Explorer Databases Analyzed in quartiles of flavonoid subclasses

Flavonoids

Total flavonoid intake was associated with a reduction in the risk of gastric adenocarcinoma only in women (HR: 0.81, 95% CI 0.70, 0.94)

Cardia HR: 0.84, 95% CI: 0.64–1.11 Noncardia HR: 0.85, 95% CI: 0.70–1.03

61

ANC

Total anthocyanin intake was not associated with a reduction in the risk of gastric adenocarcinoma

Cardia HR: 0.89, 95% CI: 0.69–1.15 Noncardia HR: 0.90, 95% CI: 0.89–1.04

61

Case-control study 230 cases and 547 controls aged 22–80 years, from Italy Food frequency questionnaires USDA database Analyzed in quintiles of flavonoid subclasses

ANC

Total anthocyanin intake was not associated with a reduction in the risk of gastric adenocarcinoma

OR: 0.91, 95% CI: 0.56–1.47

62

Meta-analysis of casecontrol or cohort studies 23 studies (out of 209): 13 case–control, 10 cohort

Flavonoids

Total flavonoid intake was not associated with a reduction in the risk of gastric adenocarcinoma

RR: 1.07, 95% CI: 0.70–1.61

63

ANC

Total anthocyanin intake was not associated with a reduction in the risk of gastric adenocarcinoma

RR: 1.03, 95% CI: 0.73–1.43

63

Case-control study 274 cases and 662 controls aged 30–79 years, from USA Food frequency questionnaires Phenol Explorer Database Analyzed in quartiles of flavonoid subclasses

Colorectal cancer Prospective cohort 42,478 male 76,364 female 26 years follow-up Food frequency questionnaires Analyzed in quintiles of flavonoid subclasses Case-control study 1632 cases and 1632 controls Food frequency questionnaires Analyzed in quartiles of flavonoid subclasses Case-control study 523 participants Food frequency questionnaires Analyzed in tertiles of flavonoid subclasses Case-control study 424 cases and 401 hospitalbased controls Food frequency questionnaires Phenol Explorer Database Analyzed in quartiles of flavonoid subclasses Metaanalysis of case-control or cohort studies 23 studies (out of 209): 13 case–control, 10 cohort Metaanalysis 18 studies (out of 1358) 9 case-control, 9 cohort

ANC

Total anthocyanin intake was associated with a reduction in the risk of colorectal only in men (RR: 0.78, 95% CI: 0.64, 0.94)

Colon RR: 0.95, 95% CI: 0.77–1.16 Rectum RR: 1.10, 95% CI: 0.83–1.45 Colorectal RR: 0.98, 95% CI: 0.81–1.19

11

Blueberries

Blueberries consumption was not associated with a reduction in the risk of colorectal cancer

Colon RR: 0.89, 95% CI: 0.766–1.20 Rectum RR: 1.22, 95% CI: 0.72–2.04 Colorectal RR: 0.95, 95% CI: 0.74–1.23

11

Flavonoids

Total flavonoid intake was not associated with a reduction in the risk of colorectal cancer

Colon OR: 0.92, 95% CI: 0.71–1.18 Rectum OR: 1.28, 95% CI: 0.96–1.72 Colorectal OR: 1.06, 95% CI: 0.85–1.32

64

ANC

Total anthocyanin intake was associated with a reduction in the risk of only colon cancer

Colon OR: 0.77, 95% CI: 0.59–0.99 Rectum OR: 0.86, 95% CI: 0.64–1.16 Colorectal OR: 0.80, 95% CI: 0.64–1.00

64

Flavonoids

Total flavonoid intake was not associated with colorectal cancer survival or recurrence

Overall survival HR: 0.97, 95% CI: 0.60– 1.56 Recurrence HR:0.87 95% CI: 0.47–1.62

65

ANC

Anthocyanin intake was not associated with colorectal cancer survival or recurrence

Overall survival HR:0.91, 95% CI: 0.58– 1.44 Recurrence HR:0.87, 95% CI: 0.48–1.57

65

Flavonoids

Total flavonoids intake was associated with a reduction in the risk of colon cancer and colorectal, but not rectum cancer

Colon OR: 0.55, 95% CI: 0.30–0.99 Rectum OR: 0.64, 95% CI: 0.32–1.29 Colorectal OR: 0.59, 95% CI: 0.35–0.99

66

ANC

Total anthocyanin intake was associated with a reduction in the risk of only colon cancer

Colon OR: 0.66, 95% CI: 0.59–0.99 Rectum OR: 1.02, 95% CI: 0.54–1.94 Colorectal OR: 0.75, 95% CI: 0.47–1.20

66

Flavonoids

Total flavonoid intake was not associated with a reduction in the risk of colorectal cancer

RR: 1.00, 95% CI: 0.90–1.11

63

ANC

Total anthocyanin intake was significantly associated with a reduction in the risk of colorectal cancer

RR: 0.68, 95% CI: 0.56–0.82

63

Flavonoids

Data suggested that total flavonoid intake was not associated with a reduced risk of colorectal cancer

OR: 0.94, 95% CI: 0.81–1.09

67

ANC

Total anthocyanin intake was associated with a reduction in the risk of gastric adenocarcinoma only in men (OR: 0.89, 95% CI: 0.82, 0.96)

Colon OR: 0.79, 95% CI: 0.61, 1.02 Rectum OR: 0.88, 95% CI: 0.67, 1.00 Colorectal OR:0.78, 95% CI: 0.61–1.01

67

Continued

TABLE 2 Epidemiological studies on the effects of ANC on esophageal, gastric, colorectal, hepatic, and pancreatic cancers—cont’d Methods

Foods/ Compounds

Results

References

Hepatic cancer Prospective cohort study 477,206 participants 9 years follow-up Food frequency questionnaires USDA and Phenol Explorer Databases Analyzed in tertiles of flavonoid subclasses Case-control study 339 cases and 360 hospitalbased controls Food frequency questionnaires USDA Database Analyzed in quintiles of flavonoid subclasses

Flavonoids

Total flavonoid intake was not associated with a reduction in the risk of liver cancer

HR: 0.65, 95% CI: 0.40–1.04

68

ANC

Total anthocyanin intake was significantly associated with a reduction in the risk of liver cancer

HR: 1.04, 95% CI: 0.90–1.21

68

Flavonoids

Data suggested that total flavonoid intake was not associated with a reduced risk of liver cancer

Hepatitis B and/or C virus positive hepatocellular carcinoma (HCC) OR: 1.22, 95% CI:0.69–2.16 HCC virus negative OR: 0.96, 95% CI: 0.43–2.12

69

ANC

Data suggested that total anthocyanin intake was not associated with a reduced risk of liver cancer

HCC virus positive OR:1.42, 95% CI: 0.81–2.47 HCC virus negative OR: 1.73, 95% CI: 0.74–4.04

69

Flavonoids

Data suggested that total flavonoid intake was not associated with a reduced risk of pancreatic cancer

HR: 0.95, 95% CI: 0.74–1.22

70

ANC

Data suggested that total anthocyanin intake was not associated with a reduced risk of pancreatic cancer

HR: 0.93, 95% CI: 0.72–1.22

70

Pancreatic cancer Prospective cohort 477,309 participants Average Follow-up of 11 years Food frequency questionnaires USDA and Phenol Explorer Databases

Epidemiological studies on the effects of ANC on esophageal, gastric, colorectal, hepatic, and pancreatic cancers. The specific methods of the study are mentioned as well as the food components, type of cancer and most significant results. CI, confidence interval; HR, hazard ratio; OR, odds ratio; RR, relative risk.

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TABLE 3 Effect of ANC on esophageal, gastric, colorectal, hepatic, and pancreatic cancers. Foods/Compounds

Gastrointestinal cancer subtype

Results

References

Black raspberries

Esophageal cancer

Urinary excretion of two, 8-epi-prostaglandin F2a and8hydroxy-20 -deoxyguanosine (8-OHdG), markers of oxidative stress, among patients with esophageal cancer

71

Strawberries

Esophageal cancer

Strawberry powder reduced the histologic grade of dysplastic premalignant lesions in 80.6% of the patients and reduced protein expression levels of iNOS, COX-2, pNFkB-p65, and pS6

72

Black raspberries

Colorectal cancer

Berries consumption reduced the proliferation rates and increased apoptosis in colon tumors but not in normalappearing tissues (crypts)

73

Black raspberries

Colorectal cancer

Black raspberries demethylated tumor suppressor genes and modulated other biomarkers of tumor development in the human colon and rectum

74

Black raspberries

Colorectal cancer

The intake of black raspberries diminished the burden of polyps decreasing cellular proliferation, DNA methylation methyl transferase 1 protein expression, and p16 promoter methylation

75

Black raspberries

Colorectal cancer

Black raspberries consumption induced significant metabolic changes and affect energy-generating pathways

76

Bilberries

Colorectal cancer

Bilberries intake was associated with a decrease in the proliferation of the tumor tissue

77

Dietary supplement from vegetable and fruits rich in color compounds

Colorectal cancer

The treatment did not change the tumor marker levels

78

Clinical human studies on the effect of ANC on esophageal, gastric, colorectal, hepatic, and pancreatic cancers. The specific foods, type of cancer, and most significant results are indicated.

apoptosis in colon tumors. Potential mechanisms were demethylation of tumor suppressor genes and modulation of other biomarkers of tumor development, through the reduction DNA methylation methyl transferase one protein expression, and p16 promoter methylation. Moreover, those changes were associated with changes in energy-generating pathways. Finally, another clinical study associated bilberries intake with a decrease in the proliferation of tumors.

Conclusions Evidence has demonstrated the effectiveness of ANC as antioxidants with potential effects on the prevention of oxidative stress and related diseases such as gastrointestinal cancer. Beneficial effects, both on oxidative stress and carcinogenesis in gastrointestinal tissues, have been described in studies using cancer cell lines in vitro and animal models in vivo. However, the absence of significant epidemiological evidence generates uncertainties about the actual impact of ANC consumption on gastrointestinal cancers. The consumption of anthocyanin-rich foods has only evidenced significant positive effects on colorectal cancer in human clinical studies. Further understanding integrating the metabolism of ANC through the gastrointestinal tract and their biological activity in the different tissues is still required. Furthermore, the need for double-blind, cross-over, placebo-control clinical studies on the efficacy of ANC, their metabolites, and foods containing them is reflected in this chapter.

Summary points l

Gastrointestinal cancer represents 27% of the deaths caused by cancer in the United States being colorectal and pancreatic cancer the most representative ones.

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l

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Oxidative stress is one of the main causes of gastrointestinal cancer, inducing DNA mutations that lead to carcinogenesis. However, the increase of oxidative stress in cancer cells is also a strategy to eliminate cancerous cells. ANC are a group of flavonoids with bright red, purple, and blue color that can be found in diverse plant foods like fruits, legumes, and cereals. ANC are antioxidant molecules able to reduce oxidative stress via direct scavenging of different radicals and the regulation of the antioxidant cellular defense response. Epidemiological studies have associated ANC consumption with a decrease in the incidence of colorectal cancer. Limited clinical research suggests that anthocyanin-rich food intake would be beneficial in the control of tumor progression in colorectal cancers.

References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69:7–34. 2. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394–424. 3. Ashktorab H, Kupfer SS, Brim H, Carethers JM. Racial disparity in gastrointestinal cancer risk. Gastroenterology 2017;153:910–23. 4. Afshin A, Sur PJ, Fay KA, Cornaby L, Ferrara G, Salama JS, et al. Health effects of dietary risks in 195 countries, 1990–2017: a systematic analysis for the global burden of disease study 2017. Lancet 2019;393:1958–72. 5. Giovannucci E. Nutritional epidemiology and cancer: a tale of two cities. Cancer Causes Control 2018;29:1007–14. 6. Fraga CG, Croft KD, Kennedy DO, Toma´s-Barbera´n FA. The effects of polyphenols and other bioactives on human health. Food Funct 2019;10:514–28. 7. Tungmunnithum D, Thongboonyou A, Pholboon A, Yangsabai A. Flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: an overview. Medicines 2018;5(3):93. 8. Rodrı´guez-Garcı´a C, Sa´nchez-Quesada C, Gaforio JJ. Dietary flavonoids as cancer chemopreventive agents: an updated review of human studies. Antioxidants 2019;8(5):137. 9. Khoo HE, Azlan A, Tang ST, Lim SM. Anthocyanidins and anthocyanins: colored pigments as food, pharmaceutical ingredients, and the potential health benefits. Food Nutr Res 2017;61(1):1361779. 10. Lin B-W, Gong C-C, Song H-F, Cui Y-Y. Effects of anthocyanins on the prevention and treatment of cancer. Br J Pharmacol 2017;174:1226–43.  Lin JH, et al. Habitual intake of flavonoid subclasses and risk of colorectal cancer in 2 large 11. Nimptsch K, Zhang X, Cassidy A, Song M, O’Reilly EJ, prospective cohorts. Am J Clin Nutr 2016;103:184–91. 12. Kurutas EB. The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. Nutr J 2016;15 (1):71. 13. Galadari S, Rahman A, Pallichankandy S, Thayyullathil F. Reactive oxygen species and cancer paradox: to promote or to suppress? Free Radic Biol Med 2017;104:144–64. 14. Rahal A, Kumar A, Singh V, Yadav B, Tiwari R, Chakraborty S, et al. Oxidative stress, prooxidants, and antioxidants: the interplay. Biomed Res Int 2014;2014:761264. 15. Halliwell B. The antioxidant paradox: less paradoxical now? Br J Clin Pharmacol 2013;75:637–44. 16. Uchida K. HNE as an inducer of COX-2. Free Radic Biol Med 2017;111:169–72. 17. Yong X, Tang B, Xiao Y-F, Xie R, Qin Y, Luo G, et al. Helicobacter pylori upregulates Nanog and Oct4 via Wnt/b-catenin signaling pathway to promote cancer stem cell-like properties in human gastric cancer. Cancer Lett 2016;374:292–303. 18. Lee DY, Jung DE, Yu SS, Lee YS, Choi BK, Lee YC. Regulation of SIRT3 signal related metabolic reprogramming in gastric cancer by Helicobacter pylori oncoprotein CagA. Oncotarget 2017;8:78365–78. 19. Wen J, Wang Y, Gao C, Zhang G, You Q, Zhang W, et al. Helicobacter pylori infection promotes aquaporin 3 expression via the ROS–HIF-1a–AQP3– ROS loop in stomach mucosa: a potential novel mechanism for cancer pathogenesis. Oncogene 2018;37:3549–61. 20. Gorrini C, Harris IS, Mak TW. Modulation of oxidative stress as an anticancer strategy. Nat Rev Drug Discov 2013;12:931–47. 21. Zhou D, Shao L, Spitz DR. Reactive oxygen species in normal and tumor stem cells. Adv Cancer Res 2014;122:1–67. 22. Nishimura M, Takaki A, Tamaki N, Maruyama T, Onishi H, Kobayashi S, et al. Serum oxidative-anti-oxidative stress balance is dysregulated in patients with hepatitis C virus-related hepatocellular carcinoma. Hepatol Res 2013;43(10):1078–92. 23. Gadd VL, Skoien R, Powell EE, Fagan KJ, Winterford C, Horsfall L, et al. The portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease. Hepatology 2014;59:1393–405. 24. Bhattacharyya A, Chattopadhyay R, Mitra S, Crowe SE. Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases. Physiol Rev 2014;94:329–54. 25. Kong SYJ, Bostick RM, Flanders WD, McClellan WM, Thyagarajan B, Gross MD, et al. Oxidative balance score, colorectal adenoma, and markers of oxidative stress and inflammation. Cancer Epidemiol Biomarkers Prev 2014;23:545–54. 26. Naito Y, Takagi T, Yoshikawa T. Molecular fingerprints of neutrophil-dependent oxidative stress in inflammatory bowel disease. J Gastroenterol 2007;42:787–98.

ANC: Oxidative stress and cancer prevention Chapter

24

279

27. Takaki A, Kawano S, Uchida D, Takahara M, Hiraoka S, Okada H. Paradoxical roles of oxidative stress response in the digestive system before and after carcinogenesis. Cancers (Basel) 2019;11(2):213. 28. Herna´ndez-Lo´pez R, Torrens-Mas M, Pons DG, Company MM, Falco´ E, Ferna´ndez T, et al. Non-tumor adjacent tissue of advanced stage from CRC shows activated antioxidant response. Free Radic Biol Med 2018;126:249–58. 29. Pericleous M, Rossi RE, Mandair D, Whyand T, Caplin ME. Nutrition and pancreatic cancer. Anticancer Res 2014;34:9–21. 30. Zhang Y, Yan W, Collins MA, Bednar F, Rakshit S, Zetter BR, et al. Interleukin-6 is required for pancreatic cancer progression by promoting MAPK signaling activation and oxidative stress resistance. Cancer Res 2013;73:6359–74. 31. Uchida D, Takaki A, Ishikawa H, Tomono Y, Kato H, Tsutsumi K, et al. Oxidative stress balance is dysregulated and represents an additional target for treating cholangiocarcinoma. Free Radic Res 2016;50:732–43. 32. Kuang Y, Sechi M, Nurra S, Ljungman M, Neamati N. Design and synthesis of novel reactive oxygen species inducers for the treatment of pancreatic ductal adenocarcinoma. J Med Chem 2018;61:1576–94. 33. Cortez R, Luna-Vital DA, Margulis D, Gonzalez de Mejia E. Natural pigments: stabilization methods of anthocyanins for food applications. Compr Rev Food Sci Food Saf 2017;16:180–98. 34. Ongkowijoyo P, Luna-Vital DA, Gonzalez de Mejia E. Extraction techniques and analysis of anthocyanins from food sources by mass spectrometry: an update. Food Chem 2018;250:113–26. 35. Sinopoli A, Calogero G, Bartolotta A. Computational aspects of anthocyanidins and anthocyanins: a review. Food Chem 2019;297:124898. 36. Bhagwat S, Haytowitz DB, Holden JM. USDA database for the flavonoid content of selected foods release 3.1. 2013https://www.ars.usda.gov/ ARSUserFiles/80400525/Data/Flav/Flav_R03-1.pdf; 2013. Accessed 29 August 2019. 37. He J, Giusti MM. Anthocyanins: natural colorants with health-promoting properties. Annu Rev Food Sci Technol 2010;1:163–87. 38. Faria A, Fernandes I, Norberto S, Mateus N, Calhau C. Interplay between anthocyanins and gut microbiota. J Agric Food Chem 2014;62:6898–902. 39. Fernandes I, Faria A, de Freitas V, Calhau C, Mateus N. Multiple-approach studies to assess anthocyanin bioavailability. Phytochem Rev 2015;14:899–919. 40. Tian L, Tan Y, Chen G, Wang G, Sun J, Ou S, et al. Metabolism of anthocyanins and consequent effects on the gut microbiota. Crit Rev Food Sci Nutr 2019;59:982–91. 41. Kay CD, Pereira-Caro G, Ludwig IA, Clifford MN, Crozier A. Anthocyanins and flavanones are more bioavailable than previously perceived: a review of recent evidence. Annu Rev Food Sci Technol 2017;8:155–80. 42. Lila MA, Burton-Freeman B, Grace M, Kalt W. Unraveling anthocyanin bioavailability for human health. Annu Rev Food Sci Technol 2016;7:375–93. 43. Mallery SR, Budendorf DE, Larsen MP, Pei P, Tong M, Holpuch AS, et al. Effects of human oral mucosal tissue, saliva, and oral microflora on intraoral metabolism and bioactivation of black raspberry anthocyanins. Cancer Prev Res 2011;4:1209–21. 44. Tan BL, Norhaizan ME, Liew W-P-P, Sulaiman Rahman H. Antioxidant and oxidative stress: a mutual interplay in age-related diseases. Front Pharmacol 2018;9:1162. 45. Di Meo S, Reed TT, Venditti P, Victor VM. Role of ROS and RNS sources in physiological and pathological conditions. Oxid Med Cell Longev 2016;2016:1245049. 46. Ndhlala A, Moyo M, Van Staden J. Natural antioxidants: fascinating or mythical biomolecules? Molecules 2010;15:6905–30. 47. Wang SY, Jiao H. Scavenging capacity of berry crops on superoxide radicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen. J Agric Food Chem 2000;48:5677–84. 48. Ullah R, Khan M, Shah SA, Saeed K, Kim MO. Natural antioxidant anthocyanins-a hidden therapeutic candidate in metabolic disorders with major focus in neurodegeneration. Nutrients 2019;11(6):1195. 49. Azevedo J, Fernandes I, Faria A, Oliveira J, Fernandes A, de Freitas V, et al. Antioxidant properties of anthocyanidins, anthocyanidin-3-glucosides and respective portisins. Food Chem 2010;119:518–23. 50. Wang L-S, Stoner GD. Anthocyanins and their role in cancer prevention. Cancer Lett 2008;269:281–90. 51. Rahman MM, Ichiyanagi T, Komiyama T, Hatano Y, Konishi T. Superoxide radical- and peroxynitrite-scavenging activity of anthocyanins; structureactivity relationship and their synergism. Free Radic Res 2006;40:993–1002. 52. Shih P-H, Yeh C-T, Yen G-C. Effects of anthocyanidin on the inhibition of proliferation and induction of apoptosis in human gastric adenocarcinoma cells. Food Chem Toxicol 2005;43:1557–66. 53. Panieri E, Saso L. Potential applications of Nrf2 inhibitors in cancer therapy. Oxid Med Cell Longev 2019;2019:8592348. 54. Bellezza I, Mierla AL, Minelli A. Nrf2 and NF-kB and their concerted modulation in cancer pathogenesis and progression. Cancers (Basel) 2010;2:483–97. 55. Mazewski C, Gonzalez de Mejia E. Impact of anthocyanins on colorectal cancer. In: Advances in plant phenolics: from chemistry to human health. vol. 1286. ACS Symposium Series; 2018. p. 339–70 (9). 56. Sun L, Subar AF, Bosire C, Dawsey SM, Kahle LL, Zimmerman TP, et al. Dietary flavonoid intake reduces the risk of head and neck but not esophageal or gastric cancer in US men and women. J Nutr 2017;147:1729–38. 57. Petrick JL, Steck SE, Bradshaw PT, Trivers KF, Abrahamson PE, Engel LS, et al. Dietary intake of flavonoids and oesophageal and gastric cancer: incidence and survival in the United States of America (USA). Br J Cancer 2015;112:1291–300. 58. Vermeulen E, Zamora-Ros R, Duell EJ, Lujan-Barroso L, Boeing H, Aleksandrova K, et al. Dietary flavonoid intake and esophageal cancer risk in the European prospective investigation into cancer and nutrition cohort. Am J Epidemiol 2013;178(4):570–81. 59. Vitelli Storelli F, Molina AJ, Zamora-Ros R, Ferna´ndez-Villa T, Roussou V, Romaguera D, et al. Flavonoids and the risk of gastric cancer: an exploratory case-control study in the MCC-Spain study. Nutrients 2019;11(5):967.

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60. Woo H, Lee J, Choi I, Kim C, Lee J, Kwon O, et al. Dietary flavonoids and gastric cancer risk in a Korean population. Nutrients 2014;6:4961–73. 61. Zamora-Ros R, Agudo A, Luja´n-Barroso L, Romieu I, Ferrari P, Knaze V, et al. Dietary flavonoid and lignan intake and gastric adenocarcinoma risk in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Am J Clin Nutr 2012;96(6):1398–408. 62. Rossi M, Rosato V, Bosetti C, Lagiou P, Parpinel M, Bertuccio P, et al. Flavonoids, proanthocyanidins, and the risk of stomach cancer. Cancer Causes Control 2010;21:1597–604. 63. Woo HD, Kim J. Dietary flavonoid intake and risk of stomach and colorectal cancer. World J Gastroenterol 2013;19:1011–9. 64. Xu M, Chen Y-M, Huang J, Fang Y-J, Huang W-Q, Yan B, et al. Flavonoid intake from vegetables and fruits is inversely associated with colorectal cancer risk: a case-control study in China. Br J Nutr 2016;116:1275–87. 65. Zamora-Ros R, Guino´ E, Henar Alonso M, Vidal C, Barenys M, Soriano A, et al. Dietary flavonoids, lignans and colorectal cancer prognosis. Sci Rep 2015;5:14148. 66. Zamora-Ros R, Not C, Guino´ E, Luja´n-Barroso L, Garcı´a RM, Biondo S, et al. Association between habitual dietary flavonoid and lignan intake and colorectal cancer in a Spanish case-control study (the Bellvitge Colorectal Cancer Study). Cancer Causes Control 2013;24:549–57. 67. He X, Sun L-M. Dietary intake of flavonoid subclasses and risk of colorectal cancer: evidence from population studies. Oncotarget 2016;7:26617–27. 68. Zamora-Ros R, Fedirko V, Trichopoulou A, Gonza´lez CA, Bamia C, Trepo E, et al. Dietary flavonoid, lignan and antioxidant capacity and risk of hepatocellular carcinoma in the European prospective investigation into cancer and nutrition study. Int J Cancer 2013;133:2429–43. 69. Lagiou P, Rossi M, Lagiou A, Tzonou A, La Vecchia C, Trichopoulos D. Flavonoid intake and liver cancer: a case-control study in Greece. Cancer Causes Control 2008;19:813–8. 70. Molina-Montes E, Sa´nchez M-J, Zamora-Ros R, Bueno-de-Mesquita HB, Wark PA, Obon-Santacana M, et al. Flavonoid and lignan intake and pancreatic cancer risk in the European prospective investigation into cancer and nutrition cohort. Int J Cancer 2016;139:1480–92. 71. Kresty LA, Frankel WL, Hammond CD, Baird ME, Mele JM, Stoner GD, et al. Transitioning from preclinical to clinical chemopreventive assessments of lyophilized black raspberries: interim results show berries modulate markers of oxidative stress in barrett’s esophagus patients. Nutr Cancer 2006;54:148–56. 72. Chen T, Yan F, Qian J, Guo M, Zhang H, Tang X, et al. Randomized phase II trial of lyophilized strawberries in patients with dysplastic precancerous lesions of the esophagus. Cancer Prev Res 2012;5:41–50. 73. Wang L-S, Sardo C, Henry C, Larue B, Rocha C, McIntyre C, Frankel W, Arnold M, Martin E, Lechner J, Stoner G. Chemoprevention of human colorectal cancer with freeze-dried black raspberries. Cancer Res 2008;68: LB-328. 74. Wang L-S, Arnold M, Huang Y-W, Sardo C, Seguin C, Martin E, et al. Modulation of genetic and epigenetic biomarkers of colorectal cancer in humans by black raspberries: a phase I pilot study. Clin Cancer Res 2011;17:598–610. 75. Wang L-S, Burke CA, Hasson H, Kuo C-T, Molmenti CLS, Seguin C, et al. A phase Ib study of the effects of black raspberries on rectal polyps in patients with familial adenomatous polyposis. Cancer Prev Res 2014;7:666–74. 76. Pan P, Skaer CW, Stirdivant SM, Young MR, Stoner GD, Lechner JF, et al. Beneficial regulation of metabolic profiles by black raspberries in human colorectal cancer patients. Cancer Prev Res 2015;8:743–50. 77. Thomasset S, Berry DP, Cai H, West K, Marczylo TH, Marsden D, et al. Pilot study of oral anthocyanins for colorectal cancer chemoprevention. Cancer Prev Res 2009;2(7):625–33. 78. Bla´zovics A, Kursinszki L, Papp N, Kleiner D, Szo˝ke E, Hegyi G, et al. Is professional prescription of a commercially derived dietary supplement in colectomysed patients necessary? Eur J Integr Med 2016;8:219–26.

Chapter 25

Caffeic Acid targets metabolism of cervical squamous cell carcinoma Malgorzata Tyszka-Czochara Jagiellonian University Medical College, Faculty of Pharmacy, Department of Food Chemistry and Nutrition, Krakow, Poland

List of abbreviations AMPK ATCC CA CDH1 CisPt EMT ETC GLS GLUT1 GLUT3 LKB-1 ME1 Met MMP-2 MMP-9 NADH NADPH OXPHOS PDH PDK PPP ROS TCA cycle TGF-b1 VEGFA VIM

50 -adenosine monophosphate-activated protein kinase American Type Culture Collection Caffeic Acid E-cadherin 1 Cisplatin Epithelial-to-Mesenchymal Transition electron transport chain Glutaminase solute carrier family 2 member 1 receptors, SLC2A1 solute carrier family 2 member 3 receptors, SLC2A3 Liver Kinase B1 Malic Enzyme 1 Metformin Matrix Metalloproteinase-2 Matrix Metalloproteinase-9 Nicotinamide Adenine Dinucleotide Nicotinamide Adenine Dinucleotide Phosphate oxidative phosphorylation Pyruvate Dehydrogenase Complex Pyruvate Dehydrogenase Kinase pentose phosphate pathway Reactive Oxygen Species tricarboxylic acids cycle, Krebs cycle Transforming Growth Factor Beta 1 Vascular Endothelial Growth Factor A Vimentin

Introduction The malignancy of the cervix is among the most common cancers in women. The incidence rate for cervical tumor is still high, especially in developing countries.1 The primary factor in cervical intraepithelial neoplasia development is the infection with high-risk Human Papilloma Viruses (HPVs) subtypes.2 Within the infected cell, the viral oncoproteins may cause abrogation of both cell cycle control checkpoints and suppressor pathways and these events may result in tumor progression. Neoplastic cells express enhanced proliferation and alleviation of apoptosis induction.3 Along with the advent of HPV vaccines, primary prevention has become more successful in Western societies. However, the currently available vaccines prevent only 70% of cervical cancer cases and cervical malignancy remains one of the main causes of cancer mortality in women worldwide.4, 5 In consequence, the prognosis in terms of patients with advanced or recurrent cervical cancer is still poor and the chance for 1-year survival does not exceed 20% at the advanced stage of tumor.6 Numerous natural compounds may exert suppressive effects on tumor cells via multiple mechanisms.7, 8 Caffeic Acid (trans-3,4-dihydroxycinnamic acid, CA) is a major representative of hydroxycinnamic acids. CA occurs in plants mainly as Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00025-0 © 2021 Elsevier Inc. All rights reserved.

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chlorogenic acid (5-Caffeoylquinic acid, an ester of CA with ()-quinic acid).9 Antitumor effects of CA have recently been demonstrated in breast cancer cells,10 malignant human keratinocytes,11 hepatocellular carcinoma,12 and human colon adenocarcinoma.13 Recent studies have focused on molecular targets regulating the peculiar metabolism of tumor cells. The recognition of specific mechanisms controlling the cancer cell metabolism may be essential for the development of novel, effective anticancer approaches. Currently, the potential use of small molecules in targeted therapies has attracted a lot of interest.14 Recently, it has been suggested that phytochemicals may interfere with cellular bioenergetics and biosynthetic processes to eliminate tumor cells.7, 8, 15 This review summarizes recent findings on the influence of CA on cervical squamous cell cancer metabolism in vitro. The presented studies particularly focus on the ability of CA to regulate metabolic checkpoints in tumor cells. The exposition of cervical cancer cells to CA leads to elucidation of oxidative stress and induction of mitochondrial-dependent apoptotic cell death. In vitro studies also show that CA targets not a single, but several molecular targets, which results in induction of massive cell death of cervical cancer cells. Most cervical cancers in patients are squamous cell carcinomas (this type accounts for 80% of total cases in humans).16 Therefore, an in vitro model, used in experiments, includes human squamous cell cervical carcinoma lines with both epithelial and mesenchymal phenotypes. American Type Culture Collection (ATCC) cell lines were derived from tumors at different grades. The experiments were conducted with the C-4I cell line (ATCC designation CRL1594, isolated from a primary in situ tumor with an epithelial phenotype), HTB-34 carcinoma line with epithelial traits, derived from a metastatic site in lymph node (ATCC designation MS751) and SiHa cells (ATCC designation HTB-35) originated from an aggressive cervical tumor with epithelial/mesenchymal characteristics.17, 18

Metabolic reprogramming confers an adaptive advantage to cancer cells The altered regulation of metabolism is a hallmark of tumors. When comparing to a differentiated quiescent cell, cancer cell metabolism is more dependent on aerobic glycolysis. The rapid shift toward glycolysis in cancer cells was first observed by Otto Warburg, who concluded that in tumors glycolysis operates actively even if oxygen is abundant in the environment (“Warburg effect”).19 Malignant cancer cells mostly utilize glucose to lactate to generate energy and proliferate. Therefore, some novel anticancer agents target inhibition of glycolysis.20, 21 In fact, cancer cells prefer aerobic glycolysis also because it generates less reactive oxygen species (ROS) than mitochondrial metabolism. The overproduction of ROS in mitochondrial electron transport chain (ETC) under some environmental conditions may lead to unbearable oxidative stress, disruption of redox homeostasis, and activation of mitochondrial-induced apoptosis.22 In general, the enhanced glycolysis dominates in rapidly proliferating cells, while the oxidative metabolism is used by more differentiated quiescent cells. The mitochondrial tricarboxylic acids cycle (TCA cycle, Krebs cycle) is a key mitochondrial process that enables generation of intermediates for biosynthesis and molecules that provide energy in cells.23 However, recent studies have shown that the regulation of metabolic processes is more complex in cancer cells. Most tumors, apart from active glycolysis, have metabolically efficient mitochondria to provide intermediates for biosynthesis via anaplerotic reactions, to generate reductive power (Nicotinamide Adenine Dinucleotide Phosphate, NADPH) and produce cofactors (Nicotinamide Adenine Dinucleotide, NADH).24 It was demonstrated that in conditions of metabolic stress, some tumors might use oxidative phosphorylation (OXPHOS) to produce energy. The studies of Rodrı´guez-Enrı´quez et al.25 and Moreno-Sa´nchez et al.26 showed that OXPHOS is active in cervical carcinoma cell lines. In dividing cancer cells, biochemical reactions operate at a moderate level, without producing ROS above the toxic threshold. In this case, compromised mitochondrial-based metabolism plays an essential role in the maintenance of cell survival.

CA hampers glucose uptake and glucose catabolism to lactate in cervical cancer cells Regarding the net efficiency of ATP generation, glycolysis is less productive than mitochondrial OXPHOS. Therefore, cancer cells, which rely on glycolysis, have to meet much higher demands for glucose than quiescent cells. The expression of GLUT receptors (solute carrier family 2 member receptors, SLC2A) is a significant rate-limiting step of aerobic glycolysis in tumors. In particular, upregulation of GLUT1 (solute carrier family 2 member 1 receptors, SLC2A1) and GLUT3 (solute carrier family 2 member 3 receptors, SLC2A3) results in enhanced glucose transport and catabolism in tumors.19 However, CA may limit glucose transport to epithelial cervical cancer cells by inhibiting GLUT-1 transporter, as shown in HTB-34 cells.27 Furthermore, CA reduces the expression of glucose transporter GLUT3 in HTB-35 cells cervical tumor cells with aggressive characteristics.28 In cancer cells, the high rate of glucose catabolism results in enhanced lactate formation and acidosis of the milieu, which additionally promotes the ability of tumor cells to spread.19 However, it was demonstrated that CA alleviates the generation of lactate in epithelial cervical cancer cells.27

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CA induces oxidative stress in mitochondria and elucidates metabolic-dependent apoptotic death in epithelial cervical cancer cells As mentioned, the TCA cycle provides intermediates for biosynthesis and energy generation in cancer cells. The mitochondrial Pyruvate Dehydrogenase (PDH) Complex catalyzes the irreversible oxidative decarboxylation of pyruvate to acetylCo-A, which subsequently fuels the TCA cycle.23 As shown in Fig. 1, PDH complex activity is precisely regulated via covalent modification by Pyruvate Dehydrogenase Kinase (PDK). Recent studies report that PDK may be a novel molecular target for anticancer intervention.36 Pharmacological PDK inhibitors, such as dichloroacetate (DCA), have been intensively tested against numerous cancer cells.29 It was shown that DCA decreased the glycolysis rate by activating PDH complex, which resulted in reduced survival of epithelial cervical adenocarcinoma HeLa cells.37 A similar effect of DCA was shown in a glioblastoma ex vivo model and even in breast and lung cancers treatment in humans. However, the clinical trials revealed toxicity of DCA in patients. Therefore, it was withdrawn from some human research at phase II.38 The data obtained in C-4I30 and HTB-3427 epithelial cervical cancer cell lines showed that CA might be another potent PDK inhibitor. CA treatment caused complete restoration of PDH complex in tumor cells, thereby enhancing the flow of pyruvate to the mitochondrial TCA cycle, instead of fueling lactate formation in the cytoplasm. The rapid shift toward mitochondrial metabolism triggered ROS generation in mitochondria and induced intolerable oxidative stress, which resulted in apoptosis (Fig. 1). A similar effect was observed in both epithelial cervical cancer cell lines used in experimental models.27, 30 Recently, it was also demonstrated that CA induced apoptosis in human breast adenocarcinoma cells.39 It should be noted that CA did not trigger excessive ROS formation and apoptosis in normal cells.28 The essence of ROS homeostasis in cancer is keeping the balance between ROS generation and the function of antioxidant systems. Some cytostatic agents, such as Cisplatin, are potent inducers of oxidative stress. ROS-elucidated apoptosis has been taken as a target for several antitumor treatments. However, tumors exhibit an elevated intracellular ROS threshold compared to normal cells due to their efficient protection, such as reduced glutathione (GSH) molecules.40 In fact, some tumors develop compensatory mechanisms to maintain oxidative stress beneath the lethal threshold even if the

Glutamine

Glutamate

Malate

Energy disruption

NADP+ Reduced glutathione NADPH

Pyruvate Gl

Cell protection against oxidative stress

lys

yco

Cell proliferation

Glutamine

is

Apoptosis Lactate

Glucose transport Glucose Lactate excretion

Caffeic acid Cervical cancer cells with epithelial phenotype

FIG. 1 Caffeic Acid (CA) induces oxidative stress and promotes apoptosis in human squamous cell cervical carcinoma in vitro. CA restrains glucose transport into the cell and increases the mitochondrial TCA cycle supply via restoration of PDH complex activity. CA targets expression of regulatory enzymes GLS and ME1, which results in alleviation of regeneration of the NADPH pool and decreases cellular antioxidative protection. Furthermore, disruption of energy homeostasis in the cell upon CA treatment leads to activation of the master regulator of energy expenditure, AMPK.14, 19, 23, 27–35 (The artworks used elements from Servier Medical Art: www.servier.fr/servier-medical-art (Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License).)

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metabolic rate is high.23 Therefore, more precise anticancer approaches should include disruption of red-ox homeostasis, not only by inducing ROS overload, but also by hampering ROS-detoxification pathways. The impairment of glutathione regeneration using pharmacological agents may be an effective strategy that increases the sensitivity of cancer cells to therapies targeting oxidative stress.29 NADPH plays a key role in reinforcing the antioxidant protection of tumor cells by the reduction of glutathione molecules.41 Under oxidative stress, cancer cells require an elevated NADPH level to avoid apoptosis. Consequently, it was demonstrated that CA, beyond a critical enhancement of mitochondrial ROS formation, substantially disturbed the regeneration of NADPH in epithelial cervical cancer cells.30 As mentioned in the previous paragraph, CA caused inhibition of glucose transport to epithelial cervical tumor cells. In conditions of limited glucose supply, generation of NADPH in the Pentose Phosphate Pathway (PPP) is insufficient to meet the high cellular demand.22, 23 Furthermore, CA limited the regeneration of the NADPH pool by downregulation of Malic Enzyme 1 (ME1). In tumor cells, the reduction of the NADP+ molecule catalyzed by ME1 is a significant way to restore NADPH (Fig. 1). Thus, it was shown that CA favored metabolic-dependent apoptosis in tumor cells through independent mechanisms, by induction of an intolerable ROS load and hampering the cellular ability to combat oxidative stress via the blockade of NADPH regeneration.30 Targeting two mechanisms at the same time resulted in massive tumor cell death.27, 30 Growing data indicate that in order to improve the therapeutic efficacy of anticancer approaches, two or more proapoptotic pathways should be targeted.20, 21, 24, 40 This concept is supported by the presented studies, highlighting the effectiveness of epithelial tumor cells treatment with CA. Furthermore, metabolic effects upon CA treatment were observed only in cancer cells, not in normal cells.28

CA impairs energy generation in cervical cancer cells In cancer cells, mitochondrial pathways may be fueled by glutamine to support biosynthesis and ATP generation. Glutamine is an important energy source for numerous cancer cell lines and some tumors in vivo. In malignant cells, glutamine is a significant substrate that provides carbon for TCA reactions.19 Therefore, glutamine catabolism was suggested as a potential target for aggressive cancer treatment.22, 29 As shown in Fig. 1, glutamine entry to the TCA cycle is controlled by the enzyme Glutaminase (GLS). As a consequence of upregulated expression of GLS, glutamine rebalances tumor cell metabolism. However, it was demonstrated that the exposition of epithelial cervical cancer cells to CA downregulated not only ME1 expression, but also GLS expression, thereby protecting mitochondrial anabolism from additional carbon supply.27, 30 What is more, the effect of CA on GLS was specific toward cervical cancer cells and no changes were measured in normal cells upon exposition to CA.28 In vivo, tumors are often poorly perfused, but, at the same time, the oxygen level in the milieu is enough to activate ETC and generate ATP in mitochondria.41 In cervical cancer cells with an epithelial phenotype, CA decreased ATP synthesis and disrupted energy homeostasis. An acute drop in the ATP/AMP ratio dramatically reduced tumor cell survival.30 These data support the finding that reliance on mitochondrial respiration may make tumor cells more vulnerable to regulatory factors that target energy balance.24, 31

Energetic stress caused by CA in cervical cancer cells activates adenosine 50 -monophosphate AMP-activated protein kinase The acute drop in the intracellular ATP level activates 50 -monophosphate AMP-activated protein kinase (AMPK). AMPK plays a crucial role in cell survival and proliferation during energetic stress. The phosphorylation and activation of AMPK by its upstream serine-threonine kinase 11 (Liver Kinase B1, LKB-1) slows down the rate of anabolic pathways and promotes alternative metabolic processes to sustain the intracellular ATP pool. AMPK has been reported to inhibit the progression of various cancers,32 including human cervical adenocarcinoma cells.42, 43 On the other hand, in some tumors AMPK may act as a pro-survival factor and its exact role is a subject of intensive ongoing research.44 Therefore, an understanding of the metabolic-dependent context and genetic background may be of critical significance to recognize the role of AMPK activation in particular tumor cells, as well as in human squamous cell cervical carcinoma.33 It was demonstrated that somatic mutations in the LKB1 gene in cervical cancer cell lines C4-I, HTB-35, and HTB-34 led to the alleviation of LKB1 protein expression.45 In this case, the classical activators of the LKB1/AMPK axis might be inefficient to eliminate cancer cells. However, a recent study suggested that tumor cells lacking functional LKB1 might be more sensitive to metabolic stress than cells with a high expression of the protein.34, 35 Our data offer some interesting findings for targeted therapy against tumor cells with such a genetic profile. As a small molecule, CA had the potency to dramatically decrease the ATP level and disrupt intracellular energy balance specifically in squamous cell cervical carcinoma cells. The drop in the ATP/AMP ratio led to inhibition of proliferation and cell death.27, 30 The obtained data show

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that energy stress, not the canonical activation of the LKB1/AMPK axis, may be the mechanism underlying cell death upon CA treatments in LKB-deficient cervical tumor cells. It has been reported before that CA activates AMPK in other tumor cell lines.46

CA affects the cervical cancer cells phenotype and migration properties under implementation of the Epithelial-to-Mesenchymal Transition process Tumor epithelial cells may acquire a mesenchymal phenotype by implementation of the EMT program. This process is accompanied by a number of changes, which enable the epithelial cells to gain the ability to move, detach from the tumor mass, and invade other tissues.47, 48 Additionally, triggering of the EMT program in cervical cancers is one of the molecular mechanisms underlying resistance of malignant cells to chemotherapy, especially using Cisplatin.6 The particular metabolic phenotype may facilitate metastatic properties of tumors and prevent anoikis.21, 41 As mentioned before, metastatic cancer cells may use metabolic pathways providing ATP without oxidative stress. The ability of circulating cancer cells to survive during invasion largely depends on such metabolic flexibility.19, 47 Therefore, it was of interest to find out whether the regulatory effects that CA exerted in cervical tumor cells also might affect the implementation of the EMT program. It has been shown before that CA inhibited EMT in cancer cell lines of various origin.11, 49 In cells that undergo the EMT process, the expression of junctional proteins related to the polarized epithelial phenotype (such as E-cadherin 1, CDH1) decreases with concomitant upregulation of mesenchymal proteins, especially those reorganizing the cellular cytoskeleton (such as Vimentin, VIM).50 EMT may be induced in cervical tumor cells upon 48 h incubation with 10 ng/mL of cytokine Transforming Growth Factor Beta 1 (TGF-b1), as shown by Cheng et al. in the HeLa cell line.51 It was demonstrated that CA restrained the metastatic phenotype induced by TGF-b1 in C-4I cells by inhibition of the EMT process.52 The mechanistic study showed that CA suppressed expression of nuclear transcription factors Snail-1, ZEB1, TWIST1, and TWIST2 (Fig. 2). The proteins play a pivotal role in tumor cell migration and invasiveness. Of those, Snail-1 has been defined as the main driver of the EMT program. Numerous in vitro and in vivo studies demonstrated that inhibition of E-cadherin expression by Snail-1 is a key process driving EMT in tumors,55 as well as in cervical cancers.56

FIG. 2 Caffeic Acid (CA) restrains implementation of the Epithelial-to-Mesenchymal Transition (EMT) in human squamous cell cervical carcinoma in vitro. CA inhibits the expression of the master inducer of the EMT program, Snail-1, and increases expression of the Snail-1 downstream protein, epithelial adhesive molecule E-cadherin. Cancer cells treatment with CA hampers the expression of metalloproteinases (MMP-2, MMP-9) and expression of the proangiogenic factor, VEGFA.47, 48, 50, 52–54 (The artworks used elements from Servier Medical Art: www.servier.fr/servier-medical-art (Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License).)

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What is more, it has been reported that E-cadherin expression decreases with a more advanced grade of cervical cancers in humans.55 CA downregulated genes encoding transcription factors, especially Snail-1, and restored its downstream protein E-cadherin along with enhanced expression of other epithelial adhesive molecules, Occludin (OCLN) and Claudin 1 (CLDN1). Furthermore, the restoration of E-cadherin expression in C-4I cells resulted in decreased cell motility, which was shown using the functional scratch test.52 CA also decreased motility of aggressive HTB-35 cells stimulated and nonstimulated with TGF-b1. Yang et al.11 demonstrated that CA inhibited the migratory capacity and increased the adhesion ratio of malignant human keratinocytes via transcriptional inactivation of Snail. At the same time, CA induced the expression of adhesive proteins, including E-cadherin, and downregulated several mesenchymal markers.11 Snail-1 is also a positive regulator of Matrix Metalloproteinase-2 (MMP-2) and Matrix Metalloproteinase-9 (MMP-9), enzymes that degrade structural tissue proteins and promote invasion. MMPs activation in tumors results in facilitating metastasis and angiogenesis.53 MMP-9 overexpression is positively correlated with the increased invasiveness of ovarian and breast malignancies, and decreased survival in patients.54 Thus far, it was reported that Chlorogenic Acid inhibited MMP-9 activity in a short-time manner.57 It was demonstrated that CA downregulated transcripts for both MMP-2 and MMP-9 proteins in C-4I cells. What is more, CA suppressed MMP-9 in two independent ways, by inhibition of gene encoding Snail-1 and via upregulation of a specific tissue inhibitor TIMP-1, which controls the degradative activity of MMP-9 (Fig. 3). It should be emphasized that in cervical tumor cells, transcription factor Snail-1 regulates expression of numerous proteins, which controls proliferation, apoptosis resistance, and angiogenesis.50 Therefore, the downregulation of Snail-1 expression caused by CA not only resulted in inhibition of the EMT process, but also contributed to antiproliferative effects in C4-I cells. Vascular Endothelial Growth Factor A (VEGFA) is a factor that induces the development of tumor-associated blood vessels and provides the way for invasion of cancer cells.47, 55 It was shown in endometrial cancers that the overexpression of VEGFA promotes angiogenesis.50 Our study has shown that CA suppresses VEGFA expression in C4-I cells.52

CA has the potency to regulate cell cycle progress in cervical cancer cells with an epithelial phenotype Anticancer drugs have great efficiency in vitro, but in in vivo conditions it is extremely difficult to induce complete death of tumor cells. The recurrence or progression after therapy may occur even if a few cells survive the treatment in patient. However, the proliferation of malignant cells can be controlled via modulation of cell cycle regulatory proteins, and several of them were demonstrated to be powerful targets for anticancer intervention.58 Mechanisms that regulate cell cycle progression may arrest the cell cycle at particular points, the so-called checkpoints. Specifically, the G1/S phase restriction FIG. 3 Cancer cells that had entered G0 phase may be less sensitive to single treatment with CisPt (upper part). Adjuvant treatment using CisPt/CA/Met regiment targets cell cycle progression, redirects quiescent cancer cells to G1 phase and the population of dividing cancer cells is more susceptible to the action of CisPt (lower part).4, 28, 31, 39, 58, 59 (The artworks used elements from Servier Medical Art: www.servier.fr/serviermedical-art (Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License).)

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point prevents replication of damaged DNA, whereas the G2/M checkpoint that occurs during mitotic cell division halts the separation of impaired chromosomes into daughter cells.59 The dysregulation of mechanisms controlling the cell cycle enables tumors to elude cellular control points and survive even in harsh environmental conditions.31 It was shown that the pharmacological activation of mechanisms controlling cell cycle checkpoints might lead to cell cycle arrest and result in tumor cell apoptosis.58 One such strategy involves the use of small molecules to arrest tumor cells specifically in the G2/M checkpoint to cancel cell division. It was demonstrated that CA treatment modified the cell cycle in the G2/M checkpoint in epithelial HTB-34 cells.27 CA has been reported before to induce cell cycle arrest in the G1 phase and induce apoptosis in breast cancer cells.39 Murad et al.13 reported that CA, by promoting specific changes in cell cycle regulation, decreased viability and stimulated apoptosis in the human colon adenocarcinoma cell culture.13 Kuo et al.60 showed that cell proliferation of human squamous carcinoma cells was suppressed by CA Phenethyl Ester through an arrest in the G1 phase, with a concomitant increase of population in the G2/M phase and activation of apoptosis.60 The findings obtained using HTB-34 cells exposed to CA were consistent with the reported data that CA is a potent regulator of cell cycle progress in cervical tumor cells.27

Applications to other conditions Cervical cancer treatment in humans using Cisplatin Cisplatin (cis-dichlorodiammineplatinum (II), CisPt) is a cytostatic drug used for cervical malignancy treatment in patients. CisPt induces oxidative stress by ROS overproduction and this is the main molecular mechanism of its action in tumor cells, but the drug also triggers other mechanisms that lead to cell death. However, chemotherapy using Cisplatin is also associated with numerous side effects.4, 5 In early-stage cervical cancers, a standard therapeutic regiment using Cisplatin may improve the survival of patients, but for advanced cervical cancers the more successful approach is highly required.1, 3, 6 Recently, combinatory therapies with Cisplatin have been introduced to increase the efficacy of treatments and minimize toxic effects.58 Two or more independent mechanisms may be targeted at the same time to sensitize cancer cells to the action of the cytostatic drug. Since the ability to develop compensatory pathways is one of the hallmarks of cancer, such an approach seems to be justified.20, 24, 29 Phytochemicals may efficiently damage cancer cells, but the use of a single compound is usually not enough to elucidate apoptotic tumor cell death.8, 14, 32 The employment of phytochemicals together with synthetic drugs may render cancer cells more vulnerable to chemotherapy, as shown in in vitro15 and in vivo studies.8

Co-treatment of cervical cancer cells with CA and the antidiabetic drug, Metformin, augments the toxic action of Cisplatin via regulation of the cell cycle—In vitro study A population of quiescent cancer cells within a solid tumor may survive upon treatment with cytostatic drugs and recur after therapy. However, the survival of quiescent cancer cells can be specifically interrupted by triggering mechanisms controlling the G0/G1 cell cycle phase transition. In such an approach, small molecule modulators may be applied to sensitize cancer cells to the treatment with radio- or chemotherapy by reentering G0 cells to the cell cycle.59 Recent reports showed that Met exerted its anticancer action via modification of the cell cycle in G0/G1 checkpoints, which resulted in induction of apoptosis and tumor cell death, and the effect was specific to malignant cells.61 Met was also shown to enhance the effect of CisPt in HeLa cells.62 The recent studies of Koraneekit et al.63 and Sirota et al.64 demonstrated that CA might be a candidate for an adjuvant that significantly increases the therapeutic efficacy of Cisplatin. Our study28 showed that the toxic action of CisPt might be supported by CA/Met treatment via targeting cell cycle regulation in aggressive cervical cancer cells.28 Specifically, CisPt/CA/Met treatment redirected G0 cells back to the cycle. At the same time, single and double treatments with Met and CA were not as effective as triple treatment.

CA and Met hamper proliferation and enhance cell death in cervical cancer cells but not in normal cells A shift in cell cycle progress sensitized malignant stationary-phase cells to the action of the chemotherapeutic drug. As a result, CisPt/Met/CA treatment caused the greatest inhibition of cervical cancer cells proliferation and survival compared to single and double treatments.28 Moreover, triple treatment selectively killed tumor cells when cocultured with normal human cells.28 This finding suggests selectivity of the employed CisPt/CA/Met regiment toward cancer cells.

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CA alleviates lactic acidosis caused by Metformin—In vitro study The enhanced lactate production was reported to be a significant metabolic side effect of Met in humans.65 Our in vitro studies demonstrated that CA counteracted the boosted lactate formation caused by Met. This effect was observed in cervical tumor cells with both epithelial and mesenchymal phenotypes, as well as in normal cells.27, 28 The alleviation of Met potency to overproduce lactate might be therapeutically used in humans, but a further in vivo study is needed.

Bioavailability of CA and perspectives of use in humans CA is an inexpensive and easily accessible chemical compound, which makes it attractive to use in societies, where the mortality rate of cervical cancer is still high. On the other hand, CA has low bioavailability in humans. This is an important limiting factor considering the clinical use of numerous naturally derived compounds.8, 9, 12, 15 Clinical trials using single oral treatments of dietary polyphenols against tumor growth show minor effects in humans.8 However, in cervical tumors, a topical formulation containing a bioactive compound at relatively high concentration might be used. Currently, a lot of interest is paid to the use of phytochemicals in treatments. Therefore, there is an urgent need for a rigorous study of mechanisms extended by these compounds, especially in tumor cells.

Summary points l

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l

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CA targets reprogrammed metabolism in human squamous cell cervical carcinoma lines with epithelial (C-4I, HTB34) and aggressive mesenchymal (HTB-35) phenotypes In particular, CA regulates mitochondrial metabolism which results in the induction of oxidative stress and apoptosis in epithelial cervical cancer cells CA targets oxidative stress via two independent mechanisms: by increasing mitochondrial metabolism and by hampering NADPH regeneration via ME1 Effect of CA is specific toward cancer cells and it neither generates oxidative stress nor apoptosis in normal cells In epithelial cervical cancer cells, PDK and GLS may be hot spots in regulation of the mitochondrial TCA cycle supply with substrates, glucose, and glutamine CA alleviates the implementation of the Epithelial-to-Mesenchymal Transition (EMT) process in cervical tumor cells by downregulation of master mesenchymal inducer Snail-1, which results in upregulation of epithelial markers, E-cadherin, Occludin, and Claudin CA suppresses expression of metalloproteinases in cervical cancer cells. CA inhibits MMP-9 by two independent mechanisms, via downregulation of transcription factor Snail-1 and by upregulation of TIMP-1, a specific inhibitor of MMP-9 CA, by acting together with the antidiabetic drug, Metformin, sensitizes cervical tumor cells to the action of Cisplatin and may be considered as a potential adjuvant for anticancer treatment

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Small Jr. W, Baco MA, Bajaj A. Cervical cancer: a global health crisis. Cancer 2017;123:2404–12. Steben M, Duarte-Franco E. Human papillomavirus infection: epidemiology and pathophysiology. Gynecol Oncol 2007;107:S2–5. Ojesina AI, Lichtenstein L, Freeman SS. Landscape of genomic alterations in cervical carcinomas. Nature 2014;506:371–5. Kumar L, Harish P, Malik PS, Khurana S. Chemotherapy and targeted therapy in the management of cervical cancer. Curr Probl Cancer 2018;42:120–8. Dasari S, Tchounwou PB. Cisplatin in cancer therapy: molecular mechanisms of action. Eur J Pharmacol 2014;740:364–78. Zhu H, Luo H, Zhang W, Shen Z, Hu X, Zhu X. Molecular mechanisms of cisplatin resistance in cervical cancer. Drug Des Devel Ther 2016;10:1885–95. Budisan L, Gulei D, Zanoaga OM, Irimie AI, Sergiu C, Braicu C, et al. Dietary intervention by phytochemicals and their role in modulating coding and non-coding genes in cancer. Int J Mol Sci 2017;18:1178. Fantini M, Benvenuto M, Masuelli L, Frajese GV, Tresoldi I, Modesti A, et al. In vitro and in vivo antitumoral effects of combinations of polyphenols, or polyphenols and anticancer drugs: perspectives on cancer treatment. Int J Mol Sci 2015;16:9236–82. Naveed M, Hejazi V, Abbas M, Kamboh AA, Khan GJ, Shumzaid M, et al. Chlorogenic acid (CGA): a pharmacological review and call for further research. Biomed Pharmacother 2018;97:67–74. Kabała-Dzik A, Rzepecka-Stojko A, Kubina R, Jastrzębska-Stojko Z˙, Stojko R, Wojtyczka R, et al. Migration rate inhibition of breast cancer cells treated by caffeic acid and caffeic acid phenethyl ester: an in vitro comparison study. Nutrients 2017;9:1144.

Caffeic Acid and metabolism of cervical cancer Chapter

25

289

11. Yang Y, Li Y, Wang K, Wang Y, Yin W, Li L. P38/NF-kB/snail pathway is involved in caffeic acid-induced inhibition of cancer stem cells-like properties and migratory capacity in malignant human keratinocyte. PLoS One 2013;8:58915. 12. Zhang Z, Wang D, Qiao S, Wu X, Cao S, Wang L, et al. Metabolic and microbial signatures in rat hepatocellular carcinoma treated with caffeic acid and chlorogenic acid. Sci Rep 2017;7:4508. 13. Murad L, Soares NC, Brand C, Monteiro MC, Teodoro AJ. Effects of caffeic and 5-caffeoylquinic acids on cell viability and cellular uptake in human colon adenocarcinoma cells. Nutr Cancer 2015;67:532–42. 14. Luengo A, Gui DY, Vander Heiden MG. Targeting metabolism for cancer therapy. Cell Chem Biol 2017;24:1161–80. 15. Joven J, Rull A, Rodriguez-Gallego E, Camps J, Riera-Borrull M, Herna´ndez-Aguilera A, et al. Multifunctional targets of dietary polyphenols in disease: a case for the chemokine network and energy metabolism. Food Chem Toxicol 2013;51:267–79. 16. Barbera L, Thomas GR. Management of early and locally advanced cervical cancer. Semin Oncol 2009;36:155–69. 17. Available online from: https://www.lgcstandards-atcc.org. 18. Carlson MW, Iyer VR, Marcotte EM. Quantitative gene expression assessment identifies appropriate cell line models for individual cervical cancer pathways. BMC Genomics 2007;10:2–13. 19. Pavlova NN, Thompson CB. The emerging hallmarks of cancer metabolism. Cell Metab 2016;23:27–47. 20. Keijer J, Bekkenkamp-Grovenstein M, Venema D, Dommels YE. Bioactive food components, cancer cell growth limitation and reversal of glycolytic metabolism. Biochim Biophys Acta 2011;1807:697–706. 21. Ganapathy-Kanniappan S, Geschwind JF. Tumor glycolysis as a target for cancer therapy: progress and prospects. Mol Cancer 2013;12:152. 22. Panieri E, Santoro MM. ROS homeostasis and metabolism: a dangerous liason in cancer cells. Cell Death Dis 2016;7:2253. 23. Lu J, Tan M, Cai Q. The Warburg effect in tumor progression: mitochondrial oxidative metabolism as an anti-metastasis mechanism. Cancer Lett 2015;356:156–64. 24. Weinberg SE, Chandel NS. Targeting mitochondria metabolism for cancer therapy. Nat Chem Biol 2015;1:9–15. 25. Rodrı´guez-Enrı´quez S, Carren˜o-Fuentes L, Gallardo-Perez JC, Saavedra E, Quezada H, Vega A, et al. Oxidative phosphorylation is impaired by prolonged hypoxia in breast and possibly in cervix carcinoma. Int J Biochem Cell Biol 2010;42:1744–51. 26. Moreno-Sa´nchez R, Marı´n-Herna´ndez A, Saavedra E, Pardo JP, Ralph SJ, Rodrı´guez-Enrı´quez S. Who controls the ATP supply in cancer cells? Biochemistry lessons to understand cancer energy metabolism. Int J Biochem Cell Biol 2014;50:10–23. 27. Tyszka-Czochara M, Konieczny P, Majka M. Caffeic acid expands anti-tumor effect of metformin in human metastatic cervical carcinoma HTB-34 cells: implications of AMPK activation and impairment of fatty acids de novo biosynthesis. Int J Mol Sci 2017;18:462. 28. Tyszka-Czochara M, Bukowska-Strakova K, Majka M. Metformin and caffeic acid regulate metabolic reprogramming in human cervical carcinoma SiHa/HTB-35 cells and augment anticancer activity of Cisplatin via cell cycle regulation. Food Chem Toxicol 2017;106:260–72. 29. Martinez-Outschoorn UE, Peiris-Pages M, Pestell RG, Sotgia F, Lisanti MP. Cancer metabolism: a therapeutic perspective. Nat Rev Clin Oncol 2017;14:113. 30. Tyszka-Czochara M, Bukowska-Strakova K, Kocemba-Pilarczyk KA, Majka M. Caffeic acid targets AMPK signaling and regulates tricarboxylic acid cycle anaplerosis while metformin downregulates HIF-1a-induced glycolytic enzymes in human cervical squamous cell carcinoma lines. Nutrients 2018;10:841. 31. Green DR, Galluzzi L, Kroemer G. Cell biology Metabolic control of cell death. Science 2014;345:1250256. 32. Marı´n-Aguilar F, Pavillard LE, Giampieri F, Bullo´n P, Cordero MD. Adenosine monophosphate (AMP)-activated protein kinase: a new target for nutraceutical compounds. Int J Mol Sci 2017;18:288. 33. Tyszka-Czochara M, Konieczny P, Majka M. Recent advances in the role of AMP-activated protein kinase in metabolic reprogramming of metastatic cancer cells: targeting cellular bioenergetics and biosynthetic pathways for anti-tumor treatment. J Physiol Pharmacol 2018;69(3):337–49. 34. Zulato E, Ciccarese F, Agnusdei V, Pinazza M, Nardo G, Iorio E, et al. LKB1 loss is associated with glutathione deficiency under oxidative stress and sensitivity of cancer cells to cytotoxic drugs and g-irradiation. Biochem Pharmacol 2018;156:479–90. 35. Herzig S, Shaw R. AMPK: guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol 2018;19:121–35. 36. Stacpoole PW. Therapeutic targeting of the pyruvate dehydrogenase complex/pyruvate dehydrogenase kinase (PDC/PDK) axis in cancer. J Natl Cancer Inst 2017;109. https://doi.org/10.1093/jnci/djx071. 37. Choi YW, Lim IK. Sensitization of metformin-cytotoxicity by dichloroacetate via reprogramming glucose metabolism in cancer cells. Cancer Lett 2014;346:300–8. 38. Maguire C, Gammer TL, Mackey JR, Fulton D, Abdulkarim B, McMurtry MS, et al. Metabolic modulation of glioblastoma with dichloroacetate. Sci Transl Med 2010;2:31ra34. 39. Kabała-Dzik A, Rzepecka-Stojko A, Kubina R, Jastrzębska-Stojko Z˙, Stojko R, Wojtyczka RD, et al. Comparison of two components of propolis: caffeic acid (CA) and caffeic acid phenethyl ester (CAPE) induce apoptosis and cell cycle arrest of breast cancer cells MDA-MB-231. Molecules 2017;22:1554. 40. Zhao Y, Hu X, Liu Y, Dong S, Wen Z, He W, et al. ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway. Mol Cancer 2017;16:79. 41. Indran IR, Tufo G, Pervaiz S, Brenner C. Recent advances in apoptosis, mitochondria and drug resistance in cancer cells. Biochim Biophys Acta 2011;1807:735–45. 42. Park SY, Park C, Park SH, Hong SH, Kim GY, Hong SH, et al. Induction of apoptosis by ethanol extract of evodia rutaecarpa in HeLa human cervical cancer cells via activation of AMP-activated protein kinase. Biosci Trends 2017;10:467–76. 43. Chen X, Li K, Zhao G. Propofol inhibits HeLa cells by impairing autophagic flux via AMP-activated protein kinase (AMPK) activation and endoplasmic reticulum stress regulated by calcium. Med Sci Monit 2018;24:2339–49.

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44. Li N, Huang D, Lu N, Luo L. Role of the LKB1/AMPK pathway in tumor invasion and metastasis of cancer cells (Review). Oncol Rep 2015;34:2821–6. 45. Wingo SN, Gallardo TD, Akbay EA, Liang MC, Contreras CM, Boren T, et al. Somatic LKB1 mutations promote cervical cancer progression. PLoS One 2009;4:5137. 46. Pistollato F, Giampieri F, Battino M. The use of plant-derived bioactive compounds to target cancer stem cells and modulate tumor microenvironment. Food Chem Toxicol 2015;75:58–70. 47. Lambert AW, Pattabiraman DR, Weinberg RA. Emerging biological principles of metastasis. Cell 2017;168:670–91. 48. Kantak RH, Kramer E. E-cadherin regulates anchorage-independent growth and survival in oral squamous cell carcinoma cells. J Biol Chem 1998;273:16953–61. 49. Dziedzic A, Kubina R, Kabała-Dzik A, Wojtyczka RD, Morawiec T, Bułdak RJ. Caffeic acid reduces the viability and migration rate of oral carcinoma cells (SCC-25) exposed to low concentrations of ethanol. Int J Mol Sci 2014;15:18725–41. 50. Lee MY, Shen MR. Epithelial-mesenchymal transition in cervical carcinoma. Am J Transl Res 2012;4:1–13. 51. Cheng K, Hao M. Metformin inhibits TGF-b1-induced epithelial-to-mesenchymal transition via PKM2 relative-mTOR/p70s6k signaling pathway in cervical carcinoma cells. Int J Mol Sci 2016;17:2000 66. 52. Tyszka-Czochara M, Lasota M, Majka M. Caffeic acid and Metformin inhibit invasive phenotype induced by TGF-b1 in C-4I and HTB-35/SiHa human cervical squamous carcinoma cells by acting on different molecular targets. Int J Mol Sci 2018;19:266. 53. Stanciu A, Zamfir-Chiru-Anton A, Stanciu M, Popescu C, Gheorghe D. Imbalance between matrix metalloproteinases and tissue inhibitors of metalloproteinases promotes invasion and metastasis of head and neck squamous cell carcinoma. Clin Lab 2017;63:1613–20. 54. Roomi M, Kalinovsky T, Rath M, Niedzwiecki A. Modulation of u-PA, MMPs and their inhibitors by a novel nutrient mixture in human female cancer cell lines. Oncol Rep 2012;28:768–76. 55. Zeisberg M, Neilson EG. Biomarkers for epithelial-mesenchymal transitions. Cell 2009;119:1429–37. 56. Laskov I, Abou-Nader P, Amin O, Philip CA, Beauchamp MC, Yasmeen A, et al. Metformin increases E-cadherin in tumors of diabetic patients with endometrial cancer and suppresses epithelial-mesenchymal transition in endometrial cancer cell lines. Int J Gynecol Cancer 2016;26:1213–21. 57. Jin U, Lee J, Kang S, Kim J, Park W, Kim J, et al. A phenolic compound, 5-caffeoylquinic acid (chlorogenic acid), is a new type and strong matrix metalloproteinase-9 inhibitor: isolation and identification from methanol extract of Euonymus alatus. Life Sci 2005;77:2760–9. 58. Horibe S, Matsuda A, Tanahashi T, Inoue J, Kawauchi S, Mizuno S, et al. Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins. Life Sci 2015;124:31–40. 59. O’Leary B, Finn RS, Turner NC. Treating cancer with selective CDK4/6 inhibitors. Nat Rev Clin Oncol 2016;13:417–30. 60. Kuo Y, Lin HP, Huo C, Su LC, Yang J, Hsiao PH, et al. Caffeic acid phenethyl ester suppresses proliferation and survival of TW2.6 human oral cancer cells via inhibition of Akt signaling. Int J Mol Sci 2013;14:8801–17. 61. Sacco F, Calderone A, Castagnoli L, Cesareni G. The cell-autonomous mechanisms underlying the activity of metformin as an anticancer drug. Br J Cancer 2016;115:1451–6. 62. Tang ZY, Sheng MJ, Qi YX, Wang LY, He DY. Metformin enhances inhibitive effects of carboplatin on HeLa cell proliferation and increases sensitivity to carboplatin by activating mitochondrial associated apoptosis signaling pathway. Eur Rev Med Pharmacol Sci 2018;22:8104–12. 63. Koraneekit A, Limpaiboon T, Sangka A, Boonsiri P, Daduang S, Daduang J. Synergistic effects of cisplatin-caffeic acid induces apoptosis in human cervical cancer cells via the mitochondrial pathways. Oncol Lett 2018;15:7397–402. 64. Sirota R, Gibson D, Kohen R. The timing of caffeic acid treatment with cisplatin determines sensitization or resistance of ovarian carcinoma cell lines. Redox Biol 2017;11:170–5. 65. Ikhlas S, Ahmad M. Metformin: insights into its anticancer potential with special reference to AMPK dependent and independent pathways. Life Sci 2017;185:53–62.

Chapter 26

Effects of caffeic acid on oxidative balance and cancer Beatriz da Silva Rosa Bonadimana, Grazielle Castagna Cezimbra Weisb, J
essica Righi da Rosab, c d e Charles Elias Assmann , Audrei de Oliveira Alves , P^amela Longhi , and Margarete Dulce Bagatinif a

Department of Biological Science: Biochemistry, Federal University of Santa Catarina, Floriano´polis, SC, Brazil, b Department of Technology and Food

Science, Rural Science Center, Federal University of Santa Maria, Santa Maria, RS, Brazil, c Department of Biochemistry and Molecular Biology, PPGBTox, CCNE, Federal University of Santa Maria, Santa Maria, RS, Brazil, d Department of Physiology and Pharmacology, Federal University of e, SC, Brazil, Santa Maria, Santa Maria, RS, Brazil, e Department Life Science and Health, University of the West of Santa Catarina, Xanxer^ f Academic Coordination, Campus Chapeco´, Federal University of Fronteira Sul, Chapeco´, SC, Brazil

Coffee Coffee belongs to the Rubiaceae family, it is an infusion of beans from the genus Coffea and is the most consumed nonalcoholic beverage worldwide, because of its sensory characteristics, nutritional properties, and historical/cultural context.1 Two major species, Coffea arabica (68% of the global production) and Coffea canephora, are cultivated in tropical areas. There are over 100 coffee species distributed throughout the world.2 The chemical composition of coffee is directly related to its quality, species, and cultivar, as well as factors such as agricultural practices and degree of roasting. The most prevalent bioactive compounds found in coffee leaves include flavonoids such as rutin, isoquercetin, quercetin, and kaempferol, as well as the alkaloid caffeine.3 There are polyphenols such as caffeic acid and chlorogenic acid and alkaloids, the principal one being caffeine.3

Coffee and oxidative balance Coffee molecules possess important activity for the regulation of oxidative stress. For example, chlorogenic acids (caffeic acid) have several health beneficial properties, not only because of their potent antioxidant capacity, but also because of hepatoprotective, hypoglycemic, antispasmodic, antiviral, antibacterial and antiinflammatory properties.4, 5 Rebollo-Hernanz et al.6 not only suggested that coffee reduces markers of oxidative and inflammatory stress, but also that chlorogenic acids, along with caffeine, were the major contributors to inflammation and oxidative stress. Jeszka-Skowron et al.7 showed that phytochemicals from coffee reduced markers of inflammation, oxidative stress, adipogenesis, and insulin resistance in vitro.

Coffee and cancer Recently, an epidemiological study from the National Institutes of Health (NIH) suggested that consuming four or more cups of caffeinated coffee per day may reduce the risk of malignant melanoma by 20%.8, 9 Lukic et al.10 reported the results of the Norwegian Women and Cancer (NOWAC) study. The authors also found that moderate consumption of filtered coffee was associated with a decreased risk of melanoma cancer. Wrze
sniok et al.9 found that caffeine modulated growth and vitality of human melanotic and amelanotic melanoma cells. Jang et al.11 found that caffeine at 10 mmol/mL had cytotoxic effects in human neuroblastoma cells. Qi et al.11a reported caffeine-induced apoptosis in human lung carcinoma cells. Okano et al.12 demonstrated that caffeine at 1–10 mmol/mL inhibited proliferation of hepatocellular carcinoma cells. Ku et al.13 reported that treatment of glioma cells with caffeine arrested the cell cycle in the G0/G1 phase and induced caspase-dependent apoptosis. In an in vitro study, Pelison et al.14 found that caffeic acid present in coffee has cytotoxic capacity against SK-MEL28, cancer cells, suggesting therapeutic potential in the treatment of this cancer. Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00026-2 © 2021 Elsevier Inc. All rights reserved.

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Berries Berries are small fruits that are commercially cultivated and commonly consumed in fresh and processed forms. The most common berries include blackberry (Rubus spp.), black raspberry (Rubus occidentalis), red raspberry (Rubus idaeus), blueberry (Vaccinium spp.), strawberry (Fragaria ananassa), cranberry (Vaccinium macrocarpon), black currant (Ribes nigrum), and chokeberry (Aronia melanocarpa).15 Most berries contain high levels of phenolic compounds, including flavonoids, condensed and hydrolyzable tannins, phenolic acids, stilbenoids, and lignans.15 The concentration of phenolic compounds varies according to many factors; the content of caffeic acid in berries ranges from 1542.65 to 14,657.98 mg/mL dry weight of fruit.15 Many studies showed that the dietary intake of berry fruits had a positive impact on human health and prevented the development of diseases such as cancer.15, 16 In this review, the effects of two berries, raspberry and blueberry, on cancer development will be discussed in more detail.

Raspberry The subgenus Idaeobatus belonging to the genus Rubus (Rosaceae) includes several species such as the most popular red raspberry (R. idaeus) and black raspberry (R. occidentalis) (Fig. 1). These plants are native to North America and Europe.16 Raspberries possess a unique phenolic profile that is characterized primarily by their anthocyanin and ellagitannin content. Other phenolic compounds such as phenolic acids, flavonols, and condensed tannins are also present in raspberries.16, 17 The total phenolic contents of red and black raspberry ethanolic extracts are 434.3  6.3 and 965.6  2.9 mg gallic acid/g, respectively.18 The total content of caffeic acid in red raspberry is 1542.65  35.11 mg/mL dry weight of fruit.15

Raspberry and oxidative balance Raspberries are unique for their high antioxidant capacities and they are consistently reported to have one of the most potent antioxidant capacities among edible berries.18 Basu and Maier18 showed that red and black raspberry ethanolic extracts (100 mg/mL) presented the highest inhibition of nitric oxide (NO) radical, 70.1%  0.6% and 64.4%  0.2%. The superoxide scavenging activity of these extracts was lower (54.4%  0.2%). The free radical scavenging capacity and reactive carbonyl species scavenging capacity of red and black raspberry were 268.5  8.6 and 2865.9  62.8 mg/mL, and 13.7% and 21.8%, respectively.19 Burton-Freeman et al.17 demonstrated in vitro that raspberry phenolic fractions reduced oxidative stress by decreasing lipid peroxidation, DNA damage, reactive oxygen species (ROS) generation and induction of antioxidant enzymes activities.

Raspberry and cancer Over the last decade, several lines of evidence have suggested that raspberries inhibit cancer of the oral cavity, esophagus, liver, colon, and skin.16, 20 Bidi et al.21 reported that red raspberry supplementation reduced the disease activity index and the risk of colorectal cancer, histological damage, and expression levels of inflammatory mediators and oncogenic genes in mice with chronic colitis. In an in vivo study, topical treatment with black raspberry extract significantly reduced cutaneous inflammation and caused marked reduction of carcinogenesis in SKH-1 mice. In all, 48 hours postexposure, investigators observed FIG. 1 Black raspberry (Rubus occidentalis) (A) and red raspberry (Rubus idaeus) (B).

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significantly reduced inflammatory responses, including reduction of skin edema, neutrophil activation, p53 protein expression levels, and markers of DNA damage; in a long-term model, black raspberry extract reduced the number and size of tumors.20 Yang et al.22 found that supplementation with red raspberry pulp polysaccharides (RPP) in mice bearing malignant melanoma-induced splenocyte proliferation and upregulated the splenic activity of immune-related enzymes. These findings suggested that RPP had antitumor activity in vivo against malignant melanoma, partly by enhancing the cellular immune response of the host organism.

Blueberry Blueberries are from the family Ericaceae, subfamily Vaccinoideae, genus Vaccinium, with approximately 450 species. These fruits are native to North America and Europe.23 Plants native to North America are also known as American blueberries, including Vaccinium corymbosum, the most commonly cultivated blueberry species. Berries native to Northern Europe are known as bilberries or European blueberries, including Vaccinium myrtillus.23 Blueberry species are also frequently grouped as highbush, lowbush, and rabbiteye, according to their height. They consist of perennial flowering plants that produce small edible blue/purple fruits (Fig. 2).23 The bioactivity of this fruit is directly related to the presence of phenolic compounds, especially anthocyanins, and other constituents such as phenolic acids and proanthocyanidins. Anthocyanin is the main flavonoid of blueberry.15 The total phenolic contents of blueberry ethanolic extract are 443.6  17 mg gallic acid/g.18 The total content of caffeic acid in blueberry is 8771.31  225.66 mg/mL dry weight of fruit.15

Blueberry and oxidative balance Hoskin et al.24 showed that spray-dried blueberries reduced ROS production and negative regulation of gene expression of inflammation markers (COX-2 and IL-1b). The authors also found milder suppression of NO production and gene expression of the enzyme nitric oxide synthetase (iNOS).24 Basu and Maier18 showed that blueberry ethanolic extract (100 mg/mL) inhibited NO radical and superoxide anion levels by 67.0%  0.6% and 54.6%  0.4%, respectively. Huang et al.25 evaluated the antioxidant role of blueberry extracted anthocyanins on antioxidant properties in endothelial cells and found that anthocyanins decreased ROS and xanthine oxidase-1 levels, as well as increased superoxide dismutase enzyme levels.

Blueberry and cancer In vitro and in vivo studies have suggested that blueberry extracts present anticancer activities against several cancer cell lines, attributable to their antioxidant, antiproliferative, and apoptotic effects.26, 27 Bunea et al.26 analyzed the antiproliferative and apoptotic properties of various blueberry cultivars in metastatic mouse melanoma (B16-F10) cells. Among the studied cultivars, Torro had the highest anthocyanin content and antioxidant activity, inhibiting the proliferation of B16-F10 melanoma murine cells in concentrations above 500 mg/mL. These authors also reported that this cultivar stimulated apoptosis and increased total LDH activity in B16-F10 cells. Diaconeasa et al.27 analyzed the antiproliferative properties of purified black currants and blueberry juices in the A2780, HeLa, and F10 cancer cell lines. In all cell lines analyzed, the treatments showed a reduction in cell viability from 100% to 45.9%, 52%, and 56% for HeLa, A2780, and B16-F10, respectively, within 24 hours. FIG. 2 Blueberry plantation grown in the state of Rio Grande do Sul, Brazil. (A), fruit bushes (B), and fruits of the cultivar Climax belonging to the rabbiteye group (C).

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Propolis Propolis is a natural resinous material collected by bees from various vegetable sources. Bees use propolis to protect the hive against microorganisms, to enclose small spaces and embalm foreign intruders. Bees gather this resinous secretion from buds, flowers, leaves, barks, and latex of plants to seal, strengthen, and disinfect their hives. Propolis can be brown, green, red, black, or white in color and sticky or brittle in texture. The plant resources are myriad, the principal ones being pine, poplar, oak, chestnut, sweet gum, birch, eucalyptus, willow, elm, acacia, and coin vine. A literature survey revealed that propolis is harvested in countries such as Thailand, Taiwan, China, India, Iraq, Turkey, Greece, Cuba, Tunisia, Croatia, Egypt, Brazil, Portugal, Mexico, and the Caribbean countries.28 Studies have reported the varied and complex chemical composition of propolis and its intimate relationship to the biodiversity of the geographical region, flora, and climate where it is collected.29 Propolis consists of a complex combination of resin (45%), wax and fatty acids (30%), essential oils (10%), pollen (5%), and organic compounds (10%).30 The most investigated and precious components of propolis are caffeic acid phenethyl ester (CAPE), an ester of caffeic acid. CAPE had several biological and pharmacological properties, including immunomodulatory, antiproliferative, antiviral, anticancer, antiinflammatory, anticarcinogenic, chemopreventive, and neuroprotective effects.31, 32

Propolis and oxidative balance CAPE possesses a variety of biological and pharmacological properties.33 It selectively induced apoptosis in cell lines, chelated metal ions, scavenged free radicals, inhibited enzymes such as lipoxygenase and cyclooxygenase (COX)-2, involved in free radical and lipid peroxidation, and suppressed the activation of nuclear transcription factor NF-kb.34–37

Propolis and cancer Propolis acts as an emergent complementary and alternative medicine and has demonstrated efficacy against cancers of the head and neck, brain and spinal cord, blood, skin, breast, pancreas, liver, colon, prostate, kidney, and bladder.28 Antitumor properties of propolis have been attributed to its antioxidant activity, augmented immune surveillance, suppression of proliferation, reduction in the cancer stem cell populations, blockade of specific oncogene signaling pathways, antiangiogenesis, modulation of the tumor microenvironments, increase of chemotherapeutic activity, and alleviation of side effects induced by drugs.38 Propolis has been studied as a possible cancer preventive medication in chronic skin inflammation. Using a UV-induced inflammatory mouse model with an increased risk of photocarcinogenesis, ethanol crude extract from an Australian propolis minimized skin inflammation by exerting immunosuppressive effects and reducing lipid peroxidation.39 It also protected against sunburn edema and contact hypersensitivity in a dose-dependent manner. Propolis suppressed typical photoimmune responses by suppressing expression of the proinflammatory cytokines IL-6 and IL-12 and stimulating expression of the antiinflammatory cytokine IL-10. A study investigated the immunomodulatory effects of propolis on mice melanoma models. Stress induced expanded the tumor area, while propolis-treated mice showed melanoma development similar to the control. Propolis administration also stimulated production of IL-2 and IL-10 that are related to immunoregulatory effects.40 CAPE was suggested to suppress ROS-induced DNA strand breakage in human melanoma A2058 cells to a greater extent than other potential protective agents.41 Formation of quinone could play an important role in CAPE-induced cell toxicity. The role of tyrosinase was investigated in CAPE toxicity. The authors found that CAPE led to negligible antiproliferative effects, apoptotic cell death, and ROS formation in shRNA plasmid-treated cells. CAPE also caused selective escalation of ROS formation and intracellular GSH depletion in melanocytic human SK-MEL-28 cells that express functional tyrosinase.42 Studies on five melanoma cell lines (B16F0, B16F10, SK-MEL-28, SK-MEL-5, and MeWo) and an in vivo efficacy study in a skin B16-F0 melanoma tumor model in C57BL/6 mice showed that CAPE (10 mg/kg/day) led to intracellular GSH depletion, increased ROS formation in B16-F0 cells, increased apoptosis in B16F0 cells, and demonstrated tumor size growth inhibition.43 The authors investigated CAPE as a selective glutathione S-transferase (GST) inhibitor in the presence of tyrosinase, which is abundant in melanoma cells. As much as 90% of CAPE was metabolized by tyrosinase after 60 minutes incubation showing 70%–84% GST inhibition. CAPE-SG conjugate and CAPE quinone demonstrated 85% GST inhibition via reversible and irreversible mechanisms.

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Apple Apple, known by the scientific name Malus domestica or Pyrus malus and belongs to the Rosaceae botanical family. The globose type has a deep depression at the insertion point of the stalk and are wider at the base than at the apex. There are about 25 species and over 7500 varieties of apples grown worldwide.44 Its origin traces to Europe and Asia. Apple planting regional studies have shown that the method of cultivation can substantially influence several chemical characteristics, including dry matter content, pH, total acidity, total sugars, and phenolic compounds.45, 46 Apples are among the most frequently consumed fruits worldwide. Their production is in the second position in the word ranking, and the largest producers located in the northern hemisphere, particularly the United States.46a

Apple and oxidative balance Apples have high concentrations of phenolic compounds, and their antioxidant action is one of the highest among fruits. The components include cinnamic acids such as quinic acid esters of caffeic and coumaric acids, flavonols such as quercetin glycoside, and dihydrochalcones such as florizine and three-flavonols.47, 48, 48a The flavonoid compounds that contribute the most to the antioxidant action of apple fruit are quercetin, florizine, and caffeic acid.49 Apple antioxidant activity must also be due to the presence of vitamin C, a powerful antioxidant that contributes around 0.4% of the total antioxidant activity. Approximately 1500 mg of vitamin C is generated from 100 g of apples.50 Phenolic compounds present in the fruit play important roles in human health due to their high antioxidant activity.50a Generally, these phenolic compounds are located in the fruit vacuoles (97%) in the epidermis and subcells of the fruit. Even thought the peel constitutes only about 10% of the fruit, it is the primary source of phenolic compounds since their concentration in the epidermis is the highest.46 The peel also contains considerable amounts of triterpenoids, including ursolic acid.51 Another important fact is that the flavonoids present in the apple have high molecular weights. This improves absorption by preventing the compounds from being absorbed in the stomach and allowing them to reach the intestine relatively intact.52 The composition of this fruit depends on factors such as nutrient availability, solar radiation, and the temperature of the site, as well as the state of ripeness of the fruit and its genetic context.53, 53a

Apple and cancer Studies have shown that the apple has several beneficial effects on health, primarily attributed to its antioxidant function, as well as to its antimutagenic and antimicrobial properties.54, 55, 55a Animal and cell culture studies showed that the action of flavonoid compounds found in apples may help to prevent chronic diseases.50 Studies showed that caffeic acid fulfilled a broad spectrum of pharmacological activities, including antiinflammatory, antioxidant, immunomodulatory, and neuroprotective activities.56 In vitro studies have shown that apple phenols are capable of inhibiting tumor cell proliferation, inducing apoptosis cell cycle arrest, suppressing angiogenesis and metastasis, modulating carcinogenic metabolism and signal transduction, and enhancing the immune system.57 A study demonstrated that the apple extract was effective in prevention and treatment of colon cancer, showing inhibitory action on growth and apoptosis induction in cancer cells.58 In another study, the apple extract inhibited the growth of breast cancer in rats, preventing breast cancer in a dosedependent manner comparable to human consumption of one to six apples a day.58a Caffeic acid has protective effects against DNA damage caused by reactive oxygen species.59 It also decreased tumor volume in a mouse model of solar-induced skin carcinogenesis.60 Flavonoids present in apples, including florizine, inhibit the activity of protein kinase C in melanoma cells, increasing melanin production in cells, thereby increasing natural protection against ultraviolet radiation. Compounds that enhance melanogenesis are important to protect human skin from solar radiation.60a Apples also help prevent the imbalance of reactive oxygen species, by virtue of their rich content of flavonoids that have broad antioxidant activity, and may be important for the prevention of several diseases, including cancer.

Grape and wine The grape (Vitis vinifera L.), family Vitaceae, was among the first domesticated fruits and is currently one of the most important economic fruit crops worldwide. It is primarily used to make wine. Nevertheless, the fruits are often consumed in natura, or are processed into raisins and grape juices as well as wines.61

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Grapes are particularly rich in polyphenols, including anthocyanins, flavonols, stilbene derivatives, and others, that have roles in plant metabolism. These compounds are responsible for several beneficial effects, including antioxidant, antiinflammatory, antidiabetic, antitumor, neuroprotective effects on the central nervous system, among others.62–65 Among the most important phenolic compounds found in grape parts are: (a) phenolic acids, including gallic acid, p-coumaric, caffeic, and ferulic acids; (b) flavonoids such as quercetin, myricetin, kaempferol-3-O-glucoside, and quercetin-3-O-glucoside; (c) polyphenols, including ( )-epicatechin-3-O-gallate, (+)-catechins, and ( )-epicatechin; (d) anthocyanins, including 3-caffeoyl-5-diglucosides of cyanidin, delphinidin, peonidin, petunidin, malvidin, 3-glucosides, 3-acetylglucosides, 3,5-diglucosides, 3-acetyl-5-diglucosides, 3-caffeoylglucosides, 3-coumaroylglucosides, and 3-coumaroyl-5-diglucosides; and (e) stilbene derivatives such as trans-resveratrol and piceatannol.64, 65 Chemical groups of polyphenols found in grape parts are summarized in Table 1.

Grape/wine and cancer The antitumor effects of grape parts extracts and some of the bioactive compounds against several cancer types, in vitro and in vivo, have been reviewed previously.64, 65 Compounds from grape inhibit cell proliferation, inducing apoptosis, and blocking cancer survival pathways. Evidence has focused on the antitumor effects of resveratrol, one of the most famous constituents of grape and also found in grape juice and wine.66 For example, a study using Duke melanoma 443 (DM443) and DM738 cell lines showed that resveratrol possessed antitumor effects manifested by decreasing cell viability, also showing selective toxicity against these cells when compared to healthy fibroblasts. Nevertheless, translation to in vivo experiments proved to be difficult.67

TABLE 1 Chemical groups of polyphenols identified in grape parts.64,

65

Different parts of grape fruit

Groups of polyphenols

Juice

Phenolic acids Flavonols Flavanols Stilbenes Anthocyanins

Pomace

Proanthocyanins Phenolic acids Flavonols Flavanols Stilbenes Anthocyanins

Raisins

Phenolic acids Flavonols Flavanols Anthocyanins

Seeds

Proanthocyanins Phenolic acids Flavonols Stilbenes

Skin

Phenolic acids Flavonols Flavanols Stilbenes Anthocyanins

Stem

Phenolic acids Flavonols Flavanols Stilbenes Anthocyanins

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The protective activity of grape extract (red grape seeds) was shown in a study that induced UVB skin damage in SKH-1 hairless mice. Solar ultraviolet radiation (UV) is the major cause of nonmelanoma skin cancer in humans and natural products may be an effective strategy in the management of cutaneous neoplasia. In fact, an investigation showed that the extract prevented the penetration of UVB into the skin of the mice, also modulating the inflammatory and apoptotic responses to UVB.68, 69 Photoprotective effects of resveratrol, one of the most important polyphenols found in grape, were shown both pre- and post-UVB irradiation in the same animal model, inhibiting tumor incidence and delaying the start of tumorigenesis.70 This molecule was also shown to induce apoptosis by modulation of antiapoptotic gene expression71 and upregulation of p53 expression.72 Recent studies have shown the antiproliferative potential of resveratrol and resveratrol-derived compounds (e-viniferin and labruscol) on melanoma cells (HT-144 and SK-Mel-28), showing no toxic effects on healthy human dermal fibroblasts. There was some differential antiproliferative activity for the three compounds, although resveratrol presented more effective action than the other oligomers.73 In another study using two cell lines derived from Merkel cell carcinoma (a rare but highly malignant tumor of the skin), resveratrol showed cytotoxicity and increased apoptosis. The combination of the molecule with chemotherapy caused a synergistic inhibition of cell proliferation; when resveratrol and irradiation were combined, a synergistic decrease in colony formation was found when compared to irradiation alone.74 Studies addressing the molecular patterns by which resveratrol acts on melanoma cells suggested that the antiproliferative properties of resveratrol are associated with the reduction of telomerase activity.75 In this context, Zhao et al.76 recently showed that resveratrol inhibited proliferation and increased apoptosis in melanoma cells by upregulating expression of the p53 gene and downregulating the Erk/PKM2/Bcl-2 axis, suggesting that this route could be used for the development of antineoplastic chemicals for the treatment of melanoma. Another investigation addressed the effect of resveratrol on a-MSH signaling, viability, and invasiveness in B16 melanoma cells, showing that this molecule inhibited pathways associated with melanoma invasiveness.77 Regarding the chemopreventive/antiproliferative effects of compounds isolated from grape, the literature appears limited with respect to the individual effects of some molecules; perhaps they could be used in combination, alone or with other agents/drugs to increase antiproliferative activity via synergism or additive effects.77a In general terms, considering the aggressiveness of melanoma and the antitumor effects of grape as well as those of some of its bioactive compounds, including resveratrol, strategies could be developed to improve treatment outcomes and to increase survival rates.

Summary points l l l l l

This chapter focuses on the effects of caffeic acid on oxidative balance and cancer. Caffeic acid is present in the chemical matrix of foods such as coffee, berries, propolis, apples, and grape. Caffeic acid is indicated as a potent anticarcinogenic agent and oxidative stress modulator. Furthermore, this molecule inhibited pathways associated with cancer invasiveness. In addition, it appears that caffeic acid may also modulate inflammatory processes.

References 1. Peng CY, et al. Characterization of Brazilian coffee based on isotope ratio mass spectrometry (d13C, d18O, d2H, and d15N) and supervised chemometrics. Food Chem 2019;297. 2. Leroy T, et al. Coffee (Coffea sp.). Methods Mol Biol 2006;344:191–209.
et al. Phytochemical overview and medicinal importance of Coffea species from the past until now. Asian Pac J Trop Med 2016;9 3. Patay EB, (12):1127–35. 4. Felberg I, et al. Effect of simultaneous consumption of soymilk and coffee on the urinary excretion of isoflavones, chlorogenic acids and metabolites in healthy adults. J Funct Foods 2015;19:688–99. 5. Moura-Nunes N, et al. The increase in human plasma antioxidant capacity after acute coffee intake is not associated with endogenous non-enzymatic antioxidant componentes. Int J Food Sci Nutr 2009;6:173–81. 6. Rebollo-Hernanz M, et al. Phenolic compounds from coffee by-products modulate adipogenesis-related inflammation, mitochondrial dysfunction, and insulin resistance in adipocytes, via insulin/PI3K/AKT signaling pathways. Food Chem Toxicol 2019;132:110672. 7. Jeszka-Skowron M, et al. Positive and negative aspects of green coffee consumption—antioxidant activity versus mycotoxins. J Sci Food Agric 2017;97(12):4022–8. 8. Loftfield E, et al. Coffee drinking and cutaneous melanoma risk in the NIH-AARP diet and health study. J Natl Cancer Inst 2015;107:421. 9. Wrze
sniok D, et al. Caffeine modulates growth and vitality of human melanotic COLO829 and amelanotic C32 melanoma cells: preliminary findings. Food Chem Toxicol 2018;120:566–70.

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10. Lukic M, et al. Coffee consumption and the risk of malignant melanoma in the Norwegian Women and cancer (NOWAC) Study. BMC Cancer 2016;16. 11. Jang M, et al. Caffeine induces apoptosis in human neuroblastoma cell line SK-N-MC. J Korean Med Sci 2002;17(5):674–8. 11a. Qi W, Martinez J, Qiao D. Caffeine induces TP53-independent G1-phase arrest and apoptosis in human lung tumor cells in a dose-dependent manner. Radiat Res 2002;157(2):166–74. 12. Okano J, et al. Caffeine inhibits the proliferation of liver cancer cells and activates the MEK/ERK/EGFR signalling pathway. Basic Clin Pharmacol Toxicol 2008;102:543–51. 13. Ku BM, et al. Caffeine inhibits cell proliferation and regulates PKA/GSK3b pathways in U87MG human glioma cells. Mol Cells 2011;31:275–9. 14. Pelison L, et al. Antiproliferative and apoptotic effects of caffeic acid on SK-Mel-28 human melanoma cancer cells. Mol Biol Rep 2019;46 (2):2085–92. 15. Kim J-S. Antioxidant activities of selected berries and their free, esterified, and insoluble-bound phenolic acid contents. Prev Nutr Food Sci 2018;23 (1):35–45. 16. Kula M, Krauze-Baranowska M. Rubus occidentalis: the black raspberry—its potential in the prevention of cancer. Nutr Cancer 2015;1–11. 17. Burton-Freeman BM, Sandhu AK, Edirisinghe I. Red raspberries and their bioactive polyphenols: cardiometabolic and neuronal health links. Adv Nutr 2016;7:44–65. 18. Basu P, Maier C. In vitro antioxidant activities and polyphenol contents of seven commercially available fruits. Pharm Res 2016;8:258–64. 19. Ma H, Johnson SL, Liu W, DaSilva NA, Meschwitz S, Dain JA, et al. Evaluation of polyphenol anthocyanin-enriched extracts of blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry for free radical scavenging, reactive carbonyl species trapping, anti-glycation, anti-bamyloid aggregation, and microglial neuroprotective effects. Int J Mol Sci 2018;19(2):461. 20. Duncan FJ, Martin JR, Wulff BC, Stoner GD, Tober KL, Oberyszyn TM, et al. Topical treatment with black raspberry extract reduces cutaneous UVBinduced carcinogenesis and inflammation. Cancer Prev Res (Phila) 2009;2:665–72. 21. Bidi S, Du M, Zhu M-J. Dietary red raspberry reduces colorectal inflammation and carcinogenic risk in mice with dextran sulfate sodium–induced colitis. J Nutr Biochem 2018;51:40–6. 22. Yang YJ, Xu H-M, Suo Y-R. Raspberry pulp polysaccharides inhibit tumor growth via immunopotentiation and enhance docetaxel chemotherapy against malignant melanoma in vivo. Food Funct 2015;6:3022–34. 23. Chu W, Cheung SCM, Lau RAW, Benzie IFF. Bilberry (Vaccinium myrtillus L.). herbal medicine: Biomolecular and clinical aspects. 2nd ed. Boca Raton, FL: CRC Press/Taylor & Francis; 2011 [chapter 4]. 24. Hoskin RT, Xiong J, Esposito DA, Lila MA. Blueberry polyphenol-protein food ingredients: the impact of spray drying on the in vivo antioxidant activity, anti-inflammatory markers, glucose metabolism and fibroblast migration. Food Chem 2019;15:187–94. 25. Huang W, Zhu Y, Li C, Sui Z, Min W. Effect of blueberry anthocyanins malvidin and glycosides on the antioxidant properties in endothelial cells. Oxid Med Cell Longev 2016;1–10. 26. Bunea A, Rugina˘ D, Scont¸a Z, Pop RM, Pintea A, Socaciu C, et al. Anthocyanin determination in blueberry extracts from various cultivars and their antiproliferative and apoptotic properties in B16-F10 metastatic murine melanoma cells. Phytochemistry 2013;95:436–44. 27. Diaconeasa Z, Leopold L, Rugina˘ D, Ayvaz H, Socaciu C. Antiproliferative and antioxidant properties of anthocyanin rich extracts from blueberry and blackcurrant juice. Int J Mol Sci 2015;16(2):2352–65. 28. Patel S. Emerging adjuvant therapy for cancer: propolis and its constituents. J Diet Suppl 2015;1–24. 29. Lo´pez BG, et al. Phytochemical markers of different types of red propolis. Food Chem 2014;146:174–80. 30. Chen YJ, et al. Caffeic acid phenethyl ester, an antioxidant from propolis, protects peripheral blood mononuclear cells of competitive cyclists against hyperthermal stress. J Food Sci 2009;74(6):H162–7. 31. Aviello A, et al. Inhibitory effect of caffeic acid phenethyl ester, a plant-derived polyphenolic compound, on rat intestinal contractility. Eur J Pharmacol 2010;640:163–7. 32. Widjaja A, et al. Enzymatic synthesis of caffeic acid phenethyl ester. J Chin Inst Chem Eng 2008;39:413–8. 33. Hsiao G, et al. Characterization of a novel and potent collagen antagonist, caffeic acid phenethyl ester, in human platelets: in vitro and in vivo studies. Cardiovasc Res 2007;75(4):782–92. 34. Bhimani RS, et al. Inhibition of oxidative stress in HeLa cells by chemopreventive agents. Cancer Res 1993;53(19):4528–33. 35. Michaluart P, et al. Inhibitory effects of caffeic acid phenethyl ester on the activity and expression of cyclooxygenase-2 in human oral epithelial cells and in a rat model of inflammation. Cancer Res 1999;59(10):2347–52. 36. Natarajan K, et al. Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B. Proc Natl Acad Sci U S A 1996;93(17):9090–5. 37. Sugiura M, et al. Inhibitory activities and inhibition specificities of caffeic acid derivatives and related compounds toward 5-lipoxygenase. Chem Pharm Bull(Tokyo) 1989;37(4):1039–43. 38. Meneghelli C, et al. Southern Brazilian autumnal propolis shows anti-angiogenic activity: an in vitro and in vivo study. Microvasc Res 2013;88:1–11. 39. Cole N, et al. Topical ‘Sydney’ propolis protects against UV-radiationinduced inflammation, lipid peroxidation and immune suppression in mouse skin. Int Arch Allergy Immunol 2010;152:87–97. 40. Missima F, et al. The effect of propolis on Th1/Th2 cytokine expression and production by melanoma-bearingmice submitted to stress. Phytother Res 2010;24(10):1501–7. 41. Chen CN, et al. Apoptosis of human melanoma cells induced by the novel compounds propolin A and propolin B from Taiwenese propolis. Cancer Lett 2007;245:218–31.

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42. Kudugunti SK, et al. Biochemical mechanism of caffeic acid phenylethyl ester (CAPE) selective toxicity towards melanoma cell lines. Chem Biol Interact 2010;188:1–14. 43. Kudugunti SK, et al. Efficacy of caffeic acid phenethyl ester (CAPE) in skin B16-F0 melanoma tumor bearing C57BL/6 mice. Invest New Drugs 2011;29:52–62. 44. Bhatti S, Jha G. Current trends and future prospects of biotechnological interventions through tissue culture in apple. Plant Cell Rep 2010;29:1215–25. 45. Drogoudi PD, et al. Peel and flesh antioxidant content and harvest quality characteristics of seven apple cultivars. Sci Hortic 2008;115:149–53. 46. Lata B. Relationship between apple peel and the whole fruit antioxidant content: year and cultivar variation. J Agric Food Chem 2007;55:663–71. 46a. Agricultural marketing resource center (AgMRC). Apples; 2018. https://www.agmrc.org/commodities-products/fruits/apples. 47. Carbone K, et al. Phenolic composition and free radical scavenging activity of different apple varieties in relation to the cultivar, tissue type and storage. Food Chem 2011;127:493–500. 48. Fromm M, et al. Characterization and quantitation of low and high molecular weight phenolic compounds in apple seeds. J Agric Food Chem 2012;60:1232–42. 48a. Mari A, Tedesco I, Nappo A, Russo G, Malorni A, Carbone V. Phenoliccompound characterisation and antiproliferative activity of “Annurca” Apple, a southernItalian cultivar. Food Chem 2010;123:157–64. 49. Lee K, et al. Major phenolics in apple and their contribution to the total antioxidant capacity. J Agric Food Chem 2003;51:6516–20. 50. Boyer J, et al. Apple phytochemicals and their health benefits. Nutr J 2004;. 50a. Bhuyan DJ, Basu A. Phenolic compounds: potential health benefits and toxicity. In: Vuong QV, editor. Utilisation of Bioactive Compounds from Agricultural and Food Production Waste (Chapter 2). vol. 2. CRC Press, Taylor & Francis Group; 2017. pp. 27–59. 51. Frighetto R, et al. Isolation of ursolic acid from apple peels by high speed counter-current chromatography. Food Chem 2008;106:767–71. 52. Koutsos A, et al. Apples and cardiovascular health—is the gut microbiota a core consideration? Nutrients 2015;7:3959–98. 53. Awad MA, et al. Flavonoid and chlorogenic acid concentrations in apple fruit: characterization of variation. Sci Hortic 2000;83:249–63. 53a. Janzantti N, Franco MRB, Wosiacki G. Efeito do processamento na composic¸a˜o de vola´teis de suco clarificado de mac¸a˜ Fuji. Ci^ enc Tecnol Aliment 2003;23:523–8. 54. Serra A, et al. Characterization of traditional and exotic apple varieties from Portugal. Part 2—antioxidant and antiproliferative activities. J Funct Foods 2010;2:46–53. 55. Trias R, et al. Bioprotection of Golden Delicious apples and Iceberg lettuce against foodborne bacterial pathogens by lactic acid bacteria. Int J Food Microbiol 2008;123:50–60. 55a. Molna´r P, et al. Biological activity of carotenoids in red paprika. Valencia orange and Golden delicious apple Phytoter Res 2005;19:700–7. 56. Chung TW, et al. Novel and therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma 53 growth and metastasis by dual mechanism. FASEP J 2004;18:1670–81. 57. Fabiani R, et al. Apple intake and cancer risk: a systematic review and meta analysis of observational studies. Public Health Nutr 2016;14:2603–17. 58. Li Y, et al. Modified apple polysaccharides could induce apoptosis in colorectal cancer cells. J Food Sci 2010;75. 58a. Liu JR, et al. Fresh apples suppress mammary carcinogenesis and proliferative activity and induce apoptosis in mammary tumors of the spraguedawley rat. J Agric Food Chem 2009;57:297–304. 59. Li Y, et al. Caffeic acid improves cell viability and protects against DNA damage: involvement of reactive oxygen species and extracellular signalregulated kinase. Braz J Med Biol Res 2015;. 60. Yang ES, et al. BRCA1 16 years later: DNA damage-induced BRCA1 Shuttling. FEBS J 2010;277:3079–85. 60a. Takekoshi S, Nagata H, Kitatani K. Flavonoids enhance melanogenesis in human melanoma cells. Tokai J Exp Clin Med 2014;39:116–21. 61. Gerrath J, Posluszny U, Melville L. Taming the wild grape: Botany and horticulture in the Vitaceae. Springer International Publishing; 2015, 194 pp. 62. Flamini R, Mattivi F, De Rosso M, Arapitsas P, Bavaresco L. Advanced knowledge of three important classes of grape phenolics: anthocyanins, stilbenes and flavonols. Int J Mol Sci 2013;14(10):19651–69. 63. Mattivi F, Guzzon R, Vrhovsek U, Stefanini M, Velasco R. Metabolite profiling of grape: flavonols and anthocyanins. J Agric Food Chem 2006;54 (20):7692–702. 64. Nassiri-Asl M, Hosseinzadeh H. Review of the pharmacological effects of Vitis vinifera (grape) and its bioactive compounds. Phytother Res 2009;23 (9):1197–204. 65. Nassiri-Asl M, Hosseinzadeh H. Review of the pharmacological effects of Vitis vinifera (grape) and its bioactive constituents: an update. Phytother Res 2016;30(9):1392–403. 66. Rauf A, Imran M, Butt MS, Nadeem M, Peters DG, Mubarak MS. Resveratrol as an anti-cancer agent: a review. Crit Rev Food Sci Nutr 2018;58 (9):1428–47. 67. Osmond GW, Augustine CK, Zipfel PA, Padussis J, Tyler DS. Enhancing melanoma treatment with resveratrol. J Surg Res 2012;172 (1):109–15. 68. Afaq F, Katiyar SK. Polyphenols: skin photoprotection and inhibition of photocarcinogenesis. Mini Rev Med Chem 2011;11(14):1200–15. 69. Filip A, Clichici S, Daicoviciu D, Catoi C, Bolfa P, Postescu ID, et al. Chemopreventive effects of Calluna vulgaris and Vitis vinifera extracts on UVBinduced skin damage in SKH-1 hairless mice. J Physiol Pharmacol 2011;62(3):385–92. 70. Aziz MH, Reagan-Shaw S, Wu J, Longley BJ, Ahmad N. Chemoprevention of skin cancer by grape constituent resveratrol: relevance to human disease? FASEB J 2005;19(9):1193–5. 71. Ivanov VN, Partridge MA, Johnson GE, Huang SX, Zhou H, Hei TK. Resveratrol sensitizes melanomas to TRAIL through modulation of antiapoptotic gene expression. Exp Cell Res 2008;314(5):1163–76.

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72. Hsieh TC, Wang Z, Hamby CV, Wu JM. Inhibition of melanoma cell proliferation by resveratrol is correlated with upregulation of quinone reductase 2 and p53. Biochem Biophys Res Commun 2005;334(1):223–30. 73. Nivelle L, Aires V, Rioult D, Martiny L, Tarpin M, Delmas D. Molecular analysis of differential antiproliferative activity of resveratrol, epsilon viniferin and labruscol on melanoma cells and normal dermal cells. Food Chem Toxicol 2018;116(Pt B):323–34. 74. Heiduschka G, Lill C, Seemann R, Brunner M, Schmid R, Houben R, et al. The effect of resveratrol in combination with irradiation and chemotherapy: study using Merkel cell carcinoma cell lines. Strahlenther Onkol 2014;190(1):75–80. 75. Platella C, Guida S, Bonmassar L, Aquino A, Bonmassar E, Ravagnan G, et al. Antitumour activity of resveratrol on human melanoma cells: a possible mechanism related to its interaction with malignant cell telomerase. Biochim Biophys Acta Gen Subj 2017;1861(11 Pt A):2843–51. 76. Zhao H, Han L, Jian Y, Ma Y, Yan W, Chen X, et al. Resveratrol induces apoptosis in human melanoma cell through negatively regulating Erk/PKM2/ Bcl-2 axis. Onco Targets Ther 2018;11:8995–9006. 77. Chen YJ, Chen YY, Lin YF, Hu HY, Liao HF. Resveratrol inhibits alpha-melanocyte-stimulating hormone signaling, viability, and invasiveness in melanoma cells. Evid Based Complement Alternat Med 2013;2013:632121. 77a. Singh CK, Siddiqui IA, El-Abd S, Mukhtar H, Ahmad N. Combination chemoprevention with grape antioxidants. Mol Nutr Food Res 2016;60 (6):1406–15.

Chapter 27

Oxidative stress and cancer: Antioxidative role of Ayurvedic plants Sahdeo Prasad and Sanjay K. Srivastava Department of Immunotherapeutics and Biotechnology and Center for Tumor Immunology and Targeted Cancer Therapy, Texas Tech University Health Sciences Center, Abilene, TX, United States

List of abbreviations AML CAT DMBA DTD EGCG GPx GSH iNOS LLC NOXs ROS SOD

acute myeloid leukemia catalase 7,12-dimethylbenz(a)anthracene DT diaphorase epigallocatechin-3-gallate glutathione peroxidase glutathione inducible nitric oxide synthase Lewis lung carcinoma NADPH oxidases reactive oxygen species superoxide dismutase

Introduction Cancer is a major health issue and the second leading cause of death worldwide after cardiovascular disease. The International Agency for Research on Cancer (IARC) estimated 17.0 million new cancer cases and 9.5 million cancer deaths worldwide in 2018. It is expected to increase to 27.5 million new cancer cases and 16.3 million cancer deaths by 2040. Also, it is observed that approximately 70% of deaths from cancer occur in low- and middle-income countries. In the United States, 1,762,450 new cancer cases and 606,880 cancer deaths were projected to occur in 2019. Although the cancer incidence rate and death rate declined at a certain extent, cancer is still a major health issue.1 In view of the seriousness of this disease, several therapeutic modalities have developed, which include chemotherapy, radiotherapy, and/or surgery. These therapeutic modalities have serious side effects and are also very expensive. The projected cost for the care of cancer is predicted to increase from 124.57 billion US dollars in 2010 to 157.77 billion dollars in 2020. However, if costs of cancer care increase annually by 2%, the projected total cost in 2020 will be 173 billion US dollars.2 Cancer is a chronic multifactorial disease. It is caused by internal factors as well as environmental factors. Internal factors include inherited mutations in genes, imbalanced hormones, and poor immunity of the body while environmental factors consist of smoking, lifestyle (consumption of fried foods, red meat, alcohol, low fruit and vegetable intake, physical inactivity), pollutants, UV exposure, infections, stress, and obesity. These factors disrupt the balance of cellular homeostasis that leads to diseases such as cancer. For example, environmental factors modulate signaling pathways, regulate transcription factors, activate proto-oncogenes, as well as induce kinases and oxidative stress in the cells. In this chapter, we will discuss the impact of oxidative stress in cancer development and further describe the role of medicinal plants, particularly Ayurvedic plants, in combating oxidative stress and cancer.

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00027-4 © 2021 Elsevier Inc. All rights reserved.

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Oxidative stress and cancer Oxidative stress is a phenomenon caused by imbalance between the systemic manifestation of free radicals or reactive oxygen species (ROS) and the level of antioxidants that neutralize them. ROS are by-products of an aerobic metabolism, mostly generated in cells by the mitochondrial respiratory chain during the process of oxidative phosphorylation (OXPHOS).3 ROS are produced primarily from complex I when the mitochondria have a high Dp (protonmotive force), and reduced the coenzyme Q pool and high NADH/NAD + ratio in the mitochondrial matrix.4 NADPH oxidases (NOXs) also contribute to the production of cellular ROS. A total of seven distinct isoforms of the NOX complex have been identified, which are NOX1, NOX2 (gp91phox), NOX3, NOX4, NOX5, DUOX1, and DUOX2, in cell membranes, mitochondria, peroxisomes, and the endoplasmic reticulum.5 The increased oxidative stress causes damage of various cellular molecules that may potentially influence homeostasis of the organism. Oxidative stress is sometimes beneficial. Acute oxidative stress drives the cellular responses for repair and regeneration, activates important signaling kinases, induces immunity, and fights with pathogens.6 However, continued oxidative stress involves the regulation of various cellular processes through modulation of signaling molecules, production of antioxidant enzymes and nonenzymes, cell growth, chronic inflammation, damage to DNA, RNA, proteins, lipids, and thus processes of malignancy.7 Thus, antioxidant systems are required to counteract oxidative stress and overcome cellular damage for the prevention of oxidative stress-mediated diseases like cancer. Plant products are one of the major sources of antioxidants, which have no or mild toxicity, abundant availability, high efficacy, and cost-effectiveness. Traditional medicines are the inherent source of antioxidants, which are being used against different ailments since centuries. Like other different traditional medicines, Ayurvedic medicine is the collection of several hundred medicinal plants.

Ayurvedic plants with antioxidative nature Accumulated evidences suggest that polyphenols of many plants exhibit antioxidant properties by neutralizing free radicals, quenching ROS, and lowering peroxides.8 Most of these plants are widely described in Ayurveda for their antioxidant and illness recovering properties. Ayurveda is an ancient Indian medical system and considered as one of the world’s oldest holistic healing systems, which is thought to have developed more than 5000 years ago in India. Ayurvedic medicine is a combination of mainly plant products, but may also include animal, metal, and mineral, with the inclusion of diet, exercise, and lifestyle. In the official Ayurvedic pharmacopeia, approximately 1200–1500 medicinal plants have been described, although over 10,000 plants are used for medicinal purposes in the Indian subcontinent since a century.9 In this chapter, we will discuss antioxidative and anticancer properties of some selected Ayurvedic plants including Emblica officinalis, Glycyrrhiza glabra, Aloe vera, Ocimum sanctum, Tinospora cordifolia, Allium cepa, Camellia sinensis, Cinnamomum verum, Curcuma longa, Terminalia bellerica, Trigonella foenum-graecum, and Zingiber officinalis (Fig. 1).

Emblica officinalis Emblica officinalis Gaertn (Euphorbeaceae), commonly known as the Indian gooseberry or Amla, is an important medicinal plant of Ayurvedic medicine and various other folk systems. The fruit of E. officinalis is one of the most important parts of the plant and has dietary and medicinal use in traditional medical systems. This plant is an excellent source of various phytochemicals, such as terpenoids, and flavonoids, minerals, vitamins, and different tannins, which are believed to possess diverse pharmacological and biological effects. The Amla fruit extract has been shown to be effective against various ailments including oxidative stress, inflammation, diabetes, cancer, osteoporosis, neurodegenerative disease, hypercholesterolemia, and infection.10 Emblica officinalis exhibits antioxidative properties by multiple mechanisms such as quenching ROS and inducing antioxidant enzymes. Reddy et al. showed that the E. officinalis fruit extract suppresses alcohol-induced oxidative damage by reducing lipid peroxidation and restoring activity of antioxidant enzymes like superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), as well as nonenzymatic antioxidant glutathione (GSH).11 Amla has also shown protective effect against arsenic-induced oxidative stress in thymocytes of mice. It restores an arsenic-induced increase of lipid peroxidation, ROS production, activity of antioxidant enzymes, and further cell viability in mice treated with arsenic, which indicates its strong antioxidant potential.12 In a hepatocyte cell line (HepG2), aqueous extracts of E. officinalis fruits have been shown to reduce the oxidative stress markers and increase antioxidant level. In a study, incubation with a nontoxic dose of E. officinalis resulted in a decrease of lipid hydroperoxide level and ROS generation. Besides these, it increased the levels of GSH, activities of antioxidant enzymes (SOD, CAT, GPx, GSH reductase, and GSH S-transferase) and antioxidant capacity.13 Amla has been shown to be more potent than vitamin C in scavenging O₂
, hydrogen peroxide,

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FIG. 1 Selected Ayurvedic plants which have shown potent anticancer effects.

Emblica officinalis

Glycyrrhiza glabra

(Indian gooseberry)

(Liquorice)

Aloe vera

Ocimum sanctum (Holy Basil)

Tinospora cordifolia

Allium cepa

Camellia sinensis

Cinnamomum verum

(Gulbel, Indian Tinospora)

(Onion)

(Tea)

(Cinnamon)

Curcuma longa

Terminalia bellerica (Baheda)

Trigonella foenumgraecum

Zingiber officinalis

(Turmeric)

(Ginger)

(Fenugreek)

and nitric oxide. It significantly preserved antioxidant enzymes MnSOD and CAT and also decreased the expression of inducible nitric oxide synthase (iNOS) and CYP2E1 protein in rat liver treated with N-nitrosodiethylamine. These antioxidative properties of Amla cause hepatoprotection in rats.14 Amla has also shown to be radioprotective in mice by reducing oxidative stress. In a study, oral treatment of Amla fruit extract (100 mg/kg) suppressed irradiation-induced elevation of lipid peroxidation and caused a reduction of GSH as well as CAT antioxidant enzyme in the mice intestine.15 In another study, oral treatment of Emblica has been shown to increase the activity of various antioxidant enzymes and the GSH system in blood, which were lowered by irradiation in mice. Treatment with Emblica also decreased the radiation-induced elevated lipid peroxides level in the serum.16 These studies indicate that Emblica fruit has properties to reduce the punitive nature of radiation and thus can be useful in decreasing the irradiation-mediated side effects during radiotherapy.

Glycyrrhiza glabra Glycyrrhiza glabra is also known as licorice, yashtimadhu, and mulethi. It has been used in Ayurvedic medicine since centuries. In Ayurvedic texts, G. glabra has been mentioned as Atirasa, Madhurasaa, Madhuka, Yastikavha, and Madhuyashtyaahvaa.17 However, this plant is listed in Chinese and other traditional medicine systems. A large number of bioactive compounds have been detected by different chromatography techniques in the extracts, roots, and leaves of G. glabra. These active constituents include triterpenes (glycyrrhetic acid, glycyrrhizin), phenols (including liquiritigenin, liquiritin, isoliquiritigenin, isoliquiritin), and many others. It has been demonstrated that licorice roots have several biological activities such as antioxidant, anti-inflammatory, anti-ulcerative, anticarcinogenic, and many other properties. These properties show its potential against various diseases including cancer, atherosclerosis, diabetes, ulcers, immunodeficiency, viral, and bacterial infections.18 Numerous studies have shown that the active components of G. glabra have potent antioxidant activity. Chin et al. found that hispaglabridin B, isoliquiritigenin, and paratocarpin B (compounds of G. glabra) are the most potent antioxidant agents.19 The compounds from G. glabra are reported to exhibit antioxidant effects by quenching ROS, reducing lipid

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peroxidation, and inducing antioxidant enzymes like SOD, CAT, GST, GPx, and GR.20 G. glabra also activates Nrf2 signaling molecules that reflect its antioxidant nature.21 Recently, licorice extract is found to alleviate the hazardous effects of cadmium chloride attributed to its antioxidant properties. An oral dose of licorice extract (3 mg/mL/kg/d) in rats has been shown to restore the CdCl2-induced elevated level of thiobarbituric acid reactive substance (TBARS) and decreased the level of SOD, CAT antioxidant enzymes, and GSH content.22 Roots of licorice are found to be potent cancer chemopreventive agents due to its various beneficial properties including its high antioxidant potential. It has been shown to inhibit the proliferation of various human cancer cells (HepG2, SW480, A549, MCF7). Glycybridin D, an active component of licorice root, at a dose of 10 mg/kg has been shown to decrease tumor mass by 39.7% on an A549 human lung carcinoma xenograft mouse model.21 Isoliquiritigenin, another component of licorice root, was demonstrated to prevent the incidence of 1,2-dimethylhydrazine-induced colon and lung tumors in mice.19 Methanol extracts of the roots of G. glabra also induced the cytotoxicity in human keratinocyte (HaCaT), lung adenocarcinoma (A549), and liver carcinoma (HepG2) cell lines.23 Besides these, the hydroalcoholic extract of G. glabra showed complete reversal of cisplatin resistance in triple negative MDA-MB-468 breast cancer cells, indicating its chemosensitizing effects.24

Aloe vera Aloe vera is commonly known as Indian aloe, true aloe, medicinal aloe, Chinese aloe and sometimes described as a “wonder plant.” In Ayurveda, Aloe vera is described as ghritkumari. Aloe vera has been used for many centuries against many diseases and disorders due to its preventive and therapeutic properties. It is also widely used in the cosmetic, pharmaceutical, and food industries, and has an estimated annual market value of $13 billion globally.25 A large number of phytochemicals have been identified in the Aloe vera leaf including various phenolic acids/polyphenols, phytosterols, fatty acids, indoles, alkanes, pyrimidines, alkaloids, organic acids, aldehydes, dicarboxylic acids, ketones, and alcohols.26 However, its antioxidant activity is found mainly in its polyphenols, indoles, and alkaloids. Many other medicinal effects of Aloe vera have been shown in its polysaccharides,27 but it is believed that these biological activities of Aloe vera are associated with a synergistic action of the compounds present in the aloe leaf.28 Aloe vera has antioxidant, anti-inflammatory, healing, antiseptic, anticancer, and antidiabetic effects. The antioxidant activity of Aloe vera is reported by using various biochemical methods in both in vitro and in vivo models. In an in vivo model, the fresh leaf pulp extract of Aloe vera increased the level of antioxidant enzymes, DT-diaphorase (DTD), SOD, CAT, GPX, and glutathione reductase (GR), as well as the glutathione content in the liver of mice.29 Topical application of Aloe vera leaf gel also exhibited antioxidant activity in 7,12-dimethylbenz(a)anthracene (DMBA)-induced skin lesions in Swiss albino mice and protected the systemic toxicity of DMBA.30 Treatment of Aloe vera extract also resulted in a significant elevation activity of key antioxidant enzymes (SOD, GST, GPx, and LDH) in EACC tumors.31 The administration of Aloe vera gel extract has been shown to be protective in X-ray exposed animals. It reduces radiation-induced ROS generation and improves hepatic and renal function in mice. Further, in vitro assays revealed that Aloe vera gel extract is effective in scavenging free radicals, which is linked to its potential in exhibiting antioxidant effects.32 These data suggest that Aloe vera may serve to boost the antioxidant system; thus, it exhibits a chemopreventive agent. Aloe vera has been also reported to be a potent chemopreventive and chemotherapeutic agent probably linked to its antioxidant nature. El-Shemy et al. showed that active principles of Aloe vera (aloesin, aloe-emodin, and barbaloin) exhibit concentration-dependent cytotoxicity against acute myeloid leukemia (AML), acute lymphocytes leukemia (ALL), and colon cancer cell lines (i.e., DLD-1 and HT2). The cytotoxic property of these active principles were associated with the internucleosomal DNA fragmentation and further apoptosis.31 In addition to its antioxidant activity, Aloe vera extract also possesses prooxidant properties in cancer cells, leading to oxidative DNA breakage. It has been shown that aqueous extract of Aloe vera generates ROS, such as superoxide anion and hydroxyl radicals, and causes DNA degradation in the presence of copper ions and further apoptosis in cancer cells.33 Aloe vera is also effective in suppressing skin carcinogenesis. In an experiment, topical and oral treatment with both gel and extract prevented tumor formation in stage-2 skin carcinogenesis of DMBA/croton oil-induced skin papillomagenesis in mice.30, 34

Ocimum sanctum Ocimum sanctum is also called Holy Basil and referred to as Tulsi in Ayurveda. It is an aromatic perennial plant and revered as an elixir of life for both its medicinal and spiritual properties. Tulsi is cultivated for religious and traditional medicine purposes, and also for its essential oil. The Tulsi plant contains numerous active compounds and the major compounds are linalol, eugenol, methylchavicol, methylcinnamat, linolen, ocimene, pinene, cineol, anethol, estragol, thymol, citral, and camphor.35 Different parts of the O. sanctum plant, mostly leaves, have a variety of pharmacological effects like

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antimicrobial, immunomodulatory, anti-stress, anti-inflammatory, antiulcer, antidiabetic, antipyretic, diuretic, hepatoprotective, chemoprotective, hypolipidemic, cardioprotective, antioxidant, antitussive, radioprotective, memory enhancing, antiarthritic, antifertility, antihypertensive, anticoagulant, anticataract, anthelmintic, and antinociceptive effects and have been used to treat gastritis, stomachache, flatulence, constipation, vomiting, and hiccup.36, 37 As stated above, the Tulsi plant has numerous biological properties including antioxidant activity. In a study, oral administration of the aqueous extract of O. sanctum (200 mg/kg body weight) in diabetic rat decreased lipid peroxidation (TBARS) and increased antioxidant SOD, CAT, GPX, GST, and GSH in plasma and rat liver, lung, kidney, and brain, which shows its antioxidant property.38 Similarly, Halim et al. found that treatment of the diabetic animals with O. sanctum, or in combination with Vitamin E, restored the levels of lipid peroxidation GPX, SOD, CAT, and GST antioxidant enzymes.39 In an animal model of Benzo(a)pyrene-induced forestomach and DMBA-initiated skin papillomagenesis, basil leaf extract was very effective in elevating antioxidant enzyme response by increasing GR, SOD, and CAT activities in the liver skin and other extrahepatic organs.40 Besides this, O. sanctum seed oil also increased the antioxidant enzymes SOD, CAT, GST, and nonenzymatic antioxidants GSH in a 20-methylcholanthrene-induced fibrosarcoma animal tumor model. It also decreased the lipid peroxidation end product, MDA, in liver. This antioxidant activity of O. sanctum seed oil is partly attributed to tumor regression and showed chemopreventive activity.41 O. sanctum leaf extracts have been shown to attenuate free radicals and inhibit oxidative damage of tissues. In a study, O. sanctum extract showed protective effect against cracker smoke-induced damage of the lung and brain tissues. Supplementation of this plant leaf extract inhibited oxidative stress as analyzed by the estimation of SOD, CAT enzyme levels and iNOS, HSP70 protein expression.42 The alcoholic aqueous extracts of different species of the Tulsi plant extract have shown reduction in tumor growth and an increase in the survival rate of mice. This extract further inhibited radiationinduced chromosomal damage and an elevation in GSH level and GST activity.43 In addition, ethanol extracts of O. sanctum had antimetastatic activity through activation of antioxidative enzymes. It inhibited metastasis of Lewis lung carcinoma (LLC) cells and reduced the tumor nodule formation in LLC-injected mice. This extract also enhances activity of antioxidant enzymes such as SOD, CAT, and GPx.44 Overall, these studies indicate that the Tulsi plant has potent antitumor, antimetastatic, and antioxidant properties.

Tinospora cordifolia Tinospora cordifolia, also called Gulbel or Indian Tinospora in English and Guduchi in Sanskrit, is a shrub that is native to India. Its root, stems, leaves, and even the whole plant are used in Ayurvedic medicine. In Ayurveda, it has shown immense application in the treatment of various diseases including diabetes, high cholesterol, allergic rhinitis, stomach upset, gout, cancer, rheumatoid arthritis, hepatitis, peptic ulcer, fever, gonorrhea, syphilis and to boost the immune system. This plant has also brought great attention to the researchers across the globe since it has various medicinal properties like antioxidant, anti-inflammatory, antidiabetic, antispasmodic, anti-stress, anti-arthritic, anti-allergic, antimalarial, anti-leprotic, hepatoprotective, immunomodulatory, and antineoplastic activities.45 A variety of active components such as alkaloids, steroids, diterpenoid lactones, aliphatics, and glycosides have been isolated from the different parts of the plant body, including the root, stem, and whole plant.46 The antioxidant property of T. cordifolia is well documented. The extract of T. cordifolia has been shown to inhibit lipid peroxidation and superoxide and hydroxyl radicals in vitro. In animals, administration of T. cordifolia reduced cyclophosphamide-induced elevated lipid peroxides in the serum and liver.47 T. cordifolia extract also showed decreased MDA formation and increased SOD, CAT, and GST in the lung; SOD and CAT in the kidney; and SOD, DTD, and GST in the forestomach of mice. The enhanced GSH level and enzyme activities involved in maintaining the antioxidant status of cells are suggestive of a chemopreventive efficacy of T. cordifolia against carcinogens.48 Besides antioxidant activity, the Tinospora plant extract has shown moderate antiproliferative activity on human cancer cell lines (MCF-7, HeLa, Caov-3, and HepG2).49 Alkaloid from T. cordifolia extract administration has also been shown to decrease the skin tumor size induced by DMBA/croton oil in mouse skin. In addition, the depleted levels of GSH, SOD, and CAT and increased DNA damage by DMBA/croton oil treatment have been found to be restored in palmatine (from T. cordifolia)-treated animals.50 These studies suggest that the Tinospora plant has the potential to be a source of natural antioxidants and nutrients, as well as a cancer chemopreventive and chemotherapeutic agent.

Other Ayurvedic plants Besides these abovementioned plants, several other medicinal plants have been documented in Ayurveda. These Ayurvedic plants have both dietary and medicinal uses and are also rich in antioxidants. For example, Allium cepa (onion; pyaaz) is prescribed as an effective remedy for several ailments in the Ayurvedic system of medicine. It has promising

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anti-inflammatory, anti-allergic, antihyperglycemic, antioxidant, anticancer, antihypertension, anti-hypercholesterolemia, and anti-asthmatic activities.51, 52 Extract of A. cepa has been shown to have antioxidant activity as revealed by the DPPH scavenging assay, H2O2, scavenging assay, and reducing power assay.53 An in vitro examination also showed cytotoxic activity of the A. cepa extract against various cancers including myeloma, glioblastoma, and breast cancer cell lines, which reflect its high efficiency as an anticancer agent.54, 55 Camellia sinensis (tea) is a widely used medicinal plant throughout India and China and is also popular in many Mediterranean countries. It is also well described in various traditional medicines such as Ayurveda. Tea leaves have shown various health benefits including promotion of cardiovascular health, cancer prevention, skin protection, antioxidant activity, high cholesterol reduction, boosting the immune system, useful in diarrhea, fatigue, and others.56 C. sinensis has exhibited cancer chemopreventive effects due to its polyphenolic antioxidant nature. Pretreatment of black tea extract to the animals restored antioxidant enzyme levels (CAT, SOD, GR, GST) and lipid peroxidation in the liver, kidney, and prostate tissues of animals treated with DMBA. Thus, it provides protection against oxidative damage induced by xenobiotics.57 Tea extract also induces apoptosis in a variety of cancers including prostate cancer, liver cancer, cervical cancer, and other cancer types.58–60 Epigallocatechin-3-gallate (EGCG), a component of green tea, has been shown to inhibit STAT3 activation and decreased phosphorylation of BCR-ABL that further caused suppression of tumor growth.61 The active component of black tea theaflavin induces cytotoxicity, cell cycle arrest, inhibits signaling molecules, and upregulates the proapoptotic proteins Bax, caspase-3, and caspase-9 and downregulates antiapoptotic protein Bcl-258; thus, C. sinensis exhibits chemopreventive and chemotherapeutic agents. Cinnamomum verum (cinnamon; dalchini) is another medicinal plant which is highly used in Ayurvedic medicines. C. verum possesses significant antiallergic, antiulcerogenic, antipyretic, and anesthetic activities.62 The bark yields an essential oil containing cinnamaldehyde and eugenol. Extract of C. verum barks were found to be potent in free radical scavenging activity. This extract also scavenged hydroxyl (OH) and superoxide radicals, indicating its antioxidant activity. The hexane extract of cinnamon has demonstrated high antiproliferative activity against various cancers including breast cancer cells. It restored antioxidant enzyme (SOD, GPx, and CAT) activity and caused apoptosis in cancer cells as indicated by the activation of caspases and suppression of AKT1 protein.63 Chen et al. identified two novel antioxidants, obtusilactone A and ( )-sesamin in Cinnamon. Both showed effective DPPH radical scavenging activity, which plays a potential role against multiple types of cancer.64 Its major components cinnamaldehyde, cinnamic acid, and cinnamyl alcohol possess antiproliferative activity in liver carcinoma cells through the downregulation of Bcl-(XL), upregulation of CD95 (APO-1), p53 and Bax proteins, as well as cleavage of PARP.65 Curcuma longa (turmeric; haldi) is a plant that has a very long history of medicinal use, dating back nearly 4000 years. In Ayurvedic practices, turmeric has been used for different medicinal purposes including rejuvenating the body, relieving gas, dispelling worms, healing wounds, improving digestion, regulating menstruation, dissolving gallstones, and relieving arthritis. Experimental studies reveal that turmeric has a potent antioxidant, anti-inflammatory, antimutagenic, antimicrobial, and anticancer properties.66 Turmeric extracts exhibit antioxidant property by scavenging free radicals, increasing antioxidant enzymes, and decreasing lipid peroxidation.67 It has been shown to reduce methotrexate-induced oxidative stress by restoring SOD, CAT, and GPx and MDA for lipid peroxidation.68 Another study has shown that a diet containing turmeric when fed to the retinol-deficient rats lowered lipid peroxidation in the liver, kidney, spleen, and brain.69 Turmeric and its active components, including curcumin, exhibit anticancer activities by inhibiting cell proliferation and inducing apoptosis of cancer cells. At the molecular level, curcumin shows anticancer activity through regulation of multiple cell signaling pathways including the cell survival pathway (Bcl-2, Bcl-xL, cFLIP, XIAP, c-IAP1), cell proliferation pathway (cyclin D1, c-myc), apoptosis pathway (caspase-8, 3, 9, PARP), tumor suppressor pathway (p53, p21), death receptor pathway (DR4, DR5), and protein kinase pathway (JNK, Akt, and AMPK).70 Terminalia bellerica (beleric, bastard myrobalank; baheda) is also known as “Bibhitaki” in traditional Ayurvedic medicine. Traditionally, it has been used for treatment of a wide range of diseases such as cough, eye and hair problems, worm infestation, and sore throat. T. bellerica is reported to have antioxidant properties through scavenging free radicals and increasing antioxidant potential.71 In a study, oxidative stress produced in rats by alloxan treatment was found to be significantly lowered by the administration of T. belerica extract. It further decreased TBARS and conjugated dienes and hydroperoxides in the blood and liver, respectively, in animals.72 Similarly, another study showed that the methanol extract of T. bellerica effectively reduces free radicals and ROS in in vitro studies and increases the antioxidant enzymes such as SOD, CAT, GST, and glutathione reductase (GR) in mice.73 Basu et al. showed that T. bellerica has excellent ROS and RNS scavenging activities. They have also found that this extract inhibits proliferation of breast (MCF-7), cervical (HeLa), and brain (U87) cancer cells by inducing G2/M arrest and apoptosis.74 T. bellerica extracts not only demonstrated growth inhibitory activity of cancer cell lines but also showed synergistic effects with cisplatin or doxorubicin in inducing apoptosis in A549 and HepG2 cells.75

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Trigonella foenum-graecum is commonly known as fenugreek and also as “methi” in Ayurveda. T. foenum-graecum seeds are traditionally used as herbal medicine for the treatment of various disorders including sore throat, diabetes, helminthiasis, inflammatory conditions, and rheumatism.76 Different parts of this plant possess antioxidant, antiinflammatory, antidiabetic, anticarcinogenic, hypocholesterolemic, antigenotoxic, antimicrobial, and gastroprotective effects.77 The antioxidant activity of T. foenum-graecum was shown by its free radical scavenging and restoration of antioxidant enzymes SOD, CAT, GST, and GPx along with reduction of lipid peroxidation.76, 78 Besides its antioxidant properties, it exhibits anticancer activity by inhibiting proliferation, inducing apoptosis, and arresting the cell cycle in cancer cells. In a study, fenugreek seeds extract inhibited the DMBA-induced mammary hyperplasia and decreased the incidence of breast cancer in rats.79The anticancer activity of fenugreek seed has been found to be modulated by various transcription factors, growth factors, protein kinases, and inflammatory mediators in cancer cells.80 Zingiber officinalis is commonly known as ginger. Ginger has been used traditionally against several disorders such as constipation, dyspepsia, diarrhea, heartburn, flatulence, nausea, vomiting, loss of appetite, infections, cough, bronchitis, cardiopathy, high blood pressure, palpitations and as a vasodilator. Chemical analysis revealed that ginger contains 6gingerol, 8-gingerol, and 6-shogaol as a main biologically active compound.81, 82 Ginger has been shown as a potent antioxidant. In a study, supplementation of ginger extract to the carcinogen treated rats reduced circulating lipid peroxidation and significantly enhanced the enzymatic (GPx, GST, GR, SOD, and CAT) and nonenzymatic antioxidants (GSH, vitamins C, E, and A).83 Ginger oil scavenged superoxide, DPPH, hydroxyl radicals and inhibited tissue lipid peroxidation and thus exhibits antioxidant activity.84 In vitro and in vivo studies have shown that ginger and its active components induce apoptosis in cancer cells.85 Along with suppression of free radicals, [6]-gingerol induces cytotoxicity in cancer cells mediated by the generation of ROS.86 The anticancer activity of ginger is further attributed to its ability to modulate several signaling molecules like NF-kB, STAT3, MAPK, PI3K, ERK1/2, Akt, TNF-a, COX-2, cyclin D1, cdk, MMP-9, survivin, cIAP-1, XIAP, Bcl-2, caspases, and other cell growth regulatory proteins.81

Conclusion Since centuries, traditional medicines like Ayurvedic medicine have been used for the treatment of various disorders. Based on the beneficial effects of these medicines, Ayurvedic plants have been recently explored for many medical uses in several chronic diseases including cancer. Cragg and Newman87 revealed that of the new drugs approved by the Food and Drug Administration (FDA) between 1983 and 1994, 39% were natural products or derived from natural products and about 60%–80% of antibiotics and anticancer drugs were derived from natural products, which indicates that traditional medicines are the source of most of the drugs. These medicinal plants not only affect the oxidative stress but also modulate various signaling pathways, which are causative factors of carcinogenesis. To date, these Ayurvedic plant extracts or their active components have been successfully evaluated in preclinical models. However, very few of them have been tested at the clinical level. Therefore, further clinical studies to determine their efficacy in patients are warranted.

Summary points l

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l l l

Oxidative stress, caused by an imbalance of ROS generation and antioxidant level, has an important role in cancer progression. Ayurveda, a traditional Indian medicine, utilizes several hundreds of medicinal plants that have been used to treat several ailments since centuries. Ayurvedic plants are rich in antioxidants that can decrease the oxidative stress and reduce the incidence of cancer. Ayurvedic plant extracts or their active components increase the antioxidant level and decrease ROS. Some of the active compounds from Ayurvedic plants have been clinically tested; however, clinical trials of other potent plant components are required.

Acknowledgments This work was supported in part by R01 grant CA129038 (to Sanjay K. Srivastava) awarded by the National Cancer Institute, NIH. Authors also appreciate the financial support from the Dodge Jones Foundation, Abilene, Texas.

Conflict of interest The authors declare no conflict of interest.

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References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69(1):7–34. 2. Mariotto AB, Yabroff KR, Shao Y, Feuer EJ, Brown ML. Projections of the cost of cancer care in the United States: 2010–2020. J Natl Cancer Inst 2011;103(2):117–28. 3. Poyton RO, Ball KA, Castello PR. Mitochondrial generation of free radicals and hypoxic signaling. Trends Endocrinol Metab 2009;20(7):332–40. 4. Murphy MP. How mitochondria produce reactive oxygen species. Biochem J 2009;417:1–13. 5. Hernandes MS, Britto LRG. NADPH oxidase and neurodegeneration. Curr Neuropharmacol 2012;10(4):321–7. 6. Serras F. The benefits of oxidative stress for tissue repair and regeneration. Fly (Austin) 2016;10(3):128–33. 7. Durackova Z. Some current insights into oxidative stress. Physiol Res 2010;59(4):459–69. 8. Boutennoun H, Boussouf L, Rawashdeh A, et al. In vitro cytotoxic and antioxidant activities of phenolic components of Algerian Achillea odorata leaves. Arab J Chem 2017;10(3):403–9. 9. Rammanohar P. Clinical evidence in the tradition of avurveda: evidence-based practice in complementary and alternative medicine. Berlin, Germany: Springer-Verlag; 201270. 10. Variya BC, Bakrania AK, Patel SS. Emblica officinalis (Amla): a review for its phytochemistry, ethnomedicinal uses and medicinal potentials with respect to molecular mechanisms. Pharmacol Res 2016;111:180–200. 11. Reddy VD, Padmavathi P, Hymavathi R, Maturu P, Varadacharyulu N. Alcohol-induced oxidative stress in rat liver microsomes: protective effect of Emblica officinalis. Pathophysiology 2014;21(2):153–9. 12. Singh MK, Yadav SS, Gupta V, Khattri S. Immunomodulatory role of Emblica officinalis in arsenic induced oxidative damage and apoptosis in thymocytes of mice. BMC Complement Altern Med 2013;13:193. 13. Shivananjappa MM, Joshi MK. Influence of Emblica officinalis aqueous extract on growth and antioxidant defense system of human hepatoma cell line (HepG2). Pharm Biol 2012;50(4):497–505. 14. Chen KH, Lin BR, Chien CT, Ho CH. Emblica officinalis Gaertn. attentuates N-nitrosodiethylamine-induced apoptosis, autophagy, and inflammation in rat livers. J Med Food 2011;14(7–8):746–55. 15. Jindal A, Soyal D, Sharma A, Goyal PK. Protective effect of an extract of Emblica officinalis against radiation-induced damage in mice. Integr Cancer Ther 2009;8(1):98–105. 16. Hari Kumar KB, Sabu MC, Lima PS, Kuttan R. Modulation of haematopoetic system and antioxidant enzymes by Emblica officinalis Gaertn and its protective role against gamma-radiation induced damages in mice. J Radiat Res 2004;45(4):549–55. 17. Anilkumar D, Hemang J, Nishteswar K. Review of glycyrrhiza glabra (Yashtimadhu): a broad spectrum herbal drug. Pharma Sci Monit 2012;12 (3):3171. 18. Sidhu P, Shankargouda S, Rath A, Hesarghatta Ramamurthy P, Fernandes B, Kumar SA. Therapeutic benefits of liquorice in dentistry. J Ayurveda Integr Med 2018. 19. Chin YW, Jung HA, Liu Y, et al. Anti-oxidant constituents of the roots and stolons of licorice (Glycyrrhiza glabra). J Agric Food Chem 2007;55 (12):4691–7. 20. Hejazi II, Khanam R, Mehdi SH, et al. New insights into the antioxidant and apoptotic potential of Glycyrrhiza glabra L. during hydrogen peroxide mediated oxidative stress: an in vitro and in silico evaluation. Biomed Pharmacother 2017;94:265–79. 21. Li K, Ji S, Song W, et al. Glycybridins A-K, bioactive phenolic compounds from Glycyrrhiza glabra. J Nat Prod 2017;80(2):334–46. 22. Mohamed NE. Effect of aqueous extract of Glycyrrhiza glabra on the biochemical changes induced by cadmium chloride in rats. Biol Trace Elem Res 2019;190(1):87–94. 23. Basar N, Oridupa OA, Ritchie KJ, et al. Comparative cytotoxicity of Glycyrrhiza glabra roots from different geographical origins against immortal human keratinocyte (HaCaT), lung adenocarcinoma (A549) and liver carcinoma (HepG2) cells. Phytother Res 2015;29(6):944–8. 24. Sharma R, Gatchie L, Williams IS, et al. Glycyrrhiza glabra extract and quercetin reverses cisplatin resistance in triple-negative MDA-MB-468 breast cancer cells via inhibition of cytochrome P450 1B1 enzyme. Bioorg Med Chem Lett 2017;27(24):5400–3. 25. Grace OM, Buerki S, Symonds MRE, et al. Evolutionary history and leaf succulence as explanations for medicinal use in aloes and the global popularity of Aloe vera. BMC Evol Biol 2015;15. 26. Hamman JH. Composition and applications of Aloe vera leaf gel. Molecules 2008;13(8):1599–616. 27. Minjares-Fuentes R, Femenia A, Comas-Serra F, Rodriguez-Gonzalez VM. Compositional and structural features of the main bioactive polysaccharides present in the Aloe vera plant. J AOAC Int 2018;101(6):1711–9. 28. Scalbert A, Williamson G. Dietary intake and bioavailability of polyphenols. J Nutr 2000;130(8) 2073s–85s. 29. Singh RP, Dhanalakshmi S, Rao AR. Chemomodulatory action of Aloe vera on the profiles of enzymes associated with carcinogen metabolism and antioxidant status regulation in mice. Phytomedicine 2000;7(3):209–19. 30. Suhail N, Bilal N, Hasan S, Banu N. Pre-exposure to chronic unpredictable stress suppresses the chemopreventive potential of Aloe vera (Av) leaf gel against 7,12-dimethylbenz(a)anthracene (DMBA) induced carcinogenesis. Nutr Cancer 2019;71(2):272–84. 31. El-Shemy HA, Aboul-Soud MA, Nassr-Allah AA, Aboul-Enein KM, Kabash A, Yagi A. Antitumor properties and modulation of antioxidant enzymes’ activity by Aloe vera leaf active principles isolated via supercritical carbon dioxide extraction. Curr Med Chem 2010;17(2):129–38. 32. Bala S, Chugh NA, Bansal SC, Garg ML, Koul A. Radiomodulatory effects of Aloe vera on hepatic and renal tissues of X-ray irradiated mice. Mutat Res 2018;811:1–15. 33. Naqvi S, Ullah MF, Hadi SM. DNA degradation by aqueous extract of Aloe vera in the presence of copper ions. Indian J Biochem Biophys 2010;47 (3):161–5.

Oxidative stress and cancer Chapter

27

309

34. Saini MR, Goyal PK, Chaudhary G. Anti-tumor activity of Aloe vera against DMBA/croton oil-induced skin Papillomagenesis in Swiss albino mice. J Environ Pathol Toxicol 2010;29(2):127–35. 35. Viyoch J, Pisutthanan N, Faikreua A, Nupangta K, Wangtorpol K, Ngokkuen J. Evaluation of in vitro antimicrobial activity of Thai basil oils and their micro-emulsion formulas against Propionibacterium acnes. Int J Cosmet Sci 2006;28(2):125–33. 36. Cohen MM. Tulsi—Ocimum sanctum: a herb for all reasons. J Ayurveda Integr Med 2014;5(4):251–9. 37. Manaharan T, Thirugnanasampandan R, Jayakumar R, Ramya G, Ramnath G, Kanthimathi MS. Antimetastatic and anti-inflammatory potentials of essential oil from edible Ocimum sanctum leaves. ScientificWorldJournal 2014;2014:239508. 38. Hussain EH, Jamil K, Rao M. Hypoglycaemic, hypolipidemic and antioxidant properties of tulsi (Ocimum sanctum linn) on streptozotocin induced diabetes in rats. Indian J Clin Biochem 2001;16(2):190–4. 39. Halim EM, Mukhopadhyay AK. Effect of Ocimum sanctum (Tulsi) and vitamin E on biochemical parameters and retinopathy in streptozotocin induced diabetic rats. Indian J Clin Biochem 2006;21(2):181–8. 40. Dasgupta T, Rao AR, Yadava PK. Chemomodulatory efficacy of basil leaf (Ocimum basilicum) on drug metabolizing and antioxidant enzymes, and on carcinogen-induced skin and forestomach papillomagenesis. Phytomedicine 2004;11(2–3):139–51. 41. Prakash J, Gupta SK. Chemopreventive activity of Ocimum sanctum seed oil. J Ethnopharmacol 2000;72(1–2):29–34. 42. Venuprasad MP, Kandikattu HK, Razack S, Amruta N, Khanum F. Chemical composition of Ocimum sanctum by LC-ESI-MS/MS analysis and its protective effects against smoke induced lung and neuronal tissue damage in rats. Biomed Pharmacother 2017;91:1–12. 43. Monga J, Sharma M, Tailor N, Ganesh N. Antimelanoma and radioprotective activity of alcoholic aqueous extract of different species of Ocimum in C (57)BL mice. Pharm Biol 2011;49(4):428–36. 44. Kim SC, Magesh V, Jeong SJ, et al. Ethanol extract of Ocimum sanctum exerts anti-metastatic activity through inactivation of matrix metalloproteinase-9 and enhancement of anti-oxidant enzymes. Food Chem Toxicol 2010;48(6):1478–82. 45. Saha S, Ghosh S. Tinospora cordifolia: one plant, many roles. Anc Sci Life 2012;31(4):151–9. 46. Upadhyay AK, Kumar K, Kumar A, Mishra HS. Tinospora cordifolia (Willd.) Hook. f. and Thoms. (Guduchi)—validation of the Ayurvedic pharmacology through experimental and clinical studies. Int J Ayurveda Res 2010;1(2):112–21. 47. Mathew S, Abraham TE. Studies on the antioxidant activities of cinnamon (Cinnamomum verum) bark extracts, through various in vitro models. Food Chem 2006;94(4):520–8. 48. Singh RP, Banerjee S, Kumar PV, Raveesha KA, Rao AR. Tinospora cordifolia induces enzymes of carcinogen/drug metabolism and antioxidant system, and inhibits lipid peroxidation in mice. Phytomedicine 2006;13(1–2):74–84. 49. Zulkhairi Jr. A, Abdah MA, NH MK, et al. Biological properties of Tinospora crispa (Akar Patawali) and its antiproliferative activities on selected human cancer cell lines. Malays J Nutr 2008;14(2):173–87. 50. Ali H, Dixit S. Extraction optimization of Tinospora cordifolia and assessment of the anticancer activity of its alkaloid palmatine. ScientificWorldJournal 2013;2013:376216. 51. Kaiser P, Youssouf MS, Tasduq SA, et al. Anti-allergic effects of herbal product from Allium cepa (bulb). J Med Food 2009;12(2):374–82. 52. Memarzia A, Amin F, Saadat S, Jalali M, Ghasemi Z, Boskabady MH. The contribution of beta-2 adrenergic, muscarinic and histamine (H1) receptors, calcium and potassium channels and cyclooxygenase pathway in the relaxant effect of Allium cepa L. on the tracheal smooth muscle. J Ethnopharmacol 2019;241:112012. 53. Tataringa G, Miron A, Paduraru I, Hancianu M, Gafitanu E, Stanescu U. Characterization of some extractive fractions isolated from raw Allium cepa L. bulbs. Rev Med Chir Soc Med Nat Iasi 2008;112(2):522–4. 54. Abdelrahman M, Mahmoud H, El-Sayed M, Tanaka S, Tran LS. Isolation and characterization of Cepa2, a natural alliospiroside A, from shallot (Allium cepa L. Aggregatum group) with anticancer activity. Plant Physiol Biochem 2017;116:167–73. 55. Fredotovic Z, Sprung M, Soldo B, et al. Chemical composition and biological activity of Allium cepa L. and Allium x cornutum (Clementi ex Visiani 1842) methanolic extracts. Molecules 2017;22(3). 56. Agarwal U, Pathak DP, Bhutani R, Kapoor G, Kant R. Review on Camellia sinensis: nature’s gift. Int J Pharmacogn Phytochem Res 2017;9 (8):1119–26. 57. Kalra N, Prasad S, Shukla Y. Antioxidant potential of black tea against 7,12-dimethylbenz(a)anthracene-induced oxidative stress in Swiss albino mice. J Environ Pathol Toxicol Oncol 2005;24(2):105–14. 58. Prasad S, Kaur J, Roy P, Kalra N, Shukla Y. Theaflavins induce G2/M arrest by modulating expression of p21waf1/cip1, cdc25C and cyclin B in human prostate carcinoma PC-3 cells. Life Sci 2007;81(17–18):1323–31. 59. Srivastava S, Singh M, Roy P, Prasad S, George J, Shukla Y. Inhibitory effect of tea polyphenols on hepatic preneoplastic foci in Wistar rats. Invest New Drugs 2009;27(6):526–33. 60. Singh M, Tyagi S, Bhui K, Prasad S, Shukla Y. Regulation of cell growth through cell cycle arrest and apoptosis in HPV 16 positive human cervical cancer cells by tea polyphenols. Invest New Drugs 2010;28(3):216–24. 61. Jung JH, Yun M, Choo EJ, et al. A derivative of epigallocatechin-3-gallate induces apoptosis via SHP-1-mediated suppression of BCR-ABL and STAT3 signalling in chronic myelogenous leukaemia. Br J Pharmacol 2015;172(14):3565–78. 62. Kurokawa M, Kumeda CA, Yamamura J, Kamiyama T, Shiraki K. Antipyretic activity of cinnamyl derivatives and related compounds in influenza virus-infected mice. Eur J Pharmacol 1998;348(1):45–51. 63. Rad SK, Kanthimathi MS, Abd Malek SN, Lee GS, Looi CY, Wong WF. Cinnamomum cassia suppresses Caspase-9 through stimulation of AKT1 in MCF-7 cells but not in MDA-MB-231 cells. PLoS One 2015;10(12)e0145216.

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64. Cheng KC, Hsueh MC, Chang HC, Lee AY, Wang HM, Chen CY. Antioxidants from the leaves of Cinnamomum kotoense. Nat Prod Commun 2010;5 (6):911–2. 65. Ng LT, Wu SJ. Antiproliferative activity of Cinnamomum cassia constituents and effects of Pifithrin-alpha on their apoptotic signaling pathways in Hep G2 cells. Evid Based Complement Alternat Med 2011;2011:492148. 66. Prasad S, Aggarwal BB. Turmeric, the golden spice: from traditional medicine to modern medicine. In: IFF B, Wachtel-Galor S, editors. Herbal medicine: biomolecular and clinical aspects. Boca Raton, FL: CRC Press; 2011. 67. Cohly HH, Taylor A, Angel MF, Salahudeen AK. Effect of turmeric, turmerin and curcumin on H2O2-induced renal epithelial (LLC-PK1) cell injury. Free Radic Biol Med 1998;24(1):49–54. 68. Moghadam AR, Tutunchi S, Namvaran-Abbas-Abad A, et al. Pre-administration of turmeric prevents methotrexate-induced liver toxicity and oxidative stress. BMC Complement Altern Med 2015;15:246. 69. Kaul S, Krishnakantha TP. Influence of retinol deficiency and curcumin/turmeric feeding on tissue microsomal membrane lipid peroxidation and fatty acids in rats. Mol Cell Biochem 1997;175(1–2):43–8. 70. Ravindran J, Prasad S, Aggarwal BB. Curcumin and cancer cells: how many ways can curry kill tumor cells selectively? AAPS J 2009;11(3):495–510. 71. Guleria S, Tiku AK, Rana S. Antioxidant activity of acetone extract/fractions of Terminalia bellerica Roxb. Fruit. Indian J Biochem Biophys 2010;47 (2):110–6. 72. Sabu MC, Kuttan R. Antidiabetic and antioxidant activity of Terminalia belerica. Roxb Indian J Exp Biol 2009;47(4):270–5. 73. Hazra B, Sarkar R, Biswas S, Mandal N. Comparative study of the antioxidant and reactive oxygen species scavenging properties in the extracts of the fruits of Terminalia chebula, Terminalia belerica and Emblica officinalis. BMC Complement Altern Med 2010;10:20. 74. Basu T, Panja S, Ghate NB, Chaudhuri D, Mandal N. Antioxidant and antiproliferative effects of different solvent fractions from Terminalia belerica Roxb. fruit on various cancer cells. Cytotechnology 2017;69(2):201–16. 75. Pinmai K, Chunlaratthanabhorn S, Ngamkitidechakul C, Soonthornchareon N, Hahnvajanawong C. Synergistic growth inhibitory effects of Phyllanthus emblica and Terminalia bellerica extracts with conventional cytotoxic agents: doxorubicin and cisplatin against human hepatocellular carcinoma and lung cancer cells. World J Gastroenterol 2008;14(10):1491–7. 76. Al-Dabbagh B, Elhaty IA, Al Hrout A, et al. Antioxidant and anticancer activities of Trigonella foenum-graecum, Cassia acutifolia and Rhazya stricta. BMC Complement Altern Med 2018;18(1):240. 77. Goyal S, Gupta N, Chatterjee S. Investigating therapeutic potential of Trigonella foenum-graecum L as our defense mechanism against several human diseases. J Toxicol 2016;2016:1250387. 78. Thirunavukkarasu V, Anuradha CV, Viswanathan P. Protective effect of fenugreek (Trigonella foenum graecum) seeds in experimental ethanol toxicity. Phytother Res 2003;17(7):737–43. 79. Amin A, Alkaabi A, Al-Falasi S, Daoud SA. Chemopreventive activities of Trigonella foenum graecum (fenugreek) against breast cancer. Cell Biol Int 2005;29(8):687–94. 80. Sung B, Prasad S, Yadav VR, Aggarwal BB. Cancer cell signaling pathways targeted by spice-derived nutraceuticals. Nutr Cancer 2012;64 (2):173–97. 81. Prasad S, Tyagi AK. Ginger and its constituents: role in prevention and treatment of gastrointestinal cancer. Gastroenterol Res Pract 2015;2015:142979. 82. Leoni A, Budriesi R, Poli F, et al. Ayurvedic preparation of Zingiber officinale roscoe: effects on cardiac and on smooth muscle parameters. Nat Prod Res 2018;32(18):2139–46. 83. Manju V, Nalini N. Chemopreventive efficacy of ginger, a naturally occurring anticarcinogen during the initiation, post-initiation stages of 1,2 dimethylhydrazine-induced colon cancer. Clin Chim Acta 2005;358(1–2):60–7. 84. Jeena K, Liju VB, Kuttan R. Antioxidant, anti-inflammatory and antinociceptive activities of essential oil from ginger. Indian J Physiol Pharmacol 2013;57(1):51–62. 85. Shukla Y, Prasad S, Tripathi C, Singh M, George J, Kalra N. In vitro and in vivo modulation of testosterone mediated alterations in apoptosis related proteins by [6]-gingerol. Mol Nutr Food Res 2007;51(12):1492–502. 86. Nigam N, Bhui K, Prasad S, George J, Shukla Y. [6]-Gingerol induces reactive oxygen species regulated mitochondrial cell death pathway in human epidermoid carcinoma A431 cells. Chem Biol Interact 2009;181(1):77–84. 87. Cragg GM, Newman DJ. Natural product drug discovery in the next millennium. Pharm Biol 2001;39(Suppl 1):8–17.

Chapter 28

Polyphenol chlorogenic acid, antioxidant profile, and breast cancer Onur Bender and Arzu Atalay Biotechnology Institute, Ankara University, Ankara, Turkey

List of abbreviations 3-CQA 4-CQA 5-CQA ABTS AGE AO APC ARE ATP BDE BHT BODIPY BW CAT CGA CK DPPH ER ERK FRAP GO GPX GSH-px GSH GSK-3b GST H2AX H2O2 HDAC6 HER2 HMGB1 HO-1 I/R ICAA ICAB ICAC IFNa-2b IUPAC JC-1 KEGG LDH

3-O-caffeoylquinic acid 4-O-caffeoylquinic acid 5-O-caffeoylquinic acid 2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) advanced glycation end product acridine orange adenomatous polyposis coli antioxidant responsive element adenosine triphosphate bond dissociation enthalpy butylohydroxytoluene boron-dipyrromethene body weight catalase chlorogenic acid creatine kinase 2,2-diphenyl-1-picrylhydrazyl estrogen receptor extracellular signal-regulated kinase ferric reducing antioxidant power gene ontology glutathione peroxidase glutathione peroxidase glutathione glycogen synthase kinase 3 beta glutathione-S-transferase H2A histone family member X hydrogen peroxide histone deacetylase 6 human epidermal growth factor receptor 2 high mobility group box protein 1 heme oxygenase 1 ischemia/reperfusion isochlorogenic acid A isochlorogenic acid B isochlorogenic acid C interferon-alpha 2b International Union of Pure and Applied Chemistry 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide Kyoto Encyclopedia of Genes and Genomes lactate dehydrogenase

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00028-6 © 2021 Elsevier Inc. All rights reserved.

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MDA MPEC MTT NF-kB NQO1 Nrf2 PI PKC PR Prx6 ROS SD SOD T-AOC TAP TBARS TNF-a Trx1 XO-1

B Antioxidants and cancer

malondialdehyde 2-methyl-6-p-methoxyphenylethynylimidazopyra-dinone (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) nuclear factor kappa B NAD(P)H Quinone dehydrogenase 1 the nuclear factor erythroid 2 (NFE2)-related factor 2 propidium iodide protein kinase C progesterone receptor peroxiredoxin 6 reactive oxygen species spin density superoxide dismutase total antioxidant capacity total antioxidant performance thiobarbituric acid reactive substances tumor necrosis factor-alpha thioredoxin xanthine oxidase 1

Introduction Throughout the evolution, plants synthesized metabolic products with many structural functions while maintaining vital activities such as coping with stress factors, protecting themselves against external threats, and communicating with their environment.1 Secondary metabolism products, also known as natural products, are small organic molecules and are essential for more stable and powerful organisms. The biogenesis of these compounds is based on the formation of the specific carbon skeleton, followed by modification and derivatization by various reactions. Today, over 100,000 plant secondary metabolites have been identified with different chemical structures and a wide range of biological activities. Polyphenols are the largest group of plant secondary metabolites. In addition to their low molecular weight and high bioavailability properties, they are easier to obtain in high amounts.1, 2 They are commonly found in human dietary products and the beneficial effects of polyphenol-rich diet on human health has been proved in many studies for many years.3–6 The best-known biological activity of polyphenols is their very strong antioxidant activity and they are the dominant source of this activity in nature. The balance of free radicals and antioxidants ensures the normal functioning of cell metabolism. The relationship between oxidative stress and cancer is important; free radicals are critical to human systems under normal conditions, but when they are released or accumulated excessively, they activate and modulate many cancer-related transcription factors such as oncogenes.7–9 In order to strike this sensitive balance, metabolism must be supported by dietary antioxidants. Breast cancer, which has the highest incidence of cancer-related deaths among women, is a highly heterogeneous disease mainly due to the expression levels of different receptor types. The relationship between oxidative stress factors and the etiology of breast cancer has widely been reviewed in the literature. As if the already quite complex breast cancer pathogenesis is not enough, free radicals cause DNA damages, and trigger mutations and disorders in cell metabolism. Because of drug resistance and cancer-specific mutations that develop during breast cancer treatment, even effective drugs were found to have limited efficacy. Different treatment strategies are being carried out for each subtype of breast cancer.10–12 Extensive studies in this area are focusing on developing a strong drug candidate that can be used for all subtypes of breast cancer, using natural compounds. Chlorogenic acids (CGAs) are cinnamic acid derivatives of the phenolic acids. The strong antioxidant properties of CGA derivatives have made them ideal candidates to investigate the effects on many diseases.13 Numerous studies have shown that CGA has antineoplastic activity on many types of cancer. In this chapter, we specifically aimed to review the antioxidant properties of CGA and its mechanism of action on breast cancer.

Chlorogenic acid The first information of chlorogenic acid as acidic components in coffee was first referred by Robiquet and Bourton in 1837, but it was officially discovered by Payen in 1846 in green coffee beans and named as chlorogenic acid. Later on, a gradual discovery course continued and in 1920 Freudenberg showed that CGA was the caffeic acid conjugate of quinic

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acid, and in 1932 Fischer and Dangschat described this structure as 3-O-caffeoylquinic acid. In the following period, a wide variety of isomers of CGA were isolated from many different sources. First of all, it is clearly wrong to use the term CGA for a single chemical structure. CGAs are combinational esters formed between certain trans-cinnamic acids (the most commons are caffeic acid, ferulic acid, sinapic acid, and p-coumaric acid) and quinic acid. Most of them are natural compounds synthesized in plants and nearly 400 CGA have been reported to date.14–16 Therefore, the terminology was very confusing. It is a confounding molecule that has been misnamed in many ways for a long time and is involved in so many different forms in the literature.17 Many CGA-related articles aimed to explain and solve this issue. According to the nomenclature of pre-IUPAC, CGA was named as 3-O-caffeoylquinic acid (3-CQA). Today, this usage is wrong. After 1976, according to the current IUPAC nomenclature, the most common and well-known conjugate of CGA is clearly and precisely named as 5-O-caffeoylquinic acid, which is the correct use. CGA is one of the most common polyphenols consumed by human diet; it is present in fruits, vegetables, and raw material of beverages. The highest amount of CGA was found in coffee beans and therefore attributed to the main component of coffee. CGA is such a compound, that is, present in different isomers and concentrations even in different coffee origins.16 To the best of our knowledge, as a functional compound, characteristic properties of CGA such as the pharmacological-pharmacokinetic actions, chemical interactions, ADME-Tox studies have widely been published.13, 18–20 Hence, the antioxidant profile of CGA and its effects on breast cancer are reviewed here by filtering the most recent and advanced studies.

Antioxidant profile of chlorogenic acid Since CGA is present in many human dietary products, highly bioavailable in metabolism and has strong antioxidant effects, research has focused on the detailed examination between CGA and antioxidant parameters. The antioxidant effects of the compounds/extracts are conventionally determined by free radical scavenging activity assays or enzyme activation measurements; however, they are now more innovatively investigated on mammalian cell culture systems or advanced animal models. In this section, the antioxidant activity of CGA has been explained comprehensively under four different categories: conventional chemical-based in vitro tests, cell culture data, in vivo studies, and computational analyses.

Antioxidant capacity of chlorogenic acid isomers with conventional in vitro tests Geometric isomerism affects biological activity. For this reason, it is necessary to analyze different isomers of the same compound with basic tests before performing advanced experiments. Thus, both the activity of the common isomer in dietary products and more effective novel drug candidate compounds can be determined. Antioxidant capacities of six different CGA isomers (3-CQA, 4-CQA, 5CQA, ICAA, ICAB, and ICAC) were investigated by conventional in vitro tests. In the DPPH radical scavenging analysis, when the radical scavenging activities of the CGA isomers were examined at varying concentrations (5, 10, 20, 40, and 60 mg/mL), all isomers displayed over 90% activity at a dose of 40 mg/mL. Below 40 mg/mL doses dicaffeoylquinic acids showed significantly higher activity than caffeoylquinic acids. The isomers in these two groups showed no significant difference among themselves. EC50 values were obtained between 7.5 mg/mL (ICAC) and 13.8 mg/mL (5-CQA) among the isomers. DPPH radical scavenging analysis also showed that all CGA isomers have significant antioxidant activities compared to the reference compound BHT (EC50 ¼ 39.62 mg/mL). In the ABTS cation radical scavenging activity analysis, dose-dependent scavenging activity of CGA isomers was analyzed at doses of 50, 75, 100, 125, and 150 mg/mL. All isomers showed activity over 90% at 150 mg/mL. The EC50 values of the isomers ranged from 67.3 to 91.4 mg/mL and all the isomers showed higher antioxidant properties than the positive control agent, vitamin E analogue Trolox (EC50 ¼ 131.1 mg mL). In FRAP analysis, CGA isomers at 25, 50, 75, 100, and 125 mg/mL doses displayed strong antioxidant activity with increasing absorbance values (OD593) in parallel with increasing concentrations. This result clearly demonstrated the electron-donating properties of CGAs on free radicals. b-Carotene Bleaching Assay test results provided evidence for the potential antioxidant activity of CGAs. According to Metal Ion Chelating Ability results, CGA isomers had an antioxidant effect and diCQAs had higher antioxidant potential than CQAs21.

Antioxidant properties of chlorogenic acid in cellular level Several studies have reported the cell protective activity of CGA by triggering intracellular antioxidant mechanisms. CGA increased HUVEC cell viability against TNF-a-induced cytotoxicity. It significantly reduced the ROS levels 1.5-fold which was produced by TNF-a treatment. This antioxidant property of CGA in endothelial cells was confirmed by analyzing the change in SOD enzyme levels, HO-1, and XO-1 protein levels. CGA normalized the TNF-a stimulated

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SOD enzyme inactivity. HO-1 protein levels increased and XO-1 protein levels decreased following CGA treatment.22 CGA was also found to be cytoprotective in PC12 cells by reducing H2O2-mediated cell damage. On the other hand, it decreased H2O2-mediated cell death by diminishing caspase 3 signal activity and apoptotic cell morphology was decreased. It reduced intracellular ROS accumulation caused by H2O2. CGA increased the expression levels of the antioxidant gene set HO-1, NQO1, Trx1, and TrxR1, which were regulated by Nrf2, thereby enabling the oxidative defense system activation against H2O2. Even more mechanistically, Nrf2 must be translocated into the nucleus for activation of the antioxidative signaling pathway. In the immunoblotting results, Nrf2 levels gradually decreased in cytosol and increased in the nucleus after CGA treatment. Thus, cellular signaling of Nrf2 due to CGA stimulation shows that the antioxidant defense and protection mechanism works flawlessly. A remarkable result was obtained by analysis of CGA activity after shRNA-mediated silencing of Nrf2 in PC12 cells. In Nrf2 silenced cells, 50 mM of CGA caused higher cell viability whereas in control cells CGA-mediated cell protection was observed at higher CGA rates. Thus, the strong relationship between CGA and the major antioxidant defense system element Nrf2, was demonstrated.23 In a study using H9C2 rat cardiomyoblast cells as a model to determine the efficacy of CGA on diabetic cardiomyopathy, CGA reduced cell death induced by high glucose treatment as much as reference drug metformin. This result was confirmed by the release test of LDH, a cardiac cell death marker. In the hyperglycemia group, LDH release was approximately four times higher than the control and it was reduced by half following dose-dependent CGA treatment. ROS formation was four times higher as a result of hyperglycemia, and CGA significantly reduced ROS levels in a dose-dependent manner (1.63-fold for 10 mM CGA, 1.88-fold for 30 mM CGA, and 2.105-fold for Metformin). Lipid peroxidation was determined by measuring the MDA levels and CGA treatment significantly reduced the elevated MDA levels in hyperglycemic cells. Protein oxidation analysis was performed by measuring protein carbonyl content and approximately fivefold increase in hyperglycemia group was found to decline in the CGA treated group. Endogenous antioxidant system changes were analyzed by SOD, GPx, GSH, and total antioxidant activity tests. In the hyperglycemia model with CGA treatment, SOD activity decreased significantly in both total SOD activity test and SOD1 and SOD2 protein expression levels. GPX enzyme activity increased with CGA treatment in total GSH and antioxidant capacity assays. In all tests, CGA was as effective as metformin. High levels of AGE production and PKC levels associated with hyperglycemia diminished following CGA administration. Likewise, CGA reduced ERK1/2 phosphorylation compared to control level in a dose-dependent manner.24 The cell culture-based antioxidant studies mentioned here were summarized in Table 1.

Antioxidant effects of chlorogenic acid in vivo In a study in I/R model rats, the antioxidant effects of CGA and its metabolite caffeic acid were measured with the in vitro MPEC analysis and superoxide anion scavenging effects of CGA (41 mM) and caffeic acid (10.1 mM) were measured. 15 mM of allopurinol, a reference XO inhibitor, was reported to exhibit a scavenging effect on superoxide anion, close to the activity of caffeic acid, whereas CGA did not reach that level. In the same study, chain-breaking activities were measured by BODIPY test and TAP values were found to increase in a similar pattern both for CGA and caffeic acid, and strong total antioxidant effects were observed starting from 1 mM. Hydrolysis of CGA to caffeic acid takes place in the small intestine where it is known to be poorly absorbed. Caco-2 is a very useful cell line model for bioactivity studies on colorectal carcinoma. The in vitro absorption test of CGA and caffeic acid was evaluated as pH-dependent, and the uptake kinetics for CGA did not change in pH 7.4 and 6, whereas caffeic acid was highly absorbed in pH 6. According to this result, it was concluded that hydrolysis of CGA is an important step in antioxidant activity on I/R damage. In histological examinations by extravasation of Evans blue, following CGA and caffeic acid uptake, in vivo antioxidant activity in rats were found to be very similar for both compounds. Compared to the I/R model, both CGA and caffeic acid treatments reduced the amount of Evans dye by half and were almost close to the control group. Thus, it was determined that even after hydrolysis of CGA, the antioxidant activity was not lost and CGA exerted as strong activity as caffeic acid.25 In another study, high HO-1 mRNA and protein levels were further increased by CGA supplementation in the liver of I/R model rats compared to the control group. On the other hand, CGA enhanced Nrf2 translocation 1.5-fold and inhibited NF-kB/p65 cellular translocation by 64.8%. In the I/R model, the increased HMGB1 translocation was dramatically reduced by CGA. HMGB1 targeting via antioxidant compounds is particularly important for the treatment of many inflammatory diseases.26 The levels of MDA, an oxidative stress marker, declined in the plasma and heart of cyclosporine-induced hypertension model rats, with CGA supplementation. While 10 and 15 mg/kg BW CGA administration showed strong antioxidant properties on the heart, 15 mg/kg BW CGA administration reduced MDA levels to normal in plasma. This activity was dosedependent and an increase was observed in heart and kidney with 15 mg/kg BW CGA.27

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TABLE 1 Antioxidant effects of CGA on mammalian cell culture systems. Cell model

Test system

Results of CGA treatment

References

TNF-a-induced HUVEC cytotoxicity model

MTT

Increased cell viability

22

TNF-a-induced HUVEC cytotoxicity model

Chemical measurement

Reduced ROS level

22

TNF-a-induced HUVEC cytotoxicity model

Chemical measurement

Normalized SOD enzyme level

22

TNF-a-induced HUVEC cytotoxicity model

Immunoblotting

Increased HO-1 protein level and decreased XO-1 protein level

22

H2O2-induced PC-12 cell damage model

MTT

Decreased H2O2-mediated cell death and apoptosis

23

H2O2-induced PC-12 cell damage model

Chemical measurement

Reduced ROS level

23

H2O2-induced PC-12 cell damage model

Real-time polymerase chain reaction

Increased HO-1, NQO1, Trx1, and TrxR1 expression

23

H2O2-induced PC-12 cell damage model

Immunoblotting

Increased level of Nrf2 in the nucleus while decreased in the cytosol

23

H2O2-induced PC-12 cell damage model

RNA interference

Higher cell viability in Nrf2 silenced cells and CGA-mediated cell protection in control cells

23

Diabetic cardiomyopathy model via H9C2 rat cardiomyoblast cells

MTT

Reduced the cell death induced by high glucose treatment

24

Diabetic cardiomyopathy model via H9C2 rat cardiomyoblast cells

LDH cytotoxicity assay

Reduced LDH release

24

Diabetic cardiomyopathy model via H9C2 rat cardiomyoblast cells

Chemical measurement

Reduced ROS, MDA and protein carbonyl content levels

24

Diabetic cardiomyopathy model via H9C2 rat cardiomyoblast cells

Chemical measurement

Decreased SOD and increased GPX enzyme levels

24

Diabetic cardiomyopathy model via H9C2 rat cardiomyoblast cells

Immunoblotting

Decreased SOD1 and SOD2 protein levels

24

Diabetic cardiomyopathy model via H9C2 rat cardiomyoblast cells

Chemical measurement

Decreased AGE production and PKC activities

24

Diabetic cardiomyopathy model via H9C2 rat cardiomyoblast cells

Immunoblotting

Reduced ERK 1/2 phosphorylation

24

Caco-2 colorectal carcinoma cell line

Analytical measurement

Strong uptake at different pH values

25

In the rat diabetes model induced by streptozotocin-nicotinamide, lipid peroxidation products increased in kidney and liver tissues according to TBARS and HP analyses, and these values were significantly reversed with 5 mg/kg CGA intake. When CGA administration was combined with another antidiabetic polyphenol tetrahydrocurcumin, the combination of 80 mg/kg tetrahydrocurcumin and 5 mg/kg CGA normalized the levels of lipid peroxidation products. During the administration of the combination to rats, the antioxidant activity was monitored by measuring SOD, GPX, and CAT enzyme

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levels. While the activity of these enzymes in diabetic rats was low, they significantly increased with CGA and tetrahydrocurcumin intake. With the combination of these two polyphenols, activity has reached enormous levels.28 In the aluminum-induced mice model, CGA uptake enhanced the Nrf2 expression by increasing Nrf2 levels in the nucleus while decreasing the levels of cytosolic Nrf2. Activation of Nrf2 also triggered expressions of other important genes related to the antioxidant signaling pathway; NQO-1, HO-1, Prx6, and Trx protein levels in the Nrf2/ARE sub-signaling pathway increased significantly compared to the control. The antioxidant defense mechanism developed by CGA against Al toxicity was executed by the accumulation of Nrf2 in the nucleus. In addition, CGA strengthened the endogenous protection mechanism against oxidative stress by increasing the antioxidant enzymes levels SOD, CAT, GPX, and GST by 52.4%, 58.2%, 42.2%, and 38.7% respectively. Moreover, CGA ameliorated mitochondrial membrane damage according to the JC-1 test and increased ATP synthesis by 46.1% and reduced hydrolysis by 31.8%. It also normalized the energy metabolism by increasing CK enzyme activity.29 Intestinal barrier dysfunction after weaning in pigs is associated with oxidative stress and increased ROS. In a study conducted on weaned pigs, antioxidant parameters that changed as a result of CGA diet were examined and remarkable results were obtained. Weaned pigs were subjected to basal diet in control group and for treatment group they were supplemented with 1000 mg/kg CGA + basal diet. Basic antioxidant tests were performed with extracted tissue homogenates. GSH-px, T-AOC, SOD, MDA, and CAT activities were measured from three parts of the small intestine. The CGA diet increased CAT activity in all three parts. GSH-Px levels increased while MDA levels decreased in jejunum and ileum. No significant changes were observed in T-AOC and SOD levels in all three parts. The CGA diet increased the expression levels of Nrf2 and HO-1 at least 1.5-fold in duodenum and jejunum, an important transcription factor for the expression of antioxidant enzymes in the small intestine, and HO-1 has a major role in regulating the level of stress-induced ROS generation. These expression changes in the antioxidant genes were also validated by enzyme levels, demonstrating the protective role of CGA against oxidative stress in small intestine barrier function problems in weaned pigs.30 In another study investigating the effects of CGA on growth performance and digestive system of weaned pigs, CGA added to the diet of weaned pigs showed the best effect at 1000 mg/kg and the antioxidant markers SOD, GSH-Px, and CAT levels were increased in pigs’ sera.31 All the data discussed in this section are summarized in Table 2.

Computational evaluations for antioxidant potential of chlorogenic acid Polyphenols usually exhibit their antioxidant effects via three reactions: (i) hydrogen atom transfer, (ii) electron transferproton transfer, and (iii) sequential proton loss-electron transfer. Computational studies of these reactions prevent many artifacts and provide more economical and controllable studies in less time. In order to determine the antioxidant capacity of CGA, BDE (bond dissociation enthalpy) and SD (spin density) analyses were performed. BDE and SD are among of the most important computational approaches in the research of antioxidant potential of polyphenols. BDE is the removal of the H atom by breaking the OdH bond and SD is the means for measuring the internal free radicals and the rate of these reactions after the H atom removal. SD operates in direct and directly proportional to BDE. BDE values of different hydroxyl groups of CGA were calculated as 30 -OH (105.35 kcal/mol) > 40 -OH (97.51 kcal/mol) > 2-OH (87.66 kcal/mol) > 1-OH (68.33 kcal/mol). Since lower BDE value is a parameter of strong antioxidant effect, this tendency is due to the presence of strong intramolecular hydrogen bonds. Furthermore, since the unmapped electrons can move more easily, the ring bearing lower number of hydroxyl groups gave a lower BDE value than the ring containing the higher number of hydroxyl groups. The SD of the O atom of the CGA radicals formed by the removal of the hydrogen atom and ranked as 30 -OH > 40 -OH > 2-OH > 1-OH. This ranking is consistent with BDE values, which can be explained by CGA’s ability to keep free radicals more stable and is another evidence for the strong antioxidant capacity of CGA. The strong antioxidant activity of CGA was found to be due to the 1-OH group because of the stronger intramolecular hydrogen bonds. At the same time, BDE values were calculated together with CGA for the nine most commonly known compounds and CGA emerged as the strongest potential antioxidant with the lowest BDE value among all tested compounds.32

Chlorogenic acid and breast cancer Cytotoxic/antiproliferative effects of chlorogenic acid on breast cancer cells Standard anticancer drug screening studies begin with determining the cytotoxicity profiles of the extracts/compounds. The cytotoxic/antiproliferative activity of CGA on ER + MCF-7 and triple-negative (ER-, PR-, HER2-) MDA-MB-231 breast cancer cell lines was reported by using the MTT test. CGA caused dose-dependent cell growth inhibition in both cell lines

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TABLE 2 In vivo antioxidant activities of CGA. Disease model

Test system

Results of CGA treatment

References

Ischemia/reperfusion injury model rats

MPEC

Superoxide anion inhibition

25

Ischemia/reperfusion injury model rats

BODIPY

Increased TAP activity

25

Ischemia/reperfusion injury model rats

Evans blue staining

Strong uptake of CGA after hydrolysis

25

Ischemia/reperfusion injury model rats

Immunoblotting

Increased HO-1 level and Nrf2 translocation. Decreased NF-kB/p65 and HMGB1 translocation

26

Hypertension model rats

Chemical measurement

Decreased MDA level on heart and plasma

27

Diabetes model rats

TBARS and HP

Decreased lipid peroxidation products

28

Diabetes model rats

Chemical measurement

Increased SOD, GPX, and CAT enzyme levels

28

Aluminum-induced mice model

Immunoblotting

Increased level of Nrf2 in the nucleus while decreased in the cytosol

29

Aluminum-induced mice model

Immunoblotting

Increased NQO-1, HO-1, Prx6, and Trx levels

29

Aluminum-induced mice model

Chemical measurement

Increased SOD, CAT, GPX, and GST enzyme levels

29

Aluminum-induced mice model

JC-1

Increased ATP synthesis and reduced hydrolysis

29

Aluminum-induced mice model

Chemical measurement

Normalized the energy metabolism via increased CK enzyme activity

29

Intestinal barrier dysfunction after weaning

Chemical measurement

Increased GSH-Px and CAT enzyme levels and decreased MDA levels in different parts of small intestine

30

Intestinal barrier dysfunction after weaning

Real-time polymerase chain reaction

Increased Nrf2 and HO-1 expression

30

Intestinal barrier dysfunction after weaning

Chemical measurement

Increased antioxidant enzyme levels

30, 31

at 20, 40, 60, 80 and 100 mg/mL doses. The IC50 values were calculated as 52.5 mg/mL for MCF-7 and 75.88 mg/mL for MDA-MB-231. After CGA treatment, the morphology of the cells was highly deteriorated and membrane structures were changed to an unhealthy and stressful state.33 In drug screening studies, conventional cell viability test concepts are end point, dye interfering, and spectrophotometer-based, which leads to limited data while investigating the cytotoxic/antiproliferative effects of the compounds. Recently developed xCELLigence cell analysis platform, which allows noninvasive, real-time, label-free, and high throughput measurement, has become a gold standard for high-quality data acquisition, especially in natural product-based drug screening studies. The effects of CGA on five different breast cancer cell lines with different receptor types (MCF-7 for ER + and PR +, SK-BR-3 for HER2 +, MDA-MB-231, MDA-MB-468, and BT-20 for triple-negative,) was examined and 8, 4, and 2 mM CGA caused irreversible death in all cell lines and 100% inhibition was observed. 1 mM CGA inhibited the growth of MCF-7, SK-BR-3, MDA-MB-468, and BT-20 cells and the cells remained in the stationary phase. A slight cellular induction was observed immediately after treatment of MDA-MB-231 cells with CGA, and after about 24 h, CGA caused an inhibitory effect on these cells. After 50 h only a slight growth inhibition was observed in the BT-20 cell line at 500 and 250 mM doses, whereas no cellular inhibition was observed in all other cell lines compared to the control. IC50 values obtained from cell index data at 72 h were 952  32.5 mM for MCF-7, 940  21.2 mM for SKBR-3, 590.5  10.6 mM for MDA-MB-231, 882.5  12.0 mM for MDA-MB-468, and 1095  121.6 mM for BT-20. The best inhibition by CGA was observed on MDA-MB-231 triple-negative breast cancer cell line.34 In another study, CGA was not cytotoxic on SKBR-3,

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TABLE 3 Antiproliferative/cytotoxic activity of CGA on breast cancer cells.

Cell line

Cell line characteristics (tumor type, source, class)

Receptor status

Test system

Results of CGA treatment

IC50 value

References

MCF-7

Invasive ductal carcinoma, Pleural effusion, Luminal

ER +, PR +

MTT and RTCA

Dose-dependent cell growth inhibition

52.5 mg/mL from MTT, 952 mM from RTCA

33, 34

MDAMB-231

Adenocarcinoma, Pleural effusion, Claudin-low

Triple negativea

MTT and RTCA

Dose-dependent cell growth inhibition

75.88 mg/mL from MTT, 590.5 mM from RTCA

33, 34

SKBR-3

Adenocarcinoma, Pleural effusion, Luminal

HER2+

RTCA

Dose-dependent cell growth inhibition

940 mM

34

MDAMB-468

Adenocarcinoma, Pleural effusion, Basal A

Triple negativea

RTCA

Dose-dependent cell growth inhibition

882.5 mM

34

BT-20

Invasive ductal carcinoma, primary breast, Basal A

Triple negativea

RTCA

Dose-dependent cell growth inhibition

1095 mM

34

Triple negative: ER , PR , HER2 .

a

T47D, MDA-MB-231, and primary breast epithelial cell line at a dose of 10 mM, and cell viability was more than 90% after treatment. When CGA was administered within oxidovanadium (IV) complex, it increased the antioxidant parameters and the complex caused 40% inhibition on SKBR-3 cell growth. The mechanism of this selective cytotoxicity was investigated and no increase in intracellular ROS levels was found following administration of CGA alone or CGA within oxidovanadium complex. Also, 30% decrease in mitochondrial membrane potential was observed. In addition, the complex triggered caspase-independent apoptotic cell death but did not activate the caspase 3/7 pathway. DNA damage was measured by H2AX phosphorylation and a 25% increase was observed. Selective cytotoxicity was confirmed by increasing LDH levels. Thus, the efficacy of an effective combinatorial agent against breast cancer was approved by combining a strong antioxidant CGA with anticancer compound vanadium.35 Breast cancer cell characteristics and antiproliferative/cytotoxicity data are compiled in Table 3.

Effects of chlorogenic acid on cell cycle distribution in breast cancer The effects of CGA on cell cycle status in breast cancer cells were determined by flow cytometry-based propidium iodide staining. 75 mg/mL CGA modulated the cell cycle distribution of MDA-MB-231 cells as 55.77% for G1 phase; 29.69% for S phase and 14.54% for G2/M phase. Untreated control cells were distributed as 45.22% for G1; 39.44% for S and 15.34% for G2/M phases. In all, 10% of MDA-MB-231 breast cancer cells shifted from the active DNA synthesis step to the G1 phase in the CGA treated group. A total of 75 mg/mL CGA inhibited the division kinetics of breast cancer cells by inducing the arrest of cells in the G1 phase. Furthermore, this transition continued at 150 mg/mL CGA treatment with a 2% increase compared to 75 mg/mL CGA, which proved the selective inhibitory potential of CGA to block the DNA replication of breast cancer cells.33

Apoptotic effects of chlorogenic acid on breast cancer The effects of CGA on apoptotic status of MDA-MB-231 cells were analyzed with flow cytometry system following acridine orange (AO) and propidium iodide (PI) labelings, which enable cell viability analysis by binding to nucleotides. In the untreated control group, distribution of cells was as follows; healthy cells 60.7%, early apoptotic cells 8.3%, late apoptotic cells 17.1%, and necrotic cells 13.8%. In the cells treated with 75 mg/mL CGA, healthy cells showed a distribution of 29.6%, early apoptotic cells 0.3%, late apoptotic cells 44.2%, and necrotic cells were 25.9%. The apoptosis-inducing

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activity of CGA on MDA-MB-231 cells was clearly observed by the 27.1% increase in the late apoptotic stage. Close results were obtained with 150 and 75 mg/mL doses without any significant change. This supports the notion that CGA selectively runs apoptosis programs without poisoning cells. Same samples were examined by DNA fragmentation analysis on agarose gel electrophoresis against dye interfering artifacts. While the healthy cells which showed a 60% distribution in the control group at AO/PI labelling, was observed as a compact DNA band on the gel, DNA from 75 and 150 mg/mL CGA treated samples displayed a smear band.33

Effects of chlorogenic acid on mitochondrial membrane potential in breast cancer Effects of CGA on mitochondrial membrane potential in breast cancer cells was investigated by JC-1 staining, which is an indicator of cell health by accumulating in mitochondria. Treatment of MDA-MB-231 cells with 75 mg/mL CGA resulted in impairment of mitochondrial integrity. In the untreated control group, mitochondria did not get structural deterioration and very compact fluorescence signals were obtained. Decreased mitochondrial membrane potential was also validated by cytochrome-c localization measurement. Data from both mitotracker staining and anti-cytochrome-c labelling showed dispersed cytochrome-c throughout the cytosol. This effect of CGA is especially important in reducing the strong metabolic activity, that is, prominent in breast cancer cells.33

Molecular simulations and validations of chlorogenic acid effects on protein kinase C Superposition analysis was performed against known PKC agonists curcumin and coumarin to determine the biophoric properties of CGA. CGA was superimposed with curcumin and coumarin with MCS scores of 0.90 and 0.77 respectively and showed a highly potent PKCa agonist phenotype. Molecular docking and interaction analyses showed that CGA is a small molecule that binds to the polar binding region of the c1b domain of PKCa and that it is strongly linked to PKC via hydrogen bonding—it makes three hydrogen bonds with Thr29, Leu41, and Leu38 residues—and hydrophobic interactions—with Pro28, Gln44, Leu37, Ser27, Tyr25, Gly26, and Thr24 residues. These data confirmed CGA as a potent ligand for PKC. Results of in vitro binding kinetics measurements supported this obtained in silico data. Trp-fluorescence intensity elevated with increasing CGA concentrations (10–50 mM) and this result verified the CGA-PKC binding dataset under in vitro conditions. The CGA-PKC complex has also successfully passed time-dependent conformational stability tests at varying temperatures. PKC is a cytosolic enzyme and is translocated from the cytosol to the cell membrane by its agonists, where it acts to induce the signaling pathways that lead to biological activity. In the immunoblotting study using cytosolic and membrane fractions of a 75 mg/mL CGA-treated MDA-MB-231 cell line, membrane fractions showed approximately fourfold elevated PKC expression levels relative to control in a time-dependent manner. CGA-induced PKC translocation data was also validated by immunofluorescence staining and PKC signal was detected in the cell membranes of CGA-treated cells with anti-PKC antibody.33

Effects of chlorogenic acid on breast cancer in vivo To investigate the chemopreventive efficacy of CGA and potentially altered gene expression levels, a breast cancer mouse model was developed via the EMT6 mouse mammary carcinoma cell line. CGA treatment resulted in the reduction of tumor volume up to 50.26% when administered as a 20 mg/kg dose. This effect was dose-dependent; 37.20% reduction for 10 mg/kg CGA and 31.86% for 5 mg/kg CGA administration were observed. Considering that the inhibition rate of Docetaxel, a cytotoxic positive control agent, was 59.92% and IFNa-2b was 40.80%, CGA was found to be chemopreventive. Determination of gene expression changes and signal pathways influenced by anticancer drug candidate compounds lead to clarification of chemopreventive mechanisms. For this reason, genomic profiling analysis and validations were performed with tissues from tumor-bearing/control mice by using gene-chip technology. Processing the data set with KEGG pathway database and GO analyses, gene sets affected by CGA treatments were identified. Gene expressions of the BDNF, CFLAR, CLN3 DDIT3, NOTCH2, RPS6, SOX9, SPN, and PPP1R13L pathways were altered. Wnt, mTOR, Notch, B cell receptor, T-cell receptor pathways were also affected. As a result of a more detailed analysis among these pathways, expression of three genes of the Wnt signaling pathway were found to change, which have the strongest relationship with breast cancer. GSK-3b and its downstream gene APC were upregulated which resulted in downregulation of the major oncogene b-catenin of the Wnt signaling pathway. b-catenin is one of the most prominent oncogenes in breast cancer and by decreasing its expression levels with CGA treatment, carcinogenesis process was impeded in vivo. In order to validate gene-chip results, qPCR analyses of these three genes were performed and it was shown that GSK-3b levels increased 2.29-fold within the 20 mg/kg CGA treated group compared to the negative control group. The inhibition activity of

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CGA on the GSK-3b levels increased in a dose-dependent manner. Like the GSK-3b levels, the dose-dependent CGA activity on the APC gene continued and APC levels increased 2.26-fold in the 20 mg/kg CGA treated group. Thus, expression changes in these two closely regulated genes with CGA treatment were validated both ontologically and experimentally. The major point was that treatment with 20 mg/kg CGA resulted in 1.66-fold reduction of b-catenin gene expression and provided evidence for tumor inhibition by directly triggering the Wnt signaling pathway.36

Applications to other cancers or conditions In this chapter, we reviewed the activities of a potent antioxidant molecule CGA on breast cancer. CGA has suppressed the cancer phenotype via many molecular ways in breast cancer and exhibited antineoplastic properties. Studies with other cancer types in the literature support the chemopreventive properties of CGA on different types of cancer. For example, in a study, CGA displayed selective antiproliferative effect on kidney cancer cells and cells underwent apoptosis via caspase, Bax and Bcl-2 signaling arrangements. It was also shown that CGA provided anticancer activity on kidney cancer via the PI3K/Akt/mTOR signaling pathway.37 The administration of CGA on lung cancer cells resulted in apoptosismediated cell death. CGA also caused activation of p38, MAPK, and JNK signaling pathways in lung cancer.38 In another study, CGA reduced the ability of lung cancer cells to migrate and colonize. CGA also acted as an HDAC6 and MMP2 inhibitor in lung cancer. Besides, it exhibited antitumor activity in vivo in the lung cancer animal model.39 CGA inhibited the growth of colon cancer cells by ROS induction. CGA also showed activity on the cell cycle by inducing colon cancer cells into S phase.40 In summary, studies investigating the anticancer efficacy of CGA have shown that it has selective activity on cell proliferation and generally exhibits a phenotype that affects various cancer-associated signaling pathways and apoptosis programs in the cells.

Summary points l l

l

l

l l l

l

This chapter discusses the antioxidant mechanism of chlorogenic acid and its anticancer roles against breast cancer. Chlorogenic acid is a potent antioxidant compound commonly found in human dietary products and activates endogenous antioxidant systems to defense and scavenge against free radicals. Chlorogenic acid enhances Nrf2 translocation from cytosol to the nucleus; thereby activating antioxidant gene sets to cytoprotection against toxicity. Chlorogenic acid significantly increases antioxidant parameters against oxidative stress in many chronic and metabolic disease models in vivo. Molecular simulations show the antioxidant activity of chlorogenic acid with its hydrogen-donating properties. Chlorogenic acid has antiproliferative activity on different receptor types of breast cancer. CGA inhibited cancer phenotype characteristics and showed activity by inhibiting b-catenin of the Wnt signaling pathway. In accordance with the cited studies, the potent antioxidant molecule CGA is a potential therapeutic against breast cancer and can be used for innovative phytotherapeutic formulations.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Bernards MA. Plant natural products: a primer. Can J Zool 2010;88:601–14. Tsao R. Chemistry and biochemistry of dietary polyphenols. Nutrients 2010;2:1231–46. Pandey KB, Rizvi SI. Plant polyphenols as dietary antioxidants in human health and disease. Oxidative Med Cell Longev 2009;2:270–8. Crozier A, Clifford MN, Ashihara H. Plant secondary metabolites: occurrence, structure and role in the human diet. New Jersey: Blackwell Publishing; 2006. Scalbert A, Manach C, Morand C, Remesy C, Jimenez L. Dietary polyphenols and the prevention of diseases. Crit Rev Food Sci Nutr 2005;45:287–306. Cirillo G, Curcio M, Vittorio O, Iemma F, Restuccia D, Spizzirri UG, et al. Polyphenol conjugates and human health: a perspective review. Crit Rev Food Sci Nutr 2016;56:326–37. Klaunig JE, Kamendulis LM, Hocevar BA. Oxidative stress and oxidative damage in carcinogenesis. Toxicol Pathol 2010;38:96–109. Ray PD, Huang BW, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal 2012;24:981–90. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact 2006;10:1–40. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394–424.

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11. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 2006;10:515–27. 12. Shao W, Brown M. Advances in estrogen receptor biology: prospects for improvements in targeted breast cancer therapy. Breast Cancer Res 2004;6:39–52. 13. Naveed M, Hejazi V, Abbas M, Kamboh AA, Khan GJ, Shumzaid M, et al. Chlorogenic acid (CGA): a pharmacological review and call for further research. Biomed Pharmacother 2018;97:67–74. 14. Clifford MN. Chlorogenic acids and other cinnamates—nature, occurrence and dietary burden. J Sci Food Agric 1999;79:362–72. 15. Clifford MN. Chlorogenic acids and other cinnamates—nature, occurrence, dietary burden, absorption and metabolism. J Sci Food Agric 2000;80:1033–43. 16. Clifford MN, Jaganath IB, Ludwig IA, Crozier A. Chlorogenic acids and the acyl-quinic acids: discovery, biosynthesis, bioavailability and bioactivity. Nat Prod Rep 2017;34:1391–421. 17. Kremr D, Bajer T, Bajerova´ P, Surmova´ S, Ventura K. Unremitting problems with chlorogenic acid nomenclature: a review. Quim Nova 2016;39:530–3. 18. Liang N, Kitts DD. Role of chlorogenic acids in controlling oxidative and inflammatory stress conditions. Nutrients 2015;8:16. 19. Niggeweg R, Michael AJ, Martin C. Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat Biotechnol 2004;22:746–54. 20. Farah A, Monteiro M, Donangelo CM, Lafay S. Chlorogenic acids from green coffee extract are highly bioavailable in humans. J Nutr 2008;138:2309–15. 21. Xu JG, Hu QP, Liu Y. Antioxidant and DNA-protective activities of chlorogenic acid isomers. J Agric Food Chem 2012;60:11625–30. 22. Huang WY, Fu L, Li CY, Xu LP, Zhang LX, Zhang WM. Quercetin, hyperin, and chlorogenic acid improve endothelial function by antioxidant, antiinflammatory, and ACE inhibitory effects. J Food Sci 2017;82:1239–46. 23. Yao J, Peng S, Xu J, Fang J. Reversing ROS-mediated neurotoxicity by chlorogenic acid involves its direct antioxidant activity and activation of Nrf2ARE signaling pathway. Biofactors 2019;45:616–26. 24. Preetha Rani MR, Anupama N, Sreelekshmi M, Raghu KG. Chlorogenic acid attenuates glucotoxicity in H9c2 cells via inhibition of glycation and PKC a upregulation and safeguarding innate antioxidant status. Biomed Pharmacother 2018;100:467–77. 25. Sato Y, Itagaki S, Kurokawa T, Ogura J, Kobayashi M, Hirano T, et al. In vitro and in vivo antioxidant properties of chlorogenic acid and caffeic acid. Int J Pharm 2011;403:136–8. 26. Yun N, Kang JW, Lee SM. Protective effects of chlorogenic acid against ischemia/reperfusion injury in rat liver: molecular evidence of its antioxidant and anti-inflammatory properties. J Nutr Biochem 2012;23:1249–55. 27. Agunloye OM, Oboh G, Ademiluyi AO, Ademosun AO, Akindahunsi AA, Oyagbemi AA, et al. Cardio-protective and antioxidant properties of caffeic acid and chlorogenic acid: mechanistic role of angiotensin converting enzyme, cholinesterase and arginase activities in cyclosporine induced hypertensive rats. Biomed Pharmacother 2019;109:450–8. 28. Pari L, Karthikesan K, Menon VP. Comparative and combined effect of chlorogenic acid and tetrahydrocurcumin on antioxidant disparities in chemical induced experimental diabetes. Mol Cell Biochem 2010;341:109–17. 29. Wang X, Xi Y, Zeng X, Zhao H, Cao J, Jiang W. Effects of chlorogenic acid against aluminium neurotoxicity in ICR mice through chelation and antioxidant actions. J Funct Foods 2018;40:365–76. 30. Chen J, Yu B, Chen D, Huang Z, Mao X, Zheng P, et al. Chlorogenic acid improves intestinal barrier functions by suppressing mucosa inflammation and improving antioxidant capacity in weaned pigs. J Nutr Biochem 2018;59:84–92. 31. Chen J, Li Y, Yu B, Chen D, Mao X, Zheng P, et al. Dietary chlorogenic acid improves growth performance of weaned pigs through maintaining antioxidant capacity and intestinal digestion and absorption function. J Anim Sci 2018;96:1108–18. 32. Saqib M, Iqbal S, Mahmood A, Akram R. Theoretical investigation for exploring the antioxidant potential of chlorogenic acid: a density functional theory study. Int J Food Prop 2016;19:745–51. 33. Deka S, Gorai S, Manna D, Trivedi V. Evidence of PKC binding and translocation to explain the anticancer mechanism of chlorogenic acid in breast cancer cells. Curr Mol Med 2017;17:79–89. 34. Bender O, Atalay A. Evaluation of anti-proliferative and cytotoxic effects of chlorogenic acid on breast cancer cell lines by real-time, label-free and high-throughput screening. Marmara Pharm J 2018;22:173–9. 35. Naso LG, Valcarcel M, Roura-Ferrer M, Kortazar D, Salado C, Lezama L, et al. Promising antioxidant and anticancer (human breast cancer) oxidovanadium (IV) complex of chlorogenic acid. Synthesis, characterization and spectroscopic examination on the transport mechanism with bovine serum albumin. J Inorg Biochem 2014;135:86–99. 36. Xu R, Kang Q, Ren J, Li Z, Xu X. Antitumor molecular mechanism of chlorogenic acid on inducting genes GSK-3 b and APC and inhibiting gene bcatenin. J Anal Methods Chem 2013;2013:951319. 37. Wang X, Liu J, Xie Z, Rao J, Xu G, Huang K, et al. Chlorogenic acid inhibits proliferation and induces apoptosis in A498 human kidney cancer cells via inactivating PI 3K/Akt/mTOR signalling pathway. J Pharm Pharmacol 2019;71:1100–9. 38. Yamagata K, Izawa Y, Onodera D, Tagami M. Chlorogenic acid regulates apoptosis and stem cell marker-related gene expression in A549 human lung cancer cells. Mol Cell Biochem 2018;441:9–19. 39. Hongtao L, Xiaoqi G, Junni L, Feng X, Guodong B, Yingping L. Chlorogenic-induced inhibition of non-small cancer cells occurs through regulation of histone deacetylase 6. Cell Mol Biol 2018;64:134–9. 40. Hou N, Liu N, Han J, Yan Y, Li J. Chlorogenic acid induces reactive oxygen species generation and inhibits the viability of human colon cancer cells. Anti-Cancer Drugs 2017;28:59–65.

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Chapter 29

Cinnamomum cassia, apoptosis, STAT3 inactivation and reactive oxygen species in cancer studies Yae Jin Yoon and Byoung-Mog Kwon Laboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea

List of abbreviations 2-DGE AP-1 BCA BIAM CA CB-PIC CETSA DARTS DCF FEMA GPx GSH H2DCFDA HCA HO-1 LRP1 MKP MOMP MT NF-kB PARP Pim-1 PKM2 PPP PTPase ROS Sec SOD STAT3 TrxR ZnPP a2M DCm

2-dimensional gel electrophoresis activator protein-1 20 -benzoyloxycinnamaldehyde biotin-conjugated iodoacetamide trans-cinnamaldehyde (E)-4-((2-(3-oxopop-1-enyl)phenoxy)methyl)pyridinium malonic acid cellular thermal shift assay drug affinity responsive target stability 20 ,70 -dichlorofluorescein Flavor and Extract Manufacturers Association glutathione peroxidase glutathione carboxy-20 ,70 -dichlorodihydrofluorescein diacetate 20 -hydroxycinnamaldehyde heme oxygenase 1 low-density lipoprotein receptor-related protein 1 MAP kinase phosphatase mitochondrial outer membrane permeabilization metallothionein nuclear factor kappa-light-chain-enhancer of activated B cells poly(ADP-ribose) polymerase proviral insertion in murine lymphomas-1 pyruvate kinase M2 pentose phosphate pathway protein tyrosine phosphatase reactive oxygen species selenocysteine superoxide dismutase signal transducer and activator of transcription 3 thioredoxin reductase zinc protoporphyrin a2-macroglobulin mitochondrial membrane potential

Introduction Cinnamon, which is obtained from the stem bark of Cinnamomum cassia Blume, has been widely used as a flavoring agent and a preservative for beverages, bakery products, sweets, chewing gum, medical products, cosmetics, and perfumes.1, 2 Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00029-8 © 2021 Elsevier Inc. All rights reserved.

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In addition to its daily use as a spice and flavoring agent, cinnamon has been used as a traditional medicine to treat dyspepsia, gastritis, blood circulation disturbances, and inflammatory diseases in Eastern and Western countries.1, 2 Cinnamon extracts consist of active compounds including essential oils, tannins, mucus, and carbohydrates.2 Approximately 45%–65% of the essential oils in cinnamon extracts are cinnamaldehyde, which functions as the main bioactive component of cinnamon extracts.3 In nature, cinnamaldehyde exists predominantly as the trans isomer, transcinnamaldehyde (CA), and is associated with its flavor and odor.2 CA contains an a,b-unsaturated carbonyl moiety, which is the electrophilic Michael acceptor and displays chemotherapeutic and chemopreventive properties by the reaction of a sulfhydryl group.4, 5 Numerous research groups have focused on Michael acceptor compounds, including curcumin, quercetin, myricetin, acrolein, crotonaldehyde, and trans-4-hydroxy-2-nonenal, as dietary ingredients.6 Notably, CA is the only a,b-unsaturated aldehyde, that is, FDA-approved for use in foods and has been given Generally Recognized As Safe status by the Flavor and Extract Manufacturers Association (FEMA) in the United States (FEMA no. 2286, 2201).4 To elucidate the structure-activity relationship of CA with its antiproliferative activity, a variety of Michael-active and Michael-inactive CA derivatives have been synthesized with substitutions at the ortho position of CA.4, 5 Michaelinactive CA derivatives, such as DHCA (loss of a,b-unsaturation), COH (replacement of the carbonyl group by reduction), and CAC (replacement of the carbonyl group by oxidation), show much less antiproliferative activity than Michael-active CA derivatives or no antiproliferative activity.4 However, MCA (4-methoxycinnamic aldehyde) with an intact Michael acceptor maintains antiproliferative activity, and FHCA (derivative of 20 -hydroxycinnamaldehyde with a 5-fluoro substitution) exhibits enhanced antiproliferative activities in human colon cancer (HCT116) and melanoma (A375P, G361, and LOX) cell lines.4, 5 CA has been shown to have cytotoxic and antiproliferative effects on various types of cancer cells, such as breast, ovarian, lung, liver, colon, melanoma, leukemia, and lymphoma.2 For decades, several CA-derived compounds have been synthesized that show diverse biological activities in cancer, diabetes, neuropathy, and cardiovascular disease. 20 -Hydroxycinnamaldehyde (HCA), a natural derivative of cinnamaldehyde, was first reported as an anticancer agent with inhibitory effects on farnesyl protein transferase.7 HCA is also a major component of cinnamon, accounting for 0.01–0.8 mg/g of commercially available cinnamon powder.8 HCA shows cytotoxic and antiproliferative activities in diverse cancer cells and tumor-suppressive effects in vivo.9 To elucidate the mode of action of HCA, several groups have investigated the direct binding targets of HCA using affinity chromatography coupled with mass spectrometry analysis and X-ray cocrystallography.8, 10–13 To the best of our knowledge, HCA has been reported to directly interact with signal transducer and activator of transcription 3 (STAT3), pyruvate kinase M2 (PKM2), proviral insertion in murine lymphomas-1 (Pim-1), low-density lipoprotein receptor-related protein 1 (LRP1), and proteasome subunits in prostate cancer, colon cancer, breast cancer, and leukemia.8, 10–13 Similar to CA, HCA contains an a,b-unsaturated carbonyl group; therefore, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), activator protein-1 (AP-1), and thioredoxin reductase (TrxR), which contain cysteine residues, have been proposed as binding targets of HCA.5, 14, 15 20 -Benzoyloxycinnamaldehyde (BCA) is a derivative of HCA that shows higher anticancer activity than CA and HCA.16 In pharmacokinetics and metabolic experiments, BCA was rapidly converted to HCA and then o-coumaric acid in Sprague-Dawley rat serum after either intravenous or oral BCA uptake and showed a half-life of approximately 2 h.17 As a promising anticancer agent, BCA was approved for clinical tests by the Korean Food and Drug Administration on January 31, 2011 and is now undergoing clinical testing. Although CA-derived compounds, especially BCA, are promising anticancer agents for treating cancer, their clinical application has been limited by poor stability, metabolic instability, inability to target cancer cells, and low therapeutic efficacy. Recently, novel strategies such as PolyCAFe micelles and PBCAE micelles have been developed that prolong the circulation time of CA-derived compounds and promote their preferential accumulation in tumors.18, 19 Fenton reaction-performing PolyCAFe micelles induce oxidative stress specifically in cancer cells and suppress tumor growth without significant toxicity to normal tissues.19 PolyCAFe micelles are self-assembled micelles based on amphiphilic PolyCAFe with BCA and iron-containing compounds.19 PolyCAFe micelles function as reactive oxygen species (ROS)-producing agents via the Fenton reaction, in which Fe2+/Fe3+ ions catalyze BCA-generated H2O2 into highly toxic hydroxyl radicals, resulting in ROS-induced apoptosis.19 Similarly, PBCAE micelles, which consist of dual pH-sensitive PBCAE copolymer, the polymeric prodrug of BCA, and zinc protoporphyrin (ZnPP), induce ROS generation and subsequent ROS-induced apoptosis.18 Specifically, BCA is covalently incorporated into the backbone via acid-cleavable acetal linkages and self-assembled from micelles, suggesting its rapid dissociation at the acidic extracellular pH of tumors.18 Moreover, ZnPP-encapsulating PBCAE micelles show synergic anticancer activity by inhibiting antioxidant heme oxygenase 1 (HO-1).18 The chemical structures of CA and CA-derived compounds are shown in Fig. 1.

O H O

O

O H

H

O OH

O

OH

Trans-cinnamaldehyde (CA)

2¢-Benzoyloxycinnamaldehyde (BCA)

2¢-Hydroxycinnamic acid (o-coumaric acid)

O

O H H3CO

OH

2¢-Hydroxycinnamaldehyde (HCA)

O

H

OH

OH

OH

4¢-Methoxycinnamaldehyde (MCA)

O F

H OH

Dihydroycinnamaldehyde (DHCA)

Cinnamyl alcohol (COH)

FIG. 1 The chemical structures of cinnamaldehyde and cinnamaldehyde-derived compounds.

Cinnamic acid (CAC)

Fluoroyhydroxycinnamaldehyde (FHCA)

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Oxidative stress-mediated apoptosis induced by cinnamaldehyde and its derivatives ROS are highly reactive molecules, and the equilibrium between the production and scavenging of ROS is maintained by a variety of antioxidant defenses.20 ROS, which includes superoxide anion radical (O2), hydrogen peroxide (H2O2), and hydroxyl radical (OH), are generated by numerous exogenous and endogenous stimuli.21 While low to modest ROS levels are essential for the regulation of normal physiological functions, excess cellular levels of ROS can damage cellular proteins, lipids, and nucleic acids.20 At high doses, ROS such as O2, NO, ONOO, OH, and H2O2 can cause deleterious effects to macromolecules and lead to cell death, such as apoptosis, necrosis, and autophagy.22 In particular, apoptosis can be triggered by ROS through activation of the extrinsic death receptor pathway or the intrinsic mitochondrial or ER pathways.22

Regulation of intrinsic and extrinsic apoptotic pathways ROS lead to the collapse of mitochondrial membrane potential (DCm) by targeting cardiolipin and mitochondrial permeability transition pore components, such as VDAC, ANT, and cyclophilin D .23 By opening Bax/Bak channels on the outer mitochondrial membrane, mitochondrial proteins, such as AIF, Endo G, and cytochrome c, are released into the cytosol, followed by the formation of the apoptosome complex and caspase activation.23 As an upstream factor, the tumor suppressor protein p53 can be activated by ROS, which induces apoptosis by upregulating the transcription of proapoptotic genes.22 Moreover, p53 is translocated into mitochondria where it binds with proapoptotic and antiapoptotic proteins, resulting in mitochondrial outer membrane permeabilization (MOMP) and the release of mitochondrial proteins into the cytosol.24 In addition to p53 activation, ROS induce JNK activation, which inhibits antiapoptotic proteins.25 Under severe oxidative stress, excess ROS trigger protein misfolding, resulting in the activation of apoptotic signaling pathways such as the IRE-1a-ASK-1-JNK pathway, ASK-1-p38 MAPK pathway, and PERK-eIF2a-ATF-4 pathway.22 The activated IRE-1a-ASK-1-JNK pathway stimulates massive Ca2+ transport from the ER to mitochondria and triggers MOMP.22 In addition to the PERK-eIF2a-ATF-4 pathway, the transcription factor CHOP is activated by the ASK-1-p38 MAPK pathway and contributes to mitochondrial apoptosis by upregulating the expression of proapoptotic proteins.22 Transmembrane death receptors activated by ROS recruit the adaptor molecule FADD to the intracellular surface of the receptor and form the DISC, followed by activation of initiator and effector caspases.26 Moreover, activated caspase 8 and 10 cause the cleavage of Bid to tBid and bind to ROMO-1, thereby triggering the activation of Bax/Bak and MOMP.26 ROS induce death receptor-mediated apoptosis by regulating diverse signaling pathways, such as the NF-kB, ASK-1, JNK, and p38 MAPK pathways.25 ROS activate ASK-1 and its downstream targets JNK and p38 MAPK by oxidizing thioredoxin and inhibiting protein phosphatases.22 Activated JNK translocates to the mitochondrial membrane and inhibits Bcl-2 localized to the mitochondria, inducing apoptosis.27 NF-kB acts as a cell survival signal and is involved in tumor promotion and progression by upregulating the expression of antiapoptotic genes including Bcl-XL, IAP, catalase, and Mn-superoxide dismutase.22 ROS inhibit TNF-R1-mediated NF-kB activation, thereby promoting apoptosis.22 CA and its derivatives increase the intracellular ROS levels of various types of cancer cells and lead to apoptotic cell death via the mitochondrial pathway (Table 1). Intracellular levels of ROS, including hydrogen peroxide, hydroxyl radicals, and peroxyl radicals are directly measured by staining with nonfluorescent carboxy-20 ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA). H2DCFDA diffuses into the cells and is hydrolyzed by intracellular esterase to DCFH, which reacts with ROS and generates fluorescent 20 ,70 -dichlorofluorescein (DCF). Therefore, the fluorescent intensity of DCF, which is analyzed by flow cytometry, fluorescence microscopy, and microplate fluorimeters, represents the intracellular level of ROS produced by the cells. An increase in intracellular ROS levels in cancer cells treated with CA and its analogs has been determined by measuring the fluorescence intensity of DCF in several cancer cell lines, such as prostate, colon, breast, liver, melanoma, and leukemia cell lines.4, 5, 13, 16, 19, 30, 32, 34, 35 Generally, CA, HCA, and BCA begin to increase the intracellular ROS level 0.5 h after their application and maintain the elevated level of ROS for up to 24 h.4, 5, 13, 16, 19, 30, 32, 34, 35 Moreover, ROS production and mitochondrial apoptosis are inhibited by antioxidant treatments, such as N-acetyl cysteine, glutathione, and vitamin E (Table 1). The apoptotic phenotype exhibits cell shrinkage, DNA fragmentation, and the phagocytosis of apoptotic bodies by macrophages. As hallmarks of apoptotic cell death, fragmentation of the nucleus and chromatic degradation is mediated by caspase activation.26 The effector caspases 3, 6, and 7 cleave cellular substrates such as poly(ADP-ribose) polymerase (PARP).26 Consistent with previous reports, CA and its derivatives induce apoptotic cell death through initiator and effector caspase activation, thereby resulting in PARP cleavage and DNA fragmentation (Table 1). In addition to caspase activation, the mitochondrial membrane potential is disrupted by CA treatment. In the HL-60 human leukemia cell line, CA induces mitochondrial membrane potential dysfunction, which is observed with flow cytometry using the fluorescent probe

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TABLE 1 The role of cinnamaldehyde and its derivatives in oxidative stress-induced apoptosis. CompoundReference

Cell lines

Effects

HCA

Human prostate cancer cells (DU145)

Increased intracellular ROS level STAT3 inactivation and sustained activation of ERK1/2 Inhibition of STAT3 inactivation and apoptotic cell death by NAC and GSH treatment

HCA28

Human colon cancer cells (SW480, SW620)

Sustained activation of p38 MAPK and JNK

HCA10

Human colon cancer cells (SW620)

Inhibition of L3-like, chymotrypsin-like, trypsin-like, and PGPHlike activities of the 26S proteasome Upregulated expression of ER stress-responsive genes, including HMOX1, HERPUD1, GADD153, and GRP78 Increased mitochondrial permeability and cytochrome c release

HCA15

Human colon cancer cells (SW620)

Suppression of AP-1 activation through inhibiting the DNA-binding activity of AP-1 Inhibition of AP-1 family, c-Fos and c-Jun expression

HCA14

Human colon cancer cells (SW620)

Sustained activation of ERK1/2 and JNK Inhibition of NF-kB transcriptional activation by ERK1/2 inhibitor treatment Inhibition of TNF-a-induced transcriptional activation of NF-kB, nuclear translocation of p50, and IkB degradation

BCA29

Human prostate cancer cells (DU145, PC3, LNCaP)

Induction of EGR1 upregulation and nuclear translocation Upregulated expression of proapoptotic genes including ATF3, NAG-1, and GADD45A EGR1 nuclear translocation induced by transient upregulation of IPO7

BCA30

Rat kidney RK3E and K-rastransformed RK3E-ras cells (RK3E and RK3E-ras)

Increased the intracellular ROS level Decreased the intracellular GSH level Decreased the protein expression of MT, g-GCS, DJ-1, and Nrf2 Inhibition of cell death by thiol antioxidant treatment (GSH monoethyl ester, NAC, and cysteine)

BCA31

H-ras12V transgenic mice

Downregulation of ROS-related genes including MT-1 and MT-2 Inhibition of tumor formation and growth

BCA32

5-fluorouracil (5-FU) and cyclophosphamide (CDDP)-resistant cells

Increased the intracellular ROS level Sustained activation of p38 MAPK Inhibition of apoptotic cell death by GSH treatment

BCA16

Human colon cancer cells (SW620) Human breast cancer cells (MDA-MB-231)

Increased the intracellular ROS level Sustained activation of EGFR and p38 MAPK Inhibition of ROS production and apoptotic cell death by NAC and GSH treatment

CA, HCA, FHCA, BCA5

Human colon cancer cells (HCT116)

Inhibition of TrxR activity at cytotoxic concentrations Induction of ARE-dependent transcriptional activity and increased protein levels of Nrf2

CA, DHCA, MCA, COH, CAC4

Human melanoma (A375)

Increased the intracellular ROS level Upregulated expression of oxidative stress-response genes including HMOX1, SRXN1, GPX2, TXNRD1, CDKN1A, DDIT3, and EGR1 Moderate depletion of cellular GSH levels Inhibition of NF-kB transcription activity and TNF-a-induced IL-8 production

CA33

Human hepatoma cells (PLC/PRF/5)

Sustained activation of ERK1/2, p38 MAPK, and JNK

34

Human hepatoma cells (PLC/PRF/5)

Increased the intracellular ROS level Inhibition of ROS production and apoptotic cell death by vitamin E treatment

13

CA

Continued

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TABLE 1 The role of cinnamaldehyde and its derivatives in oxidative stress-induced apoptosis—cont’d CompoundReference

Cell lines

Effects

CA

Human promyelocytic leukemia cells (HL-60)

Increased the intracellular ROS level Decreased the intracellular GSH and protein thiols level Increased mitochondrial permeability and cytochrome c release Inhibition of the loss of mitochondrial membrane potential and ROS generation by NAC treatment

CB-PIC36

Chemoresistant cancer cells (H460/PT, MCF7/Adr, HCT15/cos)

Downregulation of survival proteins such as survivin, Bcl-xL and Bcl-2 Decreased MDR1 expression via suppression of STAT3 and AKT signaling

CB-PIC37

Human colon cancer cells (SW620)

Increased the phosphorylation of AMPKa, ACC, and ERK1/2 in hypoxia Inhibition of HIF1a and Akt/mTOR in hypoxia

PolyCAFe micelle19

Human colon cancer cells (SW620)

Increased the intracellular ROS level Serve as nano-Fenton reactors to generate cytotoxic hydroxyl radicals Preferentially kill cancer cells

PBCAE micelle18

Human colon cancer cells (SW620)

Combined use of ROS-generating PBCAE with antioxidantsuppressing compounds ROS generation and HO-1 inhibition by ZnPP-loaded PBCAE micelles Inhibition of ROS generation by NAC treatment

35

3,30 -dihexyloxacarbocyanine iodide [DiOC6(3)].35 Moreover, mitochondrial proteins released by MOMP trigger the activation of caspases.35 In many studies on CA-induced mitochondrial apoptosis in various types of cancer cells, the protein levels of proapoptotic proteins (e.g., Bax, tBid, cytochrome c, Smac/Diablo, and Omi/HtrA2) increased, whereas those of antiapoptotic proteins (e.g., Bcl-2, Bcl-XL, Mcl-1, survivin, XIAP, cIAP-1, and cIAP-2) decreased.28, 34 The accumulation of ROS also regulates apoptotic cell death through the activation of ROS-mediated signaling pathways, including the NF-kB, ERK1/2, p38 MAPK, JNK, PI3K-Akt, STAT3, and EGFR pathways.25 After treatment with CA and its derivatives, elevated cellular levels of ROS induce the sustained activation of MAPK and EGFR; however, they inhibit NF-kB and STAT3 transcriptional activation.13, 14, 16

Regulation of antioxidant defense system Redox homeostasis is regulated by a variety of antioxidant defense systems, including enzymatic and nonenzymatic scavengers.20 Enzymatic scavengers include superoxide dismutases (SODs), catalase, glutathione peroxidase (GPx), thioredoxin, peroxiredoxin, and glutathione transferase.21, 38 Nonenzymatic scavengers include vitamin C (ascorbic acid), vitamin E (a-tocopherol), carotenoids, and glutathione (GSH).21, 38 Although cancer cells have elevated antioxidant defense mechanisms, they display high basal levels of ROS and are more sensitive to oxidative stress than normal cells.20 Thus, the difference in redox status between cancer cells and normal cells contributes to the selective killing of cancer cells by targeting ROS.20 In cancer cells, ROS are implicated in the initiation, progression, and metastasis of cancers or cell death; therefore, both prooxidant and antioxidant strategies can be applied to treat cancers.20 Various antioxidant cancer therapies, including consuming dietary antioxidants, enhancing ROS scavenging enzymes, inhibiting NADPH oxidase, and manipulating nitroxide compounds, have been applied to treat cancer.21 In contrast, ROS cause oxidative stress by disrupting redox homeostasis via the depletion of antioxidants or the generation of ROS.21 To induce high levels of ROS in cancer cells, exogenous ROSgenerating agents, such as arsenic agents, doxorubicin, paclitaxel, niclosamide, AG-221, and celecoxib, have been developed as anticancer drugs.21, 38 These ROS-enhancing agents target and regulate the activity of NADPH oxidase, cyclooxygenase, and enzymes in the electron transport chain, thereby promoting ROS generation.21 In addition to ROS production, the significant accumulation of ROS can be triggered by inhibiting ROS elimination via the antioxidant enzyme systems in cancer cells.20 Specific inhibitors targeting antioxidant enzymes have been shown to act as ROS-modulating

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agents; these inhibitors include thioredoxin, thioredoxin reductase, superoxide dismutase, catalase, and HO-1.20, 38 Moreover, the antioxidant system can be inhibited by depleting the intracellular level of GSH through its conjugation with GSH or inhibiting GSH synthesis.20 BCA increased ROS generation in K-ras-transformed cells (RK3E-ras) compared to that in isogenic nontransformed cells (RK3E), showing the association between oxidative stress and K-ras-transformed oncogenic transformation.30 Normal cells and drug-resistant cells are less sensitive to ROS stress than cancer cells due to their high intracellular GSH levels and enhanced antioxidant enzyme activities.30 The intracellular GSH level and the expression levels of metallothionein (MT), g-GCS (which catalyzes the rate-limiting step in de novo GSH synthesis), DJ-1, and Nrf2 protein increased in RK3E cells but decreased in RK3E-ras cells after BCA treatment.30 Moreover, BCA-induced cell death was reversed by treatment with the thiol antioxidants GSH-MEE (GSH monoethyl ester), NAC, and cysteine.30 Thus, the difference between normal and cancer cells contributes to the selective killing of cancer cells by the regulation of cellular ROS levels. Similarly, BCA inhibited the growth of hepatic tumors in H-ras12V transgenic mice by inhibiting the expression of MT-1 and MT-2.31 In addition, a human melanoma cell line (A375P) showed glutathione depletion in response to CA treatment.4 CA and its derivatives (HCA, BCA, and FHCA) exhibit antiproliferative activity and selectively inhibit TrxR but not glutathione reductase.5 CA, which has a Michael acceptor, has been suggested to bind to the penultimate selenocysteine residue of TrxR, rendering the enzyme irreversibly deactivated upon CA binding.5 Oxidative stress induces phase II detoxification and the expression of antioxidant enzymes, such as glutathione S-transferase, NAD(P)H quinone reductase, HO-1, g-glutamate-cysteine ligase, Trx, TrxR, and peroxiredoxin 1.39 The transcription of genes encoding phase II detoxifying enzymes is regulated by Nrf2 through its binding to antioxidant responsive elements (AREs).39 Although a sublethal dose of BCA induces an antioxidant response through Nrf2-mediated upregulation of phase II enzymes, a lethal dose of BCA shows negligible or marginal TrxR induction, resulting in significant TrxR inhibition.5

Apoptotic cell death via STAT3 inactivation Several groups have developed a screening system for STAT3-inhibitory compounds and have reported herbal medicinebased drugs with low toxicity to be potent STAT3 inhibitors.40 These natural STAT3 inhibitors include curcumin, butein, capsaicin, celastrol, diosgenin, thymoquinone, resveratrol, piperlongumine, and vitamin E.40 Anticancer therapeutics targeting the STAT3 signaling pathway target one of four elements: the SH2 domain, STAT3 dimerization, the STAT3 DNA-binding domain, and upstream tyrosine kinase and negative regulators of STAT3.41 Activation of the STAT3 pathway, an oncogenic signaling pathway, promotes tumor growth, metastasis, and angiogenesis.41 Therefore, knockdown of STAT3 expression and the inhibition of STAT3 activity induces apoptotic cell death and tumor suppression.42 Recently, Kwon BM et al. reported that HCA inhibits cancer cell proliferation and tumor growth by STAT3 inactivation and ROS generation.13 HCA inhibits STAT3 activity by directly binding STAT3 proteins and ROSmediated STAT3 inactivation.13 This report was the first study to show that HCA functions as a STAT3 inhibitor that thereby promotes apoptosis and tumor suppression. The relationship between STAT3 inactivation and ROS generation has been clearly elucidated in STAT3-activated cancer cells in particular. In addition, HCA inhibits STAT3 activity by directly binding to PKM2, which is upstream of the STAT3 signaling pathway.12 HCA decreases the phosphorylation of PKM2 at tyrosine 105, leading to a decrease in PKM2-mediated STAT3 phosphorylation at tyrosine 705 and the downregulation of STAT3 target genes, including cyclin D1 and MEK5.12 The engagement of HCA with target protein in cancer cells has been confirmed by affinity-based and label-free target identification techniques.43, 44 In a pull-down assay, biotin-labeled HCA bound to STAT3, which was inhibited by treatment with excess HCA.13 The direct binding of HCA and STAT3 was also validated by label-free methods, including drug affinity responsive target stability (DARTS) assay and cellular thermal shift assay (CETSA). Upon drug binding, the protease resistance and thermal stability of the bound target protein increased compared to those of the unbound proteins.43, 44 Consistent with previous studies on the engagement of small molecules with their targets, HCA increased the amount of pronase-resistant and thermally stable STAT3 in intact cells and cell lysates.13 Moreover, a significant decrease in STAT3 phosphorylation was detected 10 min after HCA treatment, implying that the inhibition of STAT3 phosphorylation is mediated by the direct interaction between HCA and STAT3.13 Furthermore, inactivation of STAT3 by HCA treatment inhibited dimer formation and the subsequent nuclear translocation and transcription of STAT3-regulated genes.13 In addition to the effect of direct binding between HCA and STAT3, STAT3 activity is inhibited by HCA-induced ROS generation.13 Notably, HCA binds to STAT3 regardless of intracellular ROS levels, suggesting that HCA inhibits STAT3 activity by binding directly to STAT3 and inducing ROS generation independently.

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Recent cumulative evidence has indicated that ROS influence the activation of the NF-kB, MAPK, PI3K-Akt, and Keap1-Nrf2-ARE signaling pathways.25 In particular, ROS are responsible for the sustained activation of MAP kinases, which are components of the ERK1/2, JNK, p38, and EMK1/ERK5 pathways.45 Numerous studies have reported that MAP kinase signaling pathways are activated by treatment with CA and its derivatives.13, 14, 16, 28, 32, 33 Recently, Kwon BM et al. elucidated the cross talk between STAT3 and the MAPK signaling pathway in HCA-induced apoptosis.13 In previous studies on STAT3 and ERK1/2, ERK2 was shown to increase serine phosphorylation and decrease tyrosine phosphorylation, thereby negatively regulating STAT3 activities.46, 47 Moreover, ROS-mediated ERK1/2 activation increases STAT3 phosphorylation at serine 727 but decreases STAT3 phosphorylation at tyrosine 705 by recruiting a protein tyrosine phosphatase (PTPase) or MAP kinase phosphatase (MKP).48 Consistent with previous reports, HCA increases STAT3 phosphorylation at serine 727 and decreases STAT3 phosphorylation at tyrosine 705.13 However, all these effects are abrogated by ERK1/2 inhibitor or antioxidant treatment, suggesting that ROS-induced ERK1/2 influences STAT3 phosphorylation.13 The activation of STAT3 in cancer cells contributes to the acquisition of chemoresistance; therefore, the pharmacological inhibition of STAT3 is a promising strategy to overcome chemoresistance.49 Numerous studies reported that STAT3 is activated and that the inhibition of STAT3 enhances chemosensitivity to chemotherapeutic agents in a variety of chemoresistant cancer cells.49 The CA derivative (E)-4-((2-(3-oxopop-1-enyl)phenoxy)methyl)pyridinium malonic acid (CB-PIC) induced apoptosis in drug-resistant cancer cells via the suppression of MDR1 expression and inhibition of STAT3 and Akt signaling.36 CB-PIC sensitized three resistant cancer cell lines, including paclitaxel-resistant lung cancer cells and adriamycin-resistant breast cancer and colon cancer cells.36 CB-PIC induced MDR1 inhibition by attenuating the expression of MDR1 at the protein and mRNA levels rather than MDR1 activity.36 CB-PIC inhibited STAT3-mediated MDR1 gene expression by downregulating the phosphorylation of STAT3 and Akt.36 Although the binding target of CB-PIC has not been clearly elucidated, these results suggest that STAT3 inhibitors can overcome drug resistance in chemoresistant cancer cells by effectively decreasing MDR1 expression.

Direct binding targets of cinnamaldehyde and its derivatives It has been reported that natural compounds have multiple targets with different biological activities.50 Several groups have identified direct binding targets of cinnamaldehyde and its derivatives in various cancer cell lines, such as prostate, colon, and breast cancer cells; epidermoid carcinoma; and erythroleukemia.5, 8, 10–13, 51 The protein targets of HCA and its derivatives include STAT3, PKM2, Pim-1, LRP1, proteasome subunit, tubulin, and TrxR.5, 8, 10–13, 51 The binding of compounds with their target proteins is determined by pull-down assay coupled with LC-MS/MS analysis, two-dimensional gel electrophoresis (2-DGE) coupled with MALDI-TOF analysis, X-ray cocrystallography, and the BIAM labeling assay. Although multiple targets of HCA and CA have been identified, a comprehensive understanding of the mode of action of their multicomponent and multitarget interactions is lacking (Table 2). In this review, we focus on the molecular mechanisms of the target protein underlying HCA-induced ROS generation and oxidative stress (Fig. 2).

Proteasome subunits In 2007, proteasome subunits were first reported to be protein direct binding partners of HCA in SW620 human colon cancer cells using 2-DGE coupled with MALDI-TOF analysis.10 By directly binding to proteasome subunits, HCA inhibits the L3-like, chymotrypsin-like, trypsin-like, and PGPH-like activities of the 26S subunit of the proteasome.10 Proteasome inhibitors, including bortezomib, carfilzomib, and ixazomib, produce ER stress, which triggers apoptotic cell death via the overproduction of ROS.52 Similarly, proteasome inhibition by HCA treatment induces the accumulation of misfolded proteins, resulting in ER stress-dependent ROS production.10 The involvement of ER stress in HCA-induced apoptosis was also demonstrated by the upregulated expression of the ER stress-responsive genes GADD153, ATF3, HERPUD1, and HMOX1.10

Signal transducer and activator of transcription 3 (STAT3) and pyruvate kinase M2 (PKM2) Ten years later, two more target proteins of HCA were shown to activate PKM2 or inhibit STAT3 in DU145 prostate cancer cells.12, 13 Upon the binding of HCA to STAT3, STAT3 phosphorylation is significantly reduced; however, the effects of HCA are rescued by pretreatment with antioxidants, NAC, or GSH.13 As ROS generation inhibits STAT3 activation, these findings show that high levels of ROS affect the phosphorylation of STAT3. HCA-induced ROS production leads to the activation of ERK1/2, which increases STAT3 phosphorylation at serine 727 but decreases STAT3 phosphorylation at

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TABLE 2 Direct binding targets of cinnamaldehyde and its derivatives. Compound/targetReference

Cell line

Assay

Effect

Human prostate cancer cells (DU145)

Pull down assay with biotin-conjugated HCA LC-MS analysis

Inhibition of STAT3 phosphorylation at tyrosine 705, dimerization, nuclear translocation, and transcription activity

HCA/PKM212

Human prostate cancer cells (DU145)

Pull down assay with biotin-conjugated HCA LC-MS analysis

Inhibition of PKM2 phosphorylation at tyrosine 105 and nuclear translocation Activation of PKM2 tetramer formation and increasing pyruvate kinase activity

HCA/Pim-18

Human erythroleukemia cells (HEL) Human epidermoid carcinoma cells (A431)

Pull down assay with HCA-conjugated Sepharose 4B X-ray cocrystallography

Inhibition of Pim-1 activity by altering the apo kinase structure in a manner that shielded the ligand from solvent, thereby acting as a gatekeeper loop

HCA/LRP111

Human breast cancer cells (MDA-MB-231, MCF7)

Pull down assay with biotin-conjugated HCA LC-MS analysis

Increasing the cysteine thiol oxidation status of LRP1 extracellular domains, thereby promoting a2M-ligand binding Increasing the clearance of the a2M-pepsin complexes by endocytosis and lysosomal degradation Decreasing the extracellular pepsin concentration

HCA/proteasome subunits10

Human colon cancer cells (SW620)

Pull down assay with biotin-conjugated HCA 2-Dimentional gel electrophoresis (2-DGE) and MALDI-TOF analysis

Inhibition of L3-like, chymotrypsin-like, trypsin-like, and PGPH-like activities of the 26S proteasome Induction of heat shock family and ER stressresponsive genes, reflecting the accumulation of misfolded proteins by proteasome inhibition

CA/tubulin51

Human colon cancer cells (HCT116)

BIAM labeling assay

Interacting with the sulfhydryl groups in tubulin Inducing tubulin aggregation and increasing the accumulation of insoluble tubulin

CA, HCA, FHCA, BCA/TrxR5

Human colon cancer cells (HCT116)

BIAM labeling assay

Inhibition of TrxR activity at cytotoxic concentrations Targeting the penultimate C-terminal selenocysteine residue to induce TrxR inhibition Induction of ARE-dependent transcriptional activity and increased protein levels of Nrf2

HCA/STAT3

13

tyrosine 705.13 Sustained ERK1/2 activation recruits a PTPase or MKP.48 Regardless of the presence of ROS, HCA binds to STAT3 and increases the intracellular levels of ROS.13 Moreover, knockdown of STAT3 expression or the inhibition of STAT3 activity by HCA treatment induces ROS generation and subsequent apoptotic cell death.13 The activation of STAT3 is regulated by diverse protein tyrosine kinases and protein tyrosine phosphatases.40 When analyzing the upstream signaling pathways of STAT3 activation, the phosphorylation of EGFR, JAK, and SRC was found to not be affected; however, the phosphorylation of PKM2 at tyrosine 105 was inhibited by HCA treatment.12 PKM2 is the rate-limiting enzyme of glycolysis, and the phosphorylation of PKM2 at tyrosine 105 promotes cell proliferation and tumor growth.53 Phosphorylated PKM2 at tyrosine 105 phosphorylates STAT3 at tyrosine 705, which is translocated into the nucleus and leads to the upregulation of STAT3-dependent genes, such as MEK5, cyclin D1, and c-Myc.53 In addition to the binding of HCA to STAT3, HCA directly binds to PKM2 and significantly inhibits the phosphorylation of PKM2 at tyrosine 105.12 Furthermore, HCA acts as a PKM2 activator by promoting the stable tetrameric form of PKM2.12 According to a molecular docking study, the HCA-binding site in dimeric PKM2 is similar to the

HCA

CA

aM2 + Pepsin EGF

TrxR

Tubulin aggregation

Pim-1

MAPK Akt p38

ROS

IL-6

p-ERK1/2 (T202/Y204)

pSTAT3 (S727)

PKM2 (Dimer)

pSTAT3 (Y705)

26S proteasome

LRP (thiol oxidation)

Glucose Endocytosis Misfolded protein

Bcl-2 (↓) Bax (↑) Bcl-xL(↓) Bak (↑) Mcl-1 (↓)

Cell cycle arrest

p-PKM2 (Y105)

ROS

PEP Cyclin D1 Survivin MEK5

PKM2 Pyruvate (Tetramer)

NFkB AP-1

ER stress Lysosome degradation

Cytochrome c Cell cycle progression

Lactate

Apoptosis

Caspase activation p-SNAP23 PARP cleavage Cell proliferation Apoptosis

Extracellular vesicle release

FIG. 2 Molecular mechanisms underlying HCA-induced ROS generation and oxidative stress. HCA and CA induce ROS generation and oxidative stress-mediated apoptosis through inhibiting cell signaling pathway components, including PKM2, STAT3, and Pim-1. In addition, HCA and CA induce apoptotic cell death through inhibiting the 26S proteasome, thioredoxin reductase, and pepsin or inducing tubulin aggregation. HCA, 20 -hydroxycinnamaldehyde; CA, cinnamaldehyde.

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TEPP-46-binding site, which was previously shown to be a PKM2 activator that leads to the allosteric stabilization of the PKM2 tetramer.54 Thus, HCA suppresses cell proliferation and tumor growth by inhibiting protein kinase activity and promoting the pyruvate kinase activity of PKM2, respectively.12 Recent studies have shown that ROS induce the oxidation of PKM2 at cysteine 358, which inhibits PKM2 activity and promotes the diversion of glycolytic intermediates to the pentose phosphate pathway (PPP).53 The PPP generates reduced NADPH, which facilitates the recycling of oxidized GSSG into reduced GSH and thereby generates reducing the potential for the detoxification of ROS.53 Recently, it was reported that the antitumor effects of HCA are mediated by the inhibition of tyrosine phosphorylation of PKM2-STAT3 and ROS generation.13 The high intracellular ROS levels in cancer cells treated with HCA contribute to the phosphorylation of ERK1/2, PKM2, and STAT3; however, the effects on the oxidation of PKM2 have not been elucidated.

Proviral insertion in murine lymphomas-1 (Pim-1) Pim-1 was recently identified as a molecular target of HCA in leukemia and skin cancer by screening 77 cancer-related kinases.8 HCA inhibits the activity of Pim-1 by at least 80%, and Pim-1 knockdown elicits resistance to HCA.8 When analyzing the crystal structure of Pim-1 in complex with HCA, HCA was found to bind to the Pim-1 ATP-binding pocket and alter the apo kinase structure, thereby acting as a gatekeeper loop.8 HCA induces apoptosis by inhibiting oncogenic signaling pathways that include Pim-1 via the phosphorylation of target molecules such as p21CIP1, p27KIP1, c-Myc, Bad, ASK1, and PRAS40.8 Furthermore, HCA suppressed tumor growth in both leukemia and skin cancer in a xenograft mouse model.8 Members of the Pim family of serine/threonine kinases play pivotal roles in diverse cellular processes, including signal transduction, cell cycle progression, cell metabolism, and tumor growth.27 Pim kinase, a proto-oncogene, regulates cellular redox and metabolism by switching the oncogenic activity of K-Ras to promote cell death.55 Thus, knockout of the Pim kinases or treatment with the Pim kinase inhibitor AZD1208 elevated the intracellular levels of ROS and enhanced apoptosis.55 Although Kim JE et al. did not evaluate the intracellular ROS levels in leukemia and skin cancer cells after HCA treatment, the significant inhibition of Pim-1 kinase might be involved in ROS generation and apoptotic cell death.

Low-density lipoprotein receptor-related protein 1 (LRP1) In addition to the inhibitory effects of HCA on oncogenic kinases, it influences ligand-receptor binding by modulating thiol oxidation in cell surface receptors. Hong SH et al. reported that LRP1 is a molecular target of HCA in human breast cancer cells.11 LRP1 is a large endocytic receptor that binds to over 30 ligands, including a2-macroglobulin (a2M), apolipoprotein E, tissue plasminogen activator, and blood coagulation factors.56 The LRP1 ligand a2M forms a complex with a range of proteases, including pepsin and gastricsin, which are cleared by LRP1-mediated endocytosis and lysosomal degradation.57 The extracellular a-chain of LRP1 contains ligand-binding domains consisting of cysteine-rich complement-type repeats.56 Thus, HCA binds to LRP1 and increases the cysteine thiol oxidation status of extracellular LRP1 domains, thereby promoting a2M ligand binding.11 Pepsin contributes to epithelial cell proliferation and tumor progression by digesting type III collagen; therefore, clearance of the a2M-pepsin complex by LRP1-mediated endocytosis inhibits cell invasion by decreasing the extracellular pepsin concentration.11

Thioredoxin reductase (TrxR) Recently, Chew EH et al. reported that tubulin aggregation and the inhibition of TrxR are responsible for the antiproliferative activities of CA.5 In this study, biotin-conjugated iodoacetamide (BIAM) labeling was used to investigate the direct interaction of tubulin or TrxR with CA.5 CA-induced G2/M phase arrest by dysregulating cell cycle regulatory proteins including cdk1, cdc25C, mad2, cdc20, and survivin, thereby resulting in apoptosis in HCT116 colon cancer cells.5 In addition to a decline in cell cycle regulatory proteins, CA interacts with the cysteine residues of tubulin and induces tubulin aggregation.5 CA-induced tubulin aggregation results in the accumulation of insoluble tubulin; therefore, the imbalance between soluble and insoluble tubulin contributes to disrupted microtubule polymerization and subsequent G2/M phase arrest.5 Consistent with work on the interaction between tubulin and CA, the direct binding of CA to TrxR was also validated by BIAM labeling. TrxR functions as a protein disulfide reductase and contributes to redox homeostasis, the redox regulation of transcription factor activity, and the inhibition of apoptosis.58 Mammalian TrxR contains two distinct redox-active sites: the N-terminal Cys59-Val-Asn-Val-Gly-Cys64 dithiol/disulfide active site and the C-terminus Gly-Cys497-Sec498 site.58 In previous studies on TrxR, the selenocysteine (Sec) residue, which is highly nucleophilic and thus targeted by electrophilic Michael acceptors, was shown to be important for TrxR catalytic activity.59 Thus, CA, which contains an a,b-unsaturated carbonyl group known as the Michael acceptor, inhibits

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TrxR catalytic activity by targeting the penultimate C-terminal selenocysteine residue.5 While CAC lacking the a,b-unsaturated carbonyl group does not inhibit TrxR, the hydroxyl- and benzoyloxy-substituted CA analogs HCA and BCA display greatly enhanced inhibitory activity against TrxR.5

Applications to other cancers or conditions In this chapter, we reviewed the anticancer activity of CA and its derivatives in various types of cancer cells, such as prostate cancer, colon cancer, melanoma, hepatoma, and leukemia. CA and its derivatives bind to and inhibit multiple protein targets involved in cell proliferation, migration, invasion, angiogenesis, and tumorigenesis. Notably, CA and its derivatives increase intracellular ROS levels in cancer cells, leading to oxidative stress-induced apoptotic cell death. Moreover, CA and its derivatives induce oxidative stress by inhibiting ROS elimination via the antioxidant enzyme systems of cancer cells. These data, therefore, suggest that these compounds effectively induce oxidative stress by modulating ROS levels. In addition to the anticancer activity of CA and its derivatives, their other biological activities have been studied in vascular smooth muscle cells, macrophages, T lymphocytes, microglial cells, and platelets. HCA and BCA significantly inhibit nitric oxide production through inhibition of NF-kB and AP-1 activation in macrophages, showing the antiinflammatory effect of these compounds. In addition, HCA and BCA inhibit the proliferation of T lymphocytes and induce differentiation through attenuating the cell cycle at G1 phase. Furthermore, HCA and BCA exert antineuroinflammatory effects in LPS-stimulated microglial cells by targeting LRP1, significantly inhibiting nitric oxide and pro-inflammatory cytokine production. 20 -Methoxycinnamaldehyde shows antiproliferative and antimigratory effects in TNF-a-stimulated aortic smooth muscle cells, indicating its potential use as an antiatherosclerotic agent. Eugenol and coniferaldehyde, one of the 13 compounds obtained from C. cassia, exert platelet antiaggregation and blood anticoagulation effects. Taken together, these results suggest the diverse biological activities of CA and its derivatives in cancer, inflammatory disease, diabetes, neuropathy, and cardiovascular disease.

Summary points l

l

l

l

l l

l

Cinnamaldehyde (CA) and its derivatives have antiproliferative activity in various types of cancers and are therefore promising anticancer agents. Several CA-derived compounds with increased stability, metabolic stability, ability to target cancer cells, and therapeutic efficacy have been synthesized, and BCA is undergoing clinical testing. CA and its derivatives increase the intracellular ROS levels of various types of cancer cells, which trigger apoptotic cell death through activation of the intrinsic mitochondrial and ER pathways. In addition to exogenous agents that generate ROS, CA and its derivatives induce oxidative stress by inhibiting antioxidant enzymes and depleting the intracellular level of GSH. HCA inhibits STAT3 activity by binding directly to STAT3 and inducing ROS generation independently. Sustained ERK activation induced by ROS increases serine phosphorylation and decreases tyrosine phosphorylation, thereby negatively regulating STAT3 activities, which demonstrates the cross talk between ROS and STAT3 in HCA-induced apoptosis. Binding targets of HCA and CA, such as PKM2, STAT3, Pim-1, LRP-1, 26S proteasome subunits, TrxR, and tubulin, have been identified, which contributes to the understanding of the molecular mechanisms underlying HCA-induced ROS generation and oxidative stress.

References 1. Wijesekera RO. Historical overview of the cinnamon industry. CRC Crit Rev Food Sci Nutr 1978;10(1):1–30. 2. Hong SH, Ismail IA, Kang SM, Han DC, Kwon BM. Cinnamaldehydes in Cancer chemotherapy. Phytother Res 2016;30(5):754–67. 3. Choi J, Lee KT, Ka H, Jung WT, Jung HJ, Park HJ. Constituents of the essential oil of the Cinnamomum cassia stem bark and the biological properties. Arch Pharm Res 2001;24(5):418–23. 4. Cabello CM, Bair 3rd WB, Lamore SD, Ley S, Bause AS, Azimian S, et al. The cinnamon-derived Michael acceptor cinnamic aldehyde impairs melanoma cell proliferation, invasiveness, and tumor growth. Free Radic Biol Med 2009;46(2):220–31. 5. Chew EH, Nagle AA, Zhang Y, Scarmagnani S, Palaniappan P, Bradshaw TD, et al. Cinnamaldehydes inhibit thioredoxin reductase and induce Nrf2: potential candidates for cancer therapy and chemoprevention. Free Radic Biol Med 2010;48(1):98–111. 6. Dinkova-Kostova AT, Massiah MA, Bozak RE, Hicks RJ, Talalay P. Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups. Proc Natl Acad Sci U S A 2001;98(6):3404–9.

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7. Kwon BM, Cho YK, Lee SH, Nam JY, Bok SH, Chun SK, et al. 2’-Hydroxycinnamaldehyde from stem bark of Cinnamomum cassia. Planta Med 1996;62(2):183–4. 8. Kim JE, Son JE, Jeong H, Joon Kim D, Seo SK, Lee E, et al. A novel cinnamon-related natural product with Pim-1 inhibitory activity inhibits leukemia and skin cancer. Cancer Res 2015;75(13):2716–28. 9. Lee CW, Hong DH, Han SB, Park SH, Kim HK, Kwon BM, et al. Inhibition of human tumor growth by 20 -hydroxy- and 20 -benzoyloxycinnamaldehydes. Planta Med 1999;65(3):263–6. 10. Hong SH, Kim J, Kim JM, Lee SY, Shin DS, Son KH, et al. Apoptosis induction of 20 -hydroxycinnamaldehyde as a proteasome inhibitor is associated with ER stress and mitochondrial perturbation in cancer cells. Biochem Pharmacol 2007;74(4):557–65. 11. Kang HS, Kim J, Lee HJ, Kwon BM, Lee DK, Hong SH. LRP1-dependent pepsin clearance induced by 20 -hydroxycinnamaldehyde attenuates breast cancer cell invasion. Int J Biochem Cell Biol 2014;53:15–23. 12. Yoon YJ, Kim YH, Jin Y, Chi SW, Moon JH, Han DC, et al. 20 -hydroxycinnamaldehyde inhibits cancer cell proliferation and tumor growth by targeting the pyruvate kinase M2. Cancer Lett 2018;434:42–55. 13. Yoon YJ, Kim YH, Lee YJ, Choi J, Kim CH, Han DC, et al. 2’-Hydroxycinnamaldehyde inhibits proliferation and induces apoptosis via signal transducer and activator of transcription 3 inactivation and reactive oxygen species generation. Cancer Sci 2019;110(1):366–78. 14. Lee SH, Lee CW, Lee JW, Choi MS, Son DJ, Chung YB, et al. Induction of apoptotic cell death by 20 -hydroxycinnamaldehyde is involved with ERK-dependent inactivation of NF-kappaB in TNF-alpha-treated SW620 colon cancer cells. Biochem Pharmacol 2005;70(8):1147–57. 15. Lee CW, Lee SH, Lee JW, Ban JO, Lee SY, Yoo HS, et al. 2-Hydroxycinnamaldehyde inhibits SW620 colon cancer cell growth through AP-1 inactivation. J Pharmacol Sci 2007;104(1):19–28. 16. Han DC, Lee MY, Shin KD, Jeon SB, Kim JM, Son KH, et al. 20 -Benzoyloxycinnamaldehyde induces apoptosis in human carcinoma via reactive oxygen species. J Biol Chem 2004;279(8):6911–20. 17. Lee K, Kwon BM, Kim K, Ryu J, Oh SJ, Lee KS, et al. Plasma pharmacokinetics and metabolism of the antitumour drug candidate 20 -benzoyloxycinnamaldehyde in rats. Xenobiotica 2009;39(3):255–65. 18. Park S, Kwon B, Yang W, Han E, Yoo W, Kwon BM, et al. Dual pH-sensitive oxidative stress generating micellar nanoparticles as a novel anticancer therapeutic agent. J Control Release 2014;196:19–27. 19. Kwon B, Han E, Yang W, Cho W, Yoo W, Hwang J, et al. Nano-Fenton reactors as a new class of oxidative stress amplifying anticancer therapeutic agents. ACS Appl Mater Interfaces 2016;8(9):5887–97. 20. Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov 2009;8(7):579–91. 21. Wang J, Yi J. Cancer cell killing via ROS: to increase or decrease, that is the question. Cancer Biol Ther 2008;7(12):1875–84. 22. Redza-Dutordoir M, Averill-Bates DA. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim Biophys Acta 2016;1863 (12):2977–92. 23. Singh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nat Rev Mol Cell Biol 2019;20 (3):175–93. 24. Luna-Vargas MP, Chipuk JE. The deadly landscape of pro-apoptotic BCL-2 proteins in the outer mitochondrial membrane. FEBS J 2016;283 (14):2676–89. 25. Zhang J, Wang X, Vikash V, Ye Q, Wu D, Liu Y, et al. ROS and ROS-mediated cellular signaling. Oxidative Med Cell Longev 2016;2016:4350965. 26. Ichim G, Tait SW. A fate worse than death: apoptosis as an oncogenic process. Nat Rev Cancer 2016;16(8):539–48. 27. Beharry Z, Mahajan S, Zemskova M, Lin YW, Tholanikunnel BG, Xia Z, et al. The Pim protein kinases regulate energy metabolism and cell growth. Proc Natl Acad Sci U S A 2011;108(2):528–33. 28. Nguyen HA, Kim SA. 2’-Hydroxycinnamaldehyde induces apoptosis through HSF1-mediated BAG3 expression. Int J Oncol 2017;50(1):283–9. 29. Kang HS, Ock J, Lee HJ, Lee YJ, Kwon BM, Hong SH. Early growth response protein 1 upregulation and nuclear translocation by 20 -benzoyloxycinnamaldehyde induces prostate cancer cell death. Cancer Lett 2013;329(2):217–27. 30. Ock J, Lee HA, Ismail IA, Lee HJ, Kwon BM, Suk K, et al. Differential antiproliferation effect of 20 -benzoyloxycinnamaldehyde in K-ras-transformed cells via downregulation of thiol antioxidants. Cancer Sci 2011;102(1):212–8. 31. Lee HS, Lee SY, Ha HL, Han DC, Han JM, Jeong TS, et al. 2’-Benzoyloxycinnamaldehyde inhibits tumor growth in H-ras12V transgenic mice via downregulation of metallothionein. Nutr Cancer 2009;61(5):723–34. 32. Chung YM, Yoo YD, Kim JS, Lee CY, Kim HJ. The activity of 20 -benzoyloxycinnamaldehyde against drug-resistant cancer cell lines. J Chemother 2007;19(4):428–37. 33. Wu SJ, Ng LT, Lin CC. Cinnamaldehyde-induced apoptosis in human PLC/PRF/5 cells through activation of the proapoptotic Bcl-2 family proteins and MAPK pathway. Life Sci 2005;77(8):938–51. 34. Wu SJ, Ng LT, Lin CC. Effects of vitamin E on the cinnamaldehyde-induced apoptotic mechanism in human PLC/PRF/5 cells. Clin Exp Pharmacol Physiol 2004;31(11):770–6. 35. Ka H, Park HJ, Jung HJ, Choi JW, Cho KS, Ha J, et al. Cinnamaldehyde induces apoptosis by ROS-mediated mitochondrial permeability transition in human promyelocytic leukemia HL-60 cells. Cancer Lett 2003;196(2):143–52. 36. Yun M, Lee D, Park MN, Kim EO, Sohn EJ, Kwon BM, et al. Cinnamaldehyde derivative (CB-PIC) sensitizes chemo-resistant cancer cells to druginduced apoptosis via suppression of MDR1 and its upstream STAT3 and AKT signalling. Cell Physiol Biochem 2015;35(5):1821–30. 37. Cho SY, Lee HJ, Lee HJ, Jung DB, Kim H, Sohn EJ, et al. Activation of AMP-activated protein kinase alpha and extracelluar signal-regulated kinase mediates CB-PIC-induced apoptosis in hypoxic SW620 colorectal cancer cells. Evid Based Complement Alternat Med 2013;2013:974313. 38. Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. Oxidative stress and antioxidant defense. World Allergy Organ J 2012;5(1):9–19.

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39. Rushmore TH, Morton MR, Pickett CB. The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity. J Biol Chem 1991;266(18):11632–9. 40. Wong ALA, Hirpara JL, Pervaiz S, Eu JQ, Sethi G, Goh BC. Do STAT3 inhibitors have potential in the future for cancer therapy? Expert Opin Investig Drugs 2017;26(8):883–7. 41. Chai EZ, Shanmugam MK, Arfuso F, Dharmarajan A, Wang C, Kumar AP, et al. Targeting transcription factor STAT3 for cancer prevention and therapy. Pharmacol Ther 2016;162:86–97. 42. Lee SO, Lou W, Qureshi KM, Mehraein-Ghomi F, Trump DL, Gao AC. RNA interference targeting Stat3 inhibits growth and induces apoptosis of human prostate cancer cells. Prostate 2004;60(4):303–9. 43. Lomenick B, Jung G, Wohlschlegel JA, Huang J. Target identification using drug affinity responsive target stability (DARTS). Curr Protoc Chem Biol 2011;3(4):163–80. 44. Huber KV, Olek KM, Muller AC, Tan CS, Bennett KL, Colinge J, et al. Proteome-wide drug and metabolite interaction mapping by thermal-stability profiling. Nat Methods 2015;12(11):1055–7. 45. McCubrey JA, Lahair MM, Franklin RA. Reactive oxygen species-induced activation of the MAP kinase signaling pathways. Antioxid Redox Signal 2006;8(9–10):1775–89. 46. Jain N, Zhang T, Fong SL, Lim CP, Cao X. Repression of Stat3 activity by activation of mitogen-activated protein kinase (MAPK). Oncogene 1998;17 (24):3157–67. 47. Booz GW, Day JN, Baker KM. Angiotensin II effects on STAT3 phosphorylation in cardiomyocytes: evidence for Erk-dependent Tyr705 dephosphorylation. Basic Res Cardiol 2003;98(1):33–8. 48. Jung SN, Shin DS, Kim HN, Jeon YJ, Yun J, Lee YJ, et al. Sugiol inhibits STAT3 activity via regulation of transketolase and ROS-mediated ERK activation in DU145 prostate carcinoma cells. Biochem Pharmacol 2015;97(1):38–50. 49. Han Z, Feng J, Hong Z, Chen L, Li W, Liao S, et al. Silencing of the STAT3 signaling pathway reverses the inherent and induced chemoresistance of human ovarian cancer cells. Biochem Biophys Res Commun 2013;435(2):188–94. 50. Talevi A. Multi-target pharmacology: possibilities and limitations of the "skeleton key approach" from a medicinal chemist perspective. Front Pharmacol 2015;6:205. 51. Nagle AA, Gan FF, Jones G, So CL, Wells G, Chew EH. Induction of tumor cell death through targeting tubulin and evoking dysregulation of cell cycle regulatory proteins by multifunctional cinnamaldehydes. PLoS One 2012;7(11)e50125. 52. Fribley A, Zeng Q, Wang CY. Proteasome inhibitor PS-341 induces apoptosis through induction of endoplasmic reticulum stress-reactive oxygen species in head and neck squamous cell carcinoma cells. Mol Cell Biol 2004;24(22):9695–704. 53. Tamada M, Suematsu M, Saya H. Pyruvate kinase M2: multiple faces for conferring benefits on cancer cells. Clin Cancer Res 2012;18(20):5554–61. 54. Anastasiou D, Yu Y, Israelsen WJ, Jiang JK, Boxer MB, Hong BS, et al. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis. Nat Chem Biol 2012;8(10):839–47. 55. Song JH, An N, Chatterjee S, Kistner-Griffin E, Mahajan S, Mehrotra S, et al. Deletion of Pim kinases elevates the cellular levels of reactive oxygen species and sensitizes to K-Ras-induced cell killing. Oncogene 2015;34(28):3728–36. 56. Herz J, Strickland DK. LRP: a multifunctional scavenger and signaling receptor. J Clin Invest 2001;108(6):779–84. 57. Athauda SB, Nishigai M, Arakawa H, Ikai A, Ukai M, Takahashi K. Inhibition of human pepsin and gastricsin by alpha2-macroglobulin. J Enzyme Inhib Med Chem 2003;18(3):219–24. 58. Lillig CH, Holmgren A. Thioredoxin and related molecules—from biology to health and disease. Antioxid Redox Signal 2007;9(1):25–47. 59. Sandalova T, Zhong L, Lindqvist Y, Holmgren A, Schneider G. Three-dimensional structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a selenocysteine-dependent enzyme. Proc Natl Acad Sci U S A 2001;98(17):9533–8.

Chapter 30

Antioxidative stress actions of cocoa in colonic cancer: Revisited Sonia Ramos, Luis Goya, and Maria Angeles Martı´n Department of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Ciudad Universitaria, Madrid, Spain

List of abbreviations ACF AKT AOM AP-1 ARE CAT CDK COX CRC DOC EC ERK GPx GR GSH GST HO-1 IL iNOS JNK Keap1 LDH MAPK NF-kB Nrf2 PB2 PGE2 PI3K PPAR ROS SOD t-BOOH TNFa g-GCS

aberrant crypt foci protein kinase B azoxymethane activator protein-1 antioxidant response element catalase cyclin-dependent kinase cyclooxygenase colorectal cancer deoxycholic (
)-epicatechin extracellular regulated kinase glutathione peroxidase glutathione reductase glutathione glutathione-S-transferase heme oxygenase-1 interleukin inducible nitric oxide synthase c-Jun N-terminal kinase Kelch-like ECH associating protein-1 lactate dehydrogenase mitogen-activated protein kinase nuclear factor kappa B nuclear-factor-E2-related factor 2 procyanidin B2 prostaglandins E2 PI-3-kinase, phosphatidylinositol-3-kinase poly-(ADP-ribose) polymerase reactive oxygen species superoxide dismutase tert-butyl hydroperoxide tumor necrosis factor a gamma-glutamyl cysteine synthase

Introduction Colorectal cancer (CRC) is one of the major causes of cancer-related mortality in the world.1 Indeed, CRC is the third most common cancer in the world, ranked after lung and breast cancer, and it is the second most common cause of cancer death.2 Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00030-4 © 2021 Elsevier Inc. All rights reserved.

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In 2018, 1.8 million cases of CRC have been detected and found to be responsible for 862,000 deaths.2 Environmental factors, including dietary and lifestyle, play a crucial role in their etiology even though it is also attributable to inherited and acquired genetic alterations.3 Cancer is a multistage process conventionally defined by the initiation, promotion, and progression stages. In particular, development of CRC typically follows several consecutive steps from normal epithelial cells via aberrant crypts and progressive adenoma stages to carcinomas in situ and then metastasis. Along this process, oxidative stress has the potential to affect a large array of carcinogenic pathways involved in the proliferation of initiated cells and enhanced malignant transformation.4 In fact, the gastrointestinal tract, especially the colon, is constantly exposed to reactive oxygen species (ROS), generated during normal cellular metabolism and pathological processes.5 ROS overproduction may provoke structure and function damages in colonic cells and induce somatic mutations and neoplastic transformation.4 Because of this, the suppression of oxidative stress by natural antioxidant compounds has gained interest as an effective approach in CRC prevention. Chemoprevention, defined as the use of natural or synthetic compounds to prevent, block, or reverse the development of cancers seems to be an attractive option in this field and the possible impact of several nutritional agents with antioxidant and anti-inflammatory properties has been intensively studied in recent years.6 Accordingly, cocoa and their natural flavonoid compounds have shown a potential ability to act as a highly effective antioxidant and chemopreventive agents.7 Flavanols are polyphenolic compounds extensively found in vegetables, fruits, and plant-derived beverages that present a potent antioxidant activity.8 Cocoa, the dried and fermented seeds derived from Theobroma cacao, has the highest flavanol content of all foods on a per-weight basis and is a significant contributor to the total dietary intake of flavonoids.9 Actually, for many individuals, cocoa products constitute a larger proportion of the diet than foodstuffs containing bioactive compounds with similar properties such as green tea, wine, or soybeans.10 Cocoa flavanols are powerful antioxidant agents acting directly as ROS scavengers, metal ions chelators, and free radical reaction terminators and indirectly by stimulating phase II detoxifying and antioxidant defense enzymes.11 Additionally, polyphenolic compounds can exhibit other anticarcinogenic properties independently of their conventional antioxidant activity.12 Based on these findings, cocoa polyphenols could be considered as promising candidates for colon cancer chemoprevention. Nevertheless, health effects derived from cocoa flavonoids depend on their bioavailability (absorption, distribution, metabolism, and elimination), a factor which is also influenced by their chemical structure.13 In this regard, cocoa contains high amounts of flavanols (
)-epicatechin (EC), (+)-catechin and their dimers procyanidins B2 (PB2) and B1 (Fig. 1), FIG. 1 Main flavonoids present in cocoa. Chemical structures of (
)-epicatechin and (+)catechin and their respective dimmers procyanidins B2 and B1.

OH OH

OH O

HO

O

HO

OH OH

OH

OH

OH

(+) Catechin

(–) Epicatechin

OH OH

OH HO

O

HO

O

OH OH

OH

OH

OH OH

OH

OH HO

O OH

H

O

HO

OH

OH

Procyanidin B1

OH OH

Procyanidin B2

Antioxidant effects of cocoa in colon cancer Chapter

30 339

although other polyphenols such as quercetin, isoquercitrin (quercetin 3-O-glucoside), quercetin 3-O-arabinose, hyperoside (quercetin 3-O-galactoside), naringenin, luteolin, and apigenin have also been found in minor quantities.14 Interestingly, as compared to other flavonoid-containing foodstuffs, cocoa products exhibit a high concentration of procyanidins that are poorly absorbed in the intestine and consequently their beneficial effects would be restricted to the gastrointestinal tract where they may have an important antioxidant and anticarcinogenic local function.15 In general, the evidence for chemoprevention by any bioactive substance is achieved from a combination of epidemiological, animal, and basic mechanistic studies. In view of that, the mode of action of cocoa and their flavonols has been recently investigated, especially in cell culture systems. However, it remains to be demonstrated whether these mechanisms are involved in cancer prevention in humans. In this chapter, we reviewed the different in vitro studies that have identified the potential targets and mechanisms whereby cocoa and their polyphenolic compounds could interfere with colonic cancer cells. Afterward, we showed the potential antioxidant and chemopreventive activity of cocoa in an animal model of colon cancer. Finally, some evidence from human studies are also illustrated.

Chemopreventive mechanism of cocoa polyphenols in cultured colon cancer cells In the recent past years, cocoa and their polyphenolic compounds have been widely studied for their actions against colon cancer cells and related molecular mechanisms (Table 1). All these studies have shown that the pathways responsible for the potential chemopreventive activity of cocoa and its flavonoids are mainly related to their antioxidant and anti-inflammatory properties and their ability to inhibit proliferation and to induce apoptotic cell death (Fig. 2).

TABLE 1 Effects of cocoa and cocoa polyphenols on colonic cancer cultured cellsa. Polyphenol

Result

Reference

Antioxidant

Cocoa Hexamer procyanidins Procyanidin B2 Epicatechin Catechin

# Acrylamide-induced GSH depletion, # ROS generation, " g-GCS, " GST # DOC-induced cytotoxicity, # oxidant generation, # NADPH oxidase, # Ca2+ # Acrylamide-induced GSH depletion, # ROS generation, " g-GCS, " GST # GPx, GST, GR, and Nrf2 translocation, # t-BOOH-induced ROS production and LDH ¼ GPx, GST and GR, and Nrf2 translocation, # t-BOOH-induced ROS production and LDH # Lipid peroxidation, # ROS formation, " GPx, " GR, " Nrf2, " HO-1

[16] [17, 18] [16] [19, 20] [19] [21]

Cell cycle

Polymer procyanidins Epicatechin

G2/M arrest, # ornithine decarboxylase, # S-adenosylmethionine decarboxylase S arrest

[22] [23]

Apoptosis

Hexamer procyanidins Procyanidin B2 Epicatechin

# DOC-induced caspase-3, # PPAR cleavage " Bad, " caspase-9, " caspase-3, # cytochrome c # t-BOOH-induced caspase-3 # t-BOOH-induced caspase-3

[17] [24] [19] [19]

Proliferation/ survival

Cocoa Hexamer procyanidins Procyanidin B2 Epicatechin

# Acrylamide-induced p-JNK # DOC-induced AKT, ERK, p38, and AP-1 # p-AKT, # p-p85-PI3K, # p-GSK3 " ERK, " p38 ¼ " proliferation, ¼ " p-AKT, ¼ " p-ERK ¼ proliferation, ¼ p-AKT, ¼ p-ERK

[16] [17] [24] [20] [25] [25]

Antiinflammatory

Cacao Hexamer procyanidins

# PGE2, # IL-lb, ¼IL-8, ¼ NF-kB, " COX-1 # TNF-induced IL-S. COX-2. Inos, and NF-kB activation # TNF-induced NF-kB activation and iNOS # TNF-induced IL-8, improve membrane integrity

[26] [27] [28] [29]

a The arrow indicate an increase (") or decrease (#) in the levels or activity of the different analyzed parameters. In certain cases, opposing results have been obtained since the studies were earned out in different colonic cell types and/or the final effects may depend on the dose and time of treatment with the phenolic compound.

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FIG. 2 Mechanisms involved in the potential chemopreventive effects of cocoa and its flavonoids against colorectal cancer. The arrows indicate an increase (") or decrease (#) in the levels or activity of the different analyzed parameters.

Initiation

Promotion

Progression

Normal tissue Hyperproliferation Antimutagenic effects

Adenoma Apoptotic and antiproliferative effects

Carcinoma Antiangiogenesis Antimetastasis

Antioxidative mechanisms Cell cycle arrest Radical scavenging ↓ Polyamine metabolism ↑ No-enzymatic antioxidant defenses ↓ Survival/proliferation (↓ MAPKs and AKT) ↑ Phase II enzyme activity (ARE) Antiinflammatory effects ↓ NF-κB ↓ COX-2 ↓ iNOS ↓ Interleukins and prostaglandins

Antioxidant effects Aerobic organisms cannot avoid free radical and reactive oxygen species (ROS) generation. Overproduction of ROS may lead to the formation of highly reactive oxidation products, activation of carcinogens, and formation of oxidized DNA bases and DNA strand breaks. These alterations might cause mistakes during DNA replication and genetic alterations, increase transformation frequencies, modulate transcription of redox-regulated proteins, ultimately leading to enhanced cell proliferation and tumor promotion/progression.12 In a physiological situation, cells maintain the balance between generation and neutralization of ROS through the enzymatic and nonenzymatic defenses, such as glutathione (GSH), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), nitric-oxide synthase, lipooxygenase, xanthine oxidase, etc. However, when the cellular balance is altered and cellular defenses overwhelmed, cells can be damaged as mentioned above. Cocoa and its flavonoids exert strong antioxidant effects. Thus, cocoa possesses a potent antioxidant capacity as compared with other foods or products, such as teas and red wine, and this property has been related to its flavonoid content.30 Interestingly, the antioxidant properties of cocoa and its flavonoids are partly based on their structural characteristics, including the hydroxylation of the basic flavan-ring system, especially 30 ,40 -dihydroxylation of the B-ring (catechol structure), the oligomer chain length, and the stereochemical features of the molecule.31 These structural characteristics of flavanols represent the molecular basis for their hydrogen- donating (radical-scavenging) properties and their metalchelating antioxidant properties. In addition, cocoa and its flavonoids can prevent the DNA damage caused by free radicals or carcinogenic agents acting through the modulation of enzymes related to oxidative stress (CAT, GR, GPx, SOD, etc.) and the alteration of the procarcinogenic metabolism by inhibiting phase-I drug-metabolizing enzymes (cytochrome P450) or activating phase II conjugating-enzymes (glucuronidation, sulfation, acetylation, methylation, and conjugation).

Protective effects Prevention of ROS generation and the preservation of the cellular antioxidant defenses seem to represent an important mechanism of the chemoprevention of natural polyphenols.12 In this line, intestinal Caco-2 cells pretreated with a cocoa phenolic extract or with the pure cocoa flavanols (
)-epicatechin (EC) and procyanidin B2 (PB2) at physiological concentrations (i.e., 10 mg/mL for cocoa phenolic extract and 10 mM for EC and PB2, respectively) for 20 h counteracted acrylamide-induced cytotoxicity (5 mM for 24 h) by inhibiting GSH consumption and ROS generation.16 Both cocoa phenolic extract and PB2 almost completely blocked the decrease of GSH induced by acrylamide and totally abrogated the subsequently increased ROS generation, whereas these effects were only partially restored with EC. This result suggests that the minor effect exerted by EC could be partially ascribed to the fact that EC mainly acted as a scavenger of free

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30 341

radicals. However, similar to what was reported for other polyphenols and antioxidants,12 PB2 and a cocoa phenolic extract could protect cell constituents not only by neutralizing several types of radicals but also by upregulating antioxidant defenses as well as by interacting with signaling pathways involved in cell survival. Thus, PB2 and the cocoa phenolic extract increased the levels of gamma-glutamyl cysteine synthase and glutathione-S-transferase (GST) in the mentioned experimental conditions Caco-2 cells.16 Pure PB2 and EC (1–10 mM) decreased ROS production but did not affect GSH content in Caco-2 cells, and PB2 (1–10 mM), evoked a substantial increase in GPx, GR, and GST activity after 20 h of incubation.19 Thus, pretreatment of Caco2 cells with EC and PB2 for 20 h before the oxidative insult induced by the potent prooxidant tert-butylhydroperoxide (t-BOOH at 400 mM) attenuated or blunted ROS production, respectively. In addition, EC and PB2 protected cells from necrosis, as lactate dehydrogenase (LDH) leakage decreased after 1 and up to 6 h of incubation with the prooxidant, respectively.19 All together suggests that at least two mechanisms could be involved in the protection of Caco2 cells afforded by flavanols: (1) the inherent antioxidant capacity to quench ROS and (2) the improvement of the endogenous antioxidant defenses.

Effects on phase I and II enzymes Enzymes of the phase I of drug metabolism (cytochromes P450) transform xenobiotics by adding functional groups which render these compounds more water-soluble. Phase I functionalization may be required to efficiently detoxify carcinogens.12 Phase II enzymes such as GST and sulfotransferases conjugate transformed phase I metabolites and xenobiotics to endogenous ligands like GSH, glucuronic, acetic, or sulfuric acid and enhance excretion and detoxification in form of these conjugates.12 Therefore, reduction of elevated phase I enzyme activities to physiological levels and enhanced excretion of carcinogens via upregulation of phase II enzymes are considered a strategy in chemoprevention. In addition, the transcription factor NF-E2-related factor-2 (Nrf2) and the Kelch-like ECH-associated protein 1 (Keap1) are considered as chemopreventive targets because both proteins participate in the regulation of the antioxidant response element (ARE). Thus, the modification of the protein Keap1 can lead to the accumulation of Nrf2 in the nucleus and the subsequent ARE activation.12 Cocoa and its phenolic compounds also exert their protective effect toward oxidative stress through the modulation of phase I and II enzyme activities and Nrf2. Accordingly, catechin (100 mM) increased the expression of Nrf2 and heme oxygenase-1 in a time-dependent manner in intestinal Int-407 cells.21 PB2 (1–10 mM) alone also evoked a dose-dependent increase in GPx, GR, and GST after incubating Caco-2 cells for 20 h, which could be related to an improved cell response to an oxidative challenge.19 Hence, cells treated with 10 mM PB2 for 20 h, and then submitted to oxidative stress induced by tBOOH (400 mM, for 1 or 6 h, respectively) showed a reduced ROS production, restricted activation of caspase 3 and higher viability than cells plainly submitted to the stressor.19 Furthermore, PB2 (10 mM, for 20 h) showed a protective effect against the oxidative injury induced by 400 mM t-BOOH in Caco-2 cells through the upregulation of the expression and activity of GST P1 via a mechanism that involved extracellular regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activation and Nrf2 translocation.20 Thus, PB2 treatment increased the protein levels of Nrf2 in the nucleus at 3 h, peaked at 6 h and continued elevated up to 20 h of treatment. Accordingly, this procyanidin significantly enhanced the mRNA levels and activity of GST P1 at 4–20 h of incubation, which was accompanied by an increment in the levels of protein expression at 8 and 20 h.20 Similarly, cocoa procyanidins protected Caco-2 cells from the loss of integrity induced by a lipophilic oxidant.17, 18 Interestingly, a hexameric procyanidin fraction isolated from cocoa (2.5–20 mM) interacted with the Caco-2 cell membranes preferentially at the water-lipid interface without affecting their integrity after 30 min of incubation. Moreover, the hexameric procyanidin fraction inhibited the deoxycholic (DOC)-induced cytotoxicity and partly prevented the oxidant generation following NADPH oxidase inhibition, as well as DOC-triggered increase in cellular calcium.17, 18 The limited effects on LDH release observed after 6 h of incubation for lower molecular weight procyanidins, i.e., monomer-tetramer, stress the relevance of the membrane-related effects of larger procyanidins. These differential actions have to be explained as a compromise between the incorporation of the compounds into the cells that decreases as procyanidin oligomerization increases and the adsorption to the cell surface that increases as procyanidins oligomerization increases.

Effects on apoptosis and proliferation Apoptosis and proliferation in cells are also modulated by ROS generation.12 Indeed, suppression of cell proliferation, as well as induction of differentiation and apoptosis are important approaches in cancer chemoprevention.

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Cell cycle Deregulated cell cycle and resistance to apoptosis are hallmarks of cancer.12 Cell cycle control is a highly regulated process that involves the modulation of different cell cycle regulatory proteins, such as cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, etc.12 ROS generation could induce an alteration of cell cycle-specific proteins that can affect and/or block the continuous proliferation of cancer cells. Procyanidin-enriched extracts and procyanidins inhibited Caco-2 cell growth.22 After 48 h of incubation, procyanidins extracts with a flavanol and procyanidin content of 501 mg/g, caused only 25% growth inhibition, whereas the procyanidinenriched extracts (flavanol and procyanidin content: 941 mg/g) induced a 75% growth inhibition. On the contrary, cocoa powder samples, which consisted of a flavanol and procyanidin content of 141 mg/g, showed no growth inhibitory effects in Caco-2 cells. Moreover, 50 mg/mL procyanidin-enriched extracts blocked the cell cycle at G2/M phase, without inducing apoptosis, and decreased the polyamine metabolism by inhibiting the ornithine decarboxylase and S-adenosylmethionine decarboxylase activities, which has partly been related to the accumulation of cells at the G2/M phase.22 Cocoa procyanidin hexamers (2.5–50 mM for 24–72 h) also decreased cell viability in Caco-2, HCT15, HT29, HCT116, SW480, and LoVo cells in a dose-dependent manner, showing a more prominent effect than EC or cocoa procyanidin hexamers at the same concentrations.24 Thus, cocoa procyanidin hexamers (10–30 mM, 72 h) arrested the cell cycle of Caco-2 in G2/M and induced apoptosis.24 Interestingly, hexamers did not affect the cell viability of differentiated intestinal Caco-2 cells, which highlight the different effect of the same compound depending on the proliferating state of the colon cell, being this aspect crucial in CRC prevention.24 Similarly, incubation of LoVo cancer cells with 690 and 1380 mg/mL EC for 24 h induced S phase arrest in the cell cycle progression but it did not induce apoptosis.23 Importantly, lower concentrations of EC seemed to promote a slight proliferation of LoVo cells.

Apoptosis ROS generation has been described as a critical upstream activator of the development of apoptosis.12 At molecular level, it has been widely reported the existence of two mechanisms for the activation of the programmed cell death: (1) the extrinsic pathway, which is mediated by death receptors, and (2) the intrinsic mechanism (mitochondria-mediated), that is, regulated by pro- and antiapoptotic proteins of the Bcl-2 family. Both cascades converge in a common executor mechanism involving DNA endonucleases that activate proteases (caspases) and lead to cellular death.12 Treatment of cells with natural antioxidants prevents the cytotoxicity induced by oxidative stress inducers through the ability of these compounds to restrain the increase in ROS levels and the subsequent activation of caspase-3 which leads to apoptosis induction.12 Consistent with the above, after 20 h of incubation EC or PB2 (10 mM) effectively reduced the apoptotic effects induced by t-BOOH (400 mM for 4 h) in Caco-2 cells.19 Similarly, pretreatment with 10 mM hexameric procyanidins for 30 min also delayed the DOC-induced Caco-2 cell apoptosis, as restrained caspase-3 activation, given that poly-(ADP-ribose) polymerase cleavage was observed only after 6 h incubation.17 Nevertheless, in Caco-2 cells cocoa procyanidin hexamers (20 mM, 24 h) induced apoptosis, as increased mitochondrial Bad, caspase-3 and -9 activities, and decreased mitochondrial cytochrome c levels.24

Proliferation/survival Phosphatidyl-inositol-3-kinase (PI3K)/protein kinase B (AKT), growth factor receptors/Ras/mitogen-activated protein kinases, and nuclear factor kappa B (NF-kB), which also importantly contribute to the inflammatory process (see below), constitute the most important signaling pathways regulating cell proliferation and survival.12 Cocoa phenolic compounds can interact with signaling proteins and modulate their activity. In this line, pretreatment with 10 mM hexameric procyanidins (30 min) prevented oncogenic events initiated by DOC through the interaction with Caco-2 cell membranes and inhibited the DOC-promoted activation of AKT, ERK, and p38, as well as the downstream transcription factor activator protein-1 (AP-1).17 Conversely, cocoa procyanidin hexamers (10–40 mM for 24 h) inhibited the PI3K/AKT pathway, as well as GSK-3 levels in Caco-2, which was associated with the repression of proliferative/survival routes and the induction of apoptosis.24 Interestingly, PB2 and EC (10–50 mM) did not have an obvious effect on Caco-2 and SW480 colon carcinoma cells after 24 h of incubation. However, PB2 promoted cell growth in SW480 cells by increasing p-AKT and p-ERK levels25 and activated Nrf2 translocation and increased both GST P1 protein and activity, via ERK and p38 pathways, which were also essential routes for the cytoprotective effect exerted by the flavanol in Caco-2 cells.20 This different response depending on the distinct chemical structure of the compound and the different degree of cell differentiation highlights the importance of an integrated approach to study the biological effects of phytochemicals.25

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30 343

Antiinflammatory effects The manifestation of oxidative stress by infections, immune diseases, and chronic inflammation has been associated with carcinogenesis.12 Thus, chronic inflammation is a risk for colorectal cancer,12 and most inflammation-associated colorectal cancers are characterized by the activation of the transcription factor NF-kB and inflammatory mediators such as tumor necrosis factor a (TNFa), cyclooxygenase-(COX)-2, etc., being all these proteins also related to cell proliferation, antiapoptotic activity, angiogenesis, and metastasis.12 The cocoa extract inhibited the inflammatory mediator prostaglandins E2 (PGE2) in human intestinal Caco-2 cells.26 Thus, cells incubated with a polyphenolic extract of cocoa (equivalent to 50 mM of gallic acid) for 4 h and stimulated with interleukin-(IL)-1b for 24 or 48 h showed a decrease in PGE2 synthesis, whereas IL-8 secretion and NF-kB activity remained at high levels.26 Surprisingly, in the absence of pro-inflammatory stimulus, the cocoa polyphenolic extract induced a basal PGE2 synthesis in Caco-2 cells after 24 h of incubation. This effect has been associated with induction of COX-1, which seems to be implicated in maintaining the mucosal integrity.26 More recently, pretreatment with a cocoa phenolic extract, at a physiological concentration (10 mg/mL) for 20 h, reduced the increase in inflammatory markers such as IL-8 secretion, COX-2, and inducible nitric oxide synthase (iNOS) expression induced by the pro-inflammatory agent TNFa (40 ng/mL for 24 h) in Caco-2 cells.27 In this work, cocoa phenolic extract selectively decreased both phosphorylated levels of c-Jun N-terminal kinase and nuclear translocation of NK-kB induced by TNFa, indicating that this pathway could be an important mechanism contributing to the reduction of intestinal inflammation. EC and procyanidins can inhibit NF-kB at different levels in the activation pathway. A decrease in cell oxidants that are involved in NF-kB activation is a potential mechanism of modulation by these compounds. Thus, incubation of Caco-2 cells for 30 min with 2.5–20 mM hexameric procyanidins, prior to treatment with 10 ng/mL TNFa for further 5–30 min inhibited the TNFa-induced NF-kB activation (inhibitor of kB phosphorylation and degradation, p50 and RelA nuclear translocation, and NF-kB–DNA binding), iNOS expression, and cell oxidant increase.28 These effects have been suggested to occur because hexameric procyanidins can inhibit NF-kB activation by interacting with the plasma membrane of intestinal cells, and through these interactions preferentially inhibits the binding of TNFa to its receptor and the subsequent NFkB activation.28 In addition, in HT-29 cells pretreatment with cocoa-high-molecular-weight polymeric procyanidins fractions (7 polymerization degree, 10–25 mg/mL for 24 h) were more effective than cocoa monomers or oligomerprocyanidin rich fractions to preserve the membrane integrity and reduce the levels of IL-8 in response to pro-inflammatory conditions (5 ng/mL TNFa for 6 h).29

Chemopreventive mechanism of cocoa in animal models of colon cancer Studies with colonic cell culture model have clearly demonstrated the antioxidant and chemopreventive abilities of cocoa and its flavonoids, but only experimental models for colorectal cancer could offer the opportunity to assess the contribution of this natural dietary compound to the potential prevention of CRC. To this end, carcinogen-induced rodent models have been shown to mimic many features of human non-familial colorectal cancer (nongenetic based),32 which is the most frequent and occurs sporadically. The induction of colon tumors is achieved by the administration of carcinogens such as nitrosamines, heterocyclic amines, aromatic amines, 1,2-dimethylhydrazine and azoxymethane (AOM). In particular, administration of AOM to rodents induces the development of colonic preneoplastic lesions (aberrant crypt foci, ACF) that may progress into cancer with time.33 ACF represent the earliest identifiable intermediate precancerous lesions during colon carcinogenesis in both laboratory animals and humans34 and can be identified microscopically on the surface of the colon mucosa after methylene blue staining (Fig. 3). FIG. 3 Mucosal surface of colon from control and azoxymethane (AOM)-injected rats. Mucosal of colon were stained with methylene blue and observed under light microscope (40 magnification). The presence of aberrant crypt foci (ACF) is indicated by arrows.

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B Oxidative stress and cancer

TABLE 2 Effect of dietary cocoa on aberrant crypt foci (ACF) formation in azoxymethane (AOM)-treated rats. Crypts multiplicity of ACF ACF formation

ACF/cm

1 Crypt

2 Crypts

3 Crypts

>4 Crypts

Control

0/8

0

0

0

0

0

Cocoa

0/8

0

Control + AOM

12/12

16.1  6.2

Cocoa + AOM

12/12

8.8  2.5b

2

0 a

0

0

0

a

6.3  l.9

5.8  2.8

3.0  l.4

0.87  0.23a

3.5  l.0b

3.7  l.5a

1.3  0.6b

0.14  0.05b

a

a

Values are means  SD. Means in a column without a common letter differ, P < .05.

Contrary to the strong evidence for the antioxidant and cancer preventive activity of cocoa and their components in cultured cells, there are only a few studies in rats that have demonstrated the potential chemopreventive ability of cocoa on colon carcinogenesis.35–37 In one of the studies, male Wistar rats were fed with a cocoa-enriched diet (12%) starting 2 weeks before the carcinogenic induction and throughout the experimental period (8 weeks).35 As expected, all the rats that were injected with AOM developed colonic preneoplastic lesions (aberrant crypts foci, ACF). Nevertheless, the cocoaenriched diet significantly reduced the AOM-induced ACF formation and especially those ACF with a larger number of crypts (4 crypts) which exhibit a higher tendency to progress into malignancy (Table 2). Therefore, this study showed for the first time that a cocoa-enriched diet was able to suppress the early phase of chemically induced colon carcinogenesis. More recently, two new studies using the azoxymethane/dextran sulfate sodium model in BALB/c mice have also described the antitumor effects of a 5% and 10% cocoa-rich diet on colitis-associated cancer (CAC).36, 37 The most relevant in vivo mechanisms involved in the chemopreventive effects elicited by cocoa are briefly described below. These mechanisms included the prevention of oxidative stress, cell proliferation and cell inflammation, and the ability of cocoa diet to induce cell apoptosis (Fig. 2).

Cocoa prevented AOM-induced oxidative stress in colon tissues The suppressive effect of cocoa on AOM-induced preneoplastic lesions has been associated with its antioxidative properties. AOM is metabolized in the liver to a methyl-free radical who in turn generates hydroxyl radical or hydrogen peroxide capable of oxidized DNA, RNA, lipids, or protein of colonic epithelial cells.38 As a consequence, the levels of lipid and protein peroxidation, indicative of oxidative injury, increased in the colon of animals treated with AOM.39 However, in animals fed with 12% cocoa-diet, the increased levels of protein and lipid oxidative damage induced by AOM were strongly prevented, demonstrating that cocoa possesses a potent antioxidative effect in vivo on the stressed colonic tissue.35 In particular, cocoa feeding was able to avoid oxidative stress by reverting to control values the diminished levels of GSH and the activities of GPx, GR, and GST provoked by the toxicant. Similar results were found by Panduragan et al.36 in an animal model of AOM/DSS-induced colitis-associated cancer (CAC). Furthermore, they also demonstrated that cocoa upregulated the expression of Nrf2 and its downstream targets protecting colon tissues from oxidative damage during colorectal carcinogenesis. Since Nrf2 and its antioxidant enzymes participate in the detoxification of xenobiotics, carcinogens, free radicals, and peroxides,40 it can be suggested that cocoa could prevent ACF formation by reinforcing the endogenous defense capacity in colon tissues to counteract carcinogen-induced toxicity. Consequently, the increased cellular defense in the colon of cocoa-fed animals treated with AOM seems to be an effective strategy to protect against carcinogen-induced toxicity and largely accounts for the chemoprotective activity of cocoa.

Cocoa prevented cell proliferation in AOM treated animals Besides inducing oxidative damage and genomic instability, ROS can specifically activate certain redox-sensitive signaling pathways and contribute to CRC initiation/promotion through the regulation of cellular proliferation and survival.41 Among these, PI3K/AKT and ERK/MAPKs are within the most important pathways activated in response to oxidative stress and play important roles in the carcinogenesis of many types of cancers including colon cancer.4 Accordingly, AOM treatment clearly elevated the proliferative activity of the colonic mucosa and this increase was accompanied by the phosphorylation of AKT and ERKs and the over-expression of cyclin D1, a preneoplastic marker involved in cell cycle progression.35

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However, cocoa intake prevented all these processes induced by AOM, suggesting that cocoa, by its ability to restrain oxidative stress could also inhibit the consequent activation of signaling pathways involved in proliferation and thereby the progression of preneoplasia in the colonic epithelial cells. Supporting this, a recent in vitro study has shown that flavonoids such as luteolin and quercetin have antiproliferative and proapoptotic effects in human CRC cells through the regulation of the ERK/MAPK and the PI3K pathways.42 In the same line, the expression of PCNA was increased in the colon of AOM/ DSS-induced CAC mice model while cocoa was able to reduce cell proliferation through the inactivation of p-STAT3.37

Cocoa prevented AOM-induced inflammation in colon tissues In recent years, considerable evidence has demonstrated that ROS are also involved in the link between chronic inflammation and cancer.4 Redox status has an impact on the transcription factor NF-kB which regulates the expression of the pro-inflammatory enzymes COX-2 and iNOS. Both enzymes are implicated in chronic inflammation causing a microenvironment that contributes to the development of preneoplastic lesions in the colon carcinogenesis.43 In fact, iNOS and COX-2 have been found to be increased in human CRC and AOM-induced rat colon carcinogenesis.44 A cocoa-rich diet was able to suppress the intestinal inflammation induced by AOM through the inhibition of NF-kB signaling and the downregulation of the pro-inflammatory enzyme expressions of COX-2 and iNOS.27 iNOS expression is frequently observed in dysplastic, but not in hyperplastic, ACF indicating that iNOS plays an important role in the early stages of tumor formation.45 On the other hand, tumorigenic mechanisms of COX-2 include inhibition of apoptosis via increased Bcl-2 and activation of proliferation via MAPK or PI3K/AKT signaling pathways.46 Thus, the effect of a cocoa-rich diet preventing iNOS and COX-2 expression induced by AOM seems to be related to the inhibition of ACF formation observed in the AOM group treated with cocoa. Besides, cocoa also was able to inhibit the NF-kB signaling pathways and suppress the expressions of pro-inflammatory cytokines during the early stage of colitis.37

Cocoa-induced apoptosis in AOM-treated animals During the promotion/progression phase of carcinogenesis, apoptosis is the main biological event involved in the removal of the initiated/mutated colonic epithelial cells.4 In fact, many natural dietary compounds have been shown to suppress ACF formation by increasing apoptosis.6 Accordingly, cocoa supplementation clearly induced apoptosis in the colon tissue of AOM-treated rats35 and the colon of AOM/DSS-induced mice.37 Indeed, cocoa was able to modulate the expression of proand antiapoptotic proteins (Bax and Bcl-xL, respectively) and to provoke caspase-3 activation, suggesting that cocoa induces apoptosis with the participation of the mitochondrial pathway. These data are in agreement with other results illustrating the apoptotic effect as the major mechanism for chemoprevention of different polyphenolic plant constituents.12 Consequently, the proapoptotic in vivo effect of cocoa seems to be a complementary mechanism both to reduce preneoplastic lesions induced by AOM and to prevent promotion/progression of carcinogenesis, playing thus an important role in its anticarcinogenic potential.

Human studies There is increasing evidence to support an inverse association between the intake of dietary fruit and vegetables and several cancers, including colorectal cancer.47, 48 Although the anticarcinogenic mechanism of these foods is unclear, the presence of dietary polyphenols, in particular flavonoids, maybe one of the reasons. Most studies carried out in cell cultures and experimental animal have supported the chemopreventive efficiency of cocoa polyphenols in colorectal cancer, but the outcome of epidemiologic studies on cocoa intake and risk of colon cancer is not conclusive.

Epidemiologic studies The first report in support of a potential association between flavanol/catechin intake and colorectal cancer was published in 2002 with data obtained from the Iowa Women’s Health Study (United States), where a cohort of 34,651 postmenopausal cancer-free women aged 55–69 years was followed from 1986 to 1998. Among several cancers studied, data suggested that catechin intake may protect against rectal cancer, furthermore, catechins derived primarily from fruits tended to be inversely associated with upper digestive tract cancer, whereas catechins derived from tea were inversely associated with rectal cancer.49 The Kuna tribe in Panama has been widely regarded as the most significant example in support of the preventive effect of cocoa intake in cancer. The population of this tribe inhabits the San Blas district of Panama and has cocoa as their main beverage.50 When death certificates from year 2000 to 2004 were surveyed in order to compare cause-specific

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death rates between mainland Panama and the San Blas islands, the rate of cardiovascular disease and different types of cancer, including colorectal, among island-dwelling Kuna was much lower than in mainland Panama. This natural protection was rapidly associated with the elevated intake of cocoa flavanols, which intake probably exceeds 900 mg/day, a figure that represents the highest flavonoid-rich diet of any world population.50 Just within the last 3 years, a number of epidemiological studies and meta-analysis have failed to show a protective effect of flavanols from cocoa or other sources on colon and rectal cancer. Thus, during up to 26 years of follow-up, 2519 colorectal cancer cases Nimptsch and colleagues found no support for the hypothesis that a higher habitual intake of any flavonoid subclass decreases the risk of colorectal cancer.51 Data from the European Prospective Investigation into Cancer and Nutrition (EPIC) study, which included 476,160 men and women from 10 European countries during a mean follow-up of 14 years, found that intake of flavanols was not related to colorectal, colon, or rectal cancer risks.52 Also in 2018, a massive meta-analysis performed by Chang and coworkers found no association between flavanol intake and risk of colorectal cancer, although they found an inverse association with other flavonoid subtypes.47 Considering specifically cocoa and/or chocolate flavanols, in a pioneer case–control study in Burgundy (France), chocolate was identified as a risk factor for colorectal cancer.53 An early study in North Carolina failed to detect a significantly lower prevalence of adenomatous polyps and colorectal cancer with chocolate consumption.54 In the same line, a French study from 2005 showed no significant association between a high chocolate dietary pattern and any stage of colorectal disease ranging from polyps to adenomas and colorectal cancer.55 Finally, early this year Morze et al. have reported that chocolate consumption is not related to risk for colorectal cancer.56 It is worth mentioning that any type of chocolate was included in all the above meta-analysis.

Intervention studies There are properly no human intervention studies attempting to show a correlation between cocoa intake and cancer prevention, but a few human intervention trials indicate that cocoa favorably affects intermediary factors in cancer progression.7 In this regard, several recent studies have focused on the modulation of antioxidant and anti-inflammatory status by consumption of cocoa products. In a study by Spadafranca et al.57 dark chocolate consumption significantly improved DNA resistance to oxidative stress. In this study, healthy subjects were assigned to a daily intake of 45 g of dark chocolate or white chocolate for 14 days and oxidative damage to mononuclear blood cells DNA was reduced in the dark chocolate group 2 h after consumption; 22 h later the effect disappeared. Similarly, cocoa consumption reduced NF-kB activation in peripheral blood mononuclear cells in healthy voluntaries,58 but biomarkers of inflammation, including IL-6, were unaffected in patients at high risk of cardiovascular disease consuming cocoa powder.59

Summary points - Cocoa and its main phenolic compounds regulate cellular redox status and multiple signaling pathways associated with cell proliferation, differentiation, apoptosis, and inflammation. - Animal studies have established that cocoa and its main phenolic components might prevent and/or slow down the initiation-promotion of colon cancer. - In humans, interventional studies have described favorable changes in antioxidant biomarkers. - Daily intake of small quantities of cocoa or chocolate, which provide flavanols and procyanidins, in combination with a standard dietary consumption of flavonoids, would constitute a natural approach to prevent colon cancer with insignificant toxicity. - Carefulness is obligatory to extrapolate the in vivo cellular results to in vivo animal colon cancer models and, even more notably, to humans. - Cocoa molecular mechanisms of action remain unclear and further investigations are deserved. - More well-designed epidemiological and intervention studies are needed to demonstrate the potential colorectal cancer preventive activities of cocoa in humans.

Acknowledgments This work was supported by the grants AGL2015-67087-R and RTI2018-095059-B-I00 (MINECO/FEDER, UE).

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Conflict of interest The authors declare that there are no conflicts of interest.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

Siegel R, Naishadham D, Jemal A. Cancer statistics. CA Cancer J Clin 2012;62:10–29. WHO. Cancer fact sheet. WHO Media Centre; 2019. Available from http://www.who.int/mediacentre/factsheets/fs297/en/. Benson AB. Epidemiology, disease progression, and economic burden of colorectal cancer. J Manag Care Pharm 2007;13:S5–S18. Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med 2010;49:1603–16. Klaunig JE, Kamendulis LM. The role of oxidative stress in carcinogenesis. Annu Rev Pharmacol Toxicol 2004;44:239–67. Pan M-H, Lai C-S, Wu J-C, Ho C-T. Molecular mechanisms for chemoprevention of colorectal cancer by natural dietary compounds. Mol Nutr Food Res 2011;55:32–45. Maskarinec G. Cancer protective properties of cocoa: a review of the epidemiologic evidence. Nutr Cancer 2009;61:573–9. Scalbert A, Morand C, Manach C, Remesy C. Absorption and metabolism of polyphenols in the gut and impact on health. Biomed Pharmacother 2002;56:276–82. Lamuela-Ravento´s RM, Romero-Perez AI, Andres-Lacueva C, Tornero A. Health effects of cocoa flavonoids. Food Sci Technol Int 2005;11:159–76. Cooper KA, Donovan JL, Waterhouse AL, Williamson G. Cocoa and health: a decade of research. Br J Nutr 2008;99:1–11. Vinson JA, Proch J, Bose P, Muchler S, Taffera P, Shuta D, et al. Chocolate is a powerful ex vivo and in vivo antioxidant, an antiatherosclerotic agent in an animal model, and a significant contributor to antioxidants in the European and American Diets. J Agric Food Chem 2006;54:8071–6. Ramos S. Cancer chemoprevention and chemotherapy: dietary polyphenols and signalling pathways. Mol Nutr Food Res 2008;52:507–26. Manach C, Williamson G, Morand C, Scalbert A, Remesy C. Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr 2005;81(suppl):230S–242S. Sa´nchez-Rabaneda F, Ja´uregui O, Casals I, Andres-Lacueva C, Izquierdo-Pulido M, Lamuela-Ravento´s RM. Liquid chromatographic/electrospray ionization tandem mass spectrometric study of the phenolic composition of cocoa (Theobroma cacao). J Mass Spectrom 2003;38:35–42. Ramiro-Puig E, Castell M. Cocoa: antioxidant and immunomodulator. Br J Nutr 2009;101:931–40. Rodriguez-Ramiro I, Ramos S, Bravo L, Goya L, Martı´n MA. Procyanidin B2 and a cocoa polyphenolic extract inhibit acrylamide-induced apoptosis in human Caco-2 cells by preventing oxidative stress and activation of JNK pathway. J Nutr Biochem 2011;22:1186–94. Da Silva M, Jaggers GK, Verstraeten SV, Erlejman AG, Fraga CG, Oteiza PI. Large procyanidins prevent bile-acid-induced oxidant production and membrane-initiated ERK1/2, p38, and Akt activation in Caco-2 cells. Free Radic Biol Med 2012;52:151–9. Erlejman AG, Fraga CG, Oteiza PI. Procyanidins protect Caco-2 cells from bile acid- and oxidant-induced damage. Free Radic Biol Med 2006;41:1247–56. Rodriguez-Ramiro I, Martin MA, Ramos S, Bravo L, Goya L. Comparative effects of dietary flavanols on antioxidant defences and their response to oxidant-induced stress on Caco2 cells. Eur J Nutr 2011;50:313–22. Rodrıguez-Ramiro I, Ramos S, Bravo L, Goya L, Martı´n MA. Procyanidin B2 induces Nrf2 translocation and glutathione S-transferase P1 expression via ERKs and p38-MAPK pathways and protect human colonic cells against oxidative stress. Eur J Nutr 2011;51:881–92. Cheng YT, Wu CH, Ho CY, Yen GC. Catechin protects against ketoprofen-induced oxidative damage of the gastric mucosa by up-regulating Nrf2 in vitro and in vivo. J Nutr Biochem 2013;24:475–83. Carnesecchi S, Schneider Y, Lazarus SA, Coehlo D, Gosse F, Raul F. Flavanols and procyanidins of cocoa and chocolate inhibit growth and polyamine biosynthesis of human colonic cancer cells. Cancer Lett 2002;175:147–55. Tan X, Hu D, Li S, Han Y, Zhang Y, Zhou D. Differences of four catechins in cell cycle arrest and induction of apoptosis in LoVo cells. Cancer Lett 2000;158:1–6. Choy YY, Fraga M, Mackenzie GG, Waterhouse AL, Cremonini E, Oteiza PI. The PI3K/Akt pathway is involved in procyanidin-mediated suppression of human colorectal cancer cell growth. Mol Carcinog 2016;55:2196–209. Ramos S, Rodriguez-Ramiro I, Martin MA, Goya L, Bravo L. Dietary flavanols exert different effects on antioxidant defenses and apoptosis/proliferation in Caco-2 and SW480 colon cancer cells. Toxicol In Vitro 2011;25:1771–81. Romier-Crouzet B, Van De Walle J, During A, Joly A, Rousseau C, Henry O, et al. Inhibition of inflammatory mediators by polyphenolic plant extracts in human intestinal Caco-2 cells. Food Chem Toxicol 2009;47:1221–30. Rodrıiguez-Ramiro I, Ramos S, Lopez-Oliva E, Agis-Torres A, Bravo L, Goya L, et al. Cocoa polyphenols prevent inflammation in the colon of 2 azoxymethane-treated rats and in TNF-a-stimulated Caco-2 cells. Br J Nutr 2013;110:206–15. Erlejman AG, Jaggers G, Fraga CG, Oteiza PI. TNFa-induced NF-kB activation and cell oxidant production are modulated by hexameric procyanidins in Caco-2 cells. Arch Biochem Biophys 2008;476:186–95. Bitzer ZT, Glisan SL, Dorenkott MR, Goodrich KM, Ye L, O’Keefe SF, et al. Cocoa procyanidins with different degrees of polymerization possess distinct activities in models of colonic inflammation. J Nutr Biochem 2015;26:827–31. Lee KW, Kim YJ, Lee HJ, Lee CY. Cocoa has more phenolic phytochemicals and a higher antioxidant capacity than teas and red wine. J Agric Food Chem 2003;51:7292–5.

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31. Andujar I, Recio MC, Giner RM, Rıos JL. Cocoa polyphenols and their potential benefits for human health. Oxid Med Cell Longev 2012;https://doi. org/10.1155/2012/906252. 32. Rosenberg DW, Giardina C, Tanaka T. Mouse models for the study of colon carcinogenesis. Carcinogenesis 2009;30:183–96. 33. Pritchard CC, Grady WM. Colorectal cancer molecular biology moves into clinical practice. Gut 2011;60:116–29. 34. Raju J. Azoxymethane-induced rat aberrant crypt foci: relevance in studying chemoprevention of colon cancer. World J Gastroenterol 2008;14:6632–5. 35. Rodrıiguez-Ramiro I, Ramos S, Lopez-Oliva E, Agis-Torres A, Gomez-Juaristi M, Mateos R, et al. Cocoa-rich diet prevents azoxymethane-induced colonic preneoplastic lesions in rats by restraining oxidative stress and cell proliferation and inducing apoptosis. Mol Nutr Food Res 2011;55:1895–9. 36. Pandurangan AK, Saadatdoust Z, Hamzah H, Ismail A. Dietary cocoa protects against colitis-associated cancer by activating the Nrf2/Keap1 pathway. Biofactors 2015;41:1–14. 37. Saadatdoust Z, Pandurangan AK, Sadagopan SKA, Esa NM, Ismail A, Mustafa MR. Dietary cocoa inhibits colitis associated cancer: a crucial involvement of the IL-6/STAT3 pathway. J Nutr Biochem 2015;26:1547–58. 38. Chen J, Huang X-F. The signal pathways in azoxymethane-induced colon cancer and preventive implications. Cancer Biol Therap 2009;8:1313–7. 39. Ashokkumar P, Sudhandiran G. Protective role of luteolin on the status of lipid peroxidation and antioxidant defense against azoxymethane-induced experimental colon carcinogenesis. Biomed Pharmacother 2008;62:590–7. 40. Masella R, Di Benedetto R, Varı` R, Filesi C, Giovannini C. Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione-related enzymes. J Nutr Biochem 2005;16:577–86. 41. Storz P. Reactive oxygen species in tumor progression. Front Biosci 2005;10:1881–96. 42. Xavier CP, Lima CF, Preto A, Seruca R, Fernandes-Ferreira M, Pereira-Wilson C. Luteolin, quercetin and ursolic acid are potent inhibitors of proliferation and inducers of apoptosis in both KRAS and BRAF mutated human colorectal cancer cells. Cancer Lett 2009;281:162–70. 43. Itzkowitz SH, Yio X. Colorectal cancer in inflammatory bowel disease: the role of inflammation. Am J Physiol Gastrointest Liver Physiol 2004;287: G7–G17. 44. Half E, Arber N. Colon cancer: preventive agents and the present status of chemoprevention. Expert Opin Pharmacother 2009;10:211–9. 45. Romier B, Van De Walle J, During A, Larondelle Y, Schneider YJ. Modulation of signalling nuclear factor-kB activation pathway by polyphenols in human intestinal Caco-2 cells. Br J Nutr 2008;100:542–51. 46. Surh YJ, Chun KS, Cha HH, Han SS, Keum YS, Park KK, et al. Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-kappa B activation. Mutat Res 2001;481:243–68. 47. Chang H, Lei L, Zhou Y, Ye F, Zhao G. Dietary flavonoids and the risk of colorectal cancer: an updated meta-analysis of epidemiological studies. Nutrients 2018;10:950–63. 48. Feng YL, Shu L, Zheng PF, Zhang XY, Si CJ, Yu XL, et al. Dietary patterns and colorectal cancer risk: a meta-analysis. Eur J Cancer Prev 2017;26:201–11. 49. Arts I, Jacobs Jr. D, Gross M, Harnack L, Folsom A. Dietary catechins and cancer incidence among postmenopausal women: the Iowa Women’s Health Study (United States). Cancer Causes Control 2002;13:373–82. 50. Bayard V, Chamorro F, Motta J, Hollenberg NK. Does flavanol intake influence mortality from nitric oxide-dependent processes? Ischemic heart disease, stroke, diabetes mellitus, and cancer in Panama. Int J Med Sci 2007;4:53–8. 51. Nimptsch K, Zhang X, Cassidy A, Song M, O’Reilly EJ, Lin JH, et al. Habitual intake of flavonoid subclasses and risk of colorectal cancer in 2 large prospective cohorts. Am J Clin Nutr 2016;103:184–91. 52. Zamora-Ros R, Cayssials V, Jenab M, Rothwell JA, Fedirko V, Aleksandrova K, et al. Dietary intake of total polyphenol and polyphenol classes and the risk of colorectal cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Eur J Epidemiol 2018;33:1063–75. 53. Boutron-Ruault MC, Senesse P, Faivre J, Chatelain N, Belghiti C, Meance S. Foods as risk factors for colorectal cancer: a case-control study in Burgundy (France). Eur J Cancer Prev 1999;8:229–35. 54. McKelvey W, Greenland S, Sandler RS. A second look at the relation between colorectal adenomas and consumption of foods containing partially hydrogenated oils. Epidemiology 2000;11:469–73. 55. Rouillier P, Senesse P, Cottet V, Valleau A, Faivre J, Boutron-Ruault MC. Dietary patterns and the adenomacarcinoma sequence of colorectal cancer. Eur J Nutr 2005;44:311–8. 56. Morze J, Schwedhelm C, Bencic A, Hoffmann G, Boeing H, Przybylowicz K, et al. Chocolate and risk of chronic disease: a systematic review and dose response meta-analysis. Eur J Nutr 2019;59:389–97. https://doi.org/10.1007/s00394-019-01914-9. 57. Spadafranca A, Martinez Conesa C, Sirini S, Testolin G. Effect of dark chocolate on plasma epicatechin levels. DNA resistance to oxidative stress and total antioxidant activity in healthy subjects. Br J Nutr 2010;103:1008–114. 58. Va´zquez-Agell M, Urpı´-Sarda M, Sacanella E, Camino-Lo´pez S, Chiva-Blanch G, Llorente-Cortes V, et al. Cocoa consumption reduces NF-kB activation in peripheral blood mononuclear cells in humans. Nutr Metab Cardiovasc Dis 2013;23:257–63. 59. Monagas M, Khan N, Andres-Lacueva C, Casas R, Urpı´-Sarda M, Llorach R, et al. Effect of cocoa powder on the modulation of inflammatory biomarkers in patients at high risk of cardiovascular disease. Am J Clin Nutr 2009;90:1144–50.

Chapter 31

Medicinal plants, antioxidant potential, and cancer Emmanuel Mfotie Njoya Institute of Pharmacy, Martin-Luther University of Halle-Wittenberg, Halle (Saale), Germany Department of Biochemistry, Faculty of Science, University of Yaound e I, Yaound e, Cameroon

List of abbreviations 1 O2 AA ABTS ATP CAT COX DNA DPPH FRAP GPX H2O2 HO2% HOCl iNOS IR LOX MDA NADH NADPH NO NO% O2%2 OCl% OH%

ONOO2 ORAC PG PMS RNS ROS RSS SOD TBARS XO

singlet oxygen arachidonic acid 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) adenosine triphosphate catalase cyclooxygenase deoxyribonucleic acid 2,2-diphenyl-1-picrylhydrazyl ferric reducing ability of plasma glutathione peroxidase hydrogen peroxide perhydroxyl radical hypochlorous acid inducible nitric oxide synthase ionizing radiation lipoxygenase malondialdehyde nicotinamide adenine dinucleotide (reduced) nicotinamide adenine dinucleotide phosphate (reduced) nitric oxide nitric oxide radical superoxide anion radical hypochlorite radical hydroxyl radical peroxynitrite oxygen radical absorbance capacity prostaglandin phenazine methosulfate reactive nitrogen species reactive oxygen species reactive sulfur species superoxide dismutase thiobarbituric acid reactive substances xanthine oxidase

Introduction Free radicals are involved in oxidative stress and the occurrence of diverse metabolic disorders within the organism including the initiation of cancer or its progression. Cancer is a chronic disease characterized by abnormal and uncontrolled Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00031-6 © 2021 Elsevier Inc. All rights reserved.

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cell division. The formation of premalignant cells is caused either by internal factors such as inherited mutations, hormones, immune conditions or external factors such as smoking, alcohol abuse, exposure to radiation, overweight and obesity, lack of physical activity, environmental pollutants, low fruit and vegetable intake, chronic infections, and oxidative stress.1 These causative factors may act together or in sequence to initiate or promote carcinogenesis. The development of most cancers requires multiple steps which include initiation, promotion, tumor progression leading to metastasis and may occur many years later.2 Cancer is a major public health burden in both developed and developing countries. According to estimates from the International Agency for Research on Cancer (IARC), there were 14.1 million new cancer cases with 8.2 million cancer-related deaths in 2012. By 2030, the global burden is expected to grow to 21.4 million new cancer cases and 13.2 million cancer deaths simply due to the growth and aging of the population and deaths from infectious diseases in developing countries.3 The treatment of cancer includes the application of chemotherapy, radiotherapy, or chirurgical intervention. However, the use of medicinal plants with their diverse composition in secondary metabolites represents alternative prophylactic and therapeutic approach which can be efficiently used to control cancer progression. Medicinal plants are rich source of antioxidants which have the potential to counteract oxidative stress or DNA and protein damage in tissues and prevent the living organisms from chronic diseases such as cancer, cardiovascular diseases, diabetes, and aging. In fact, due to biotic and abiotic stress factors, plants are able to produce secondary metabolites which may exhibit strong antioxidant activities depending on the part of the plant collected. Phytochemicals such as flavonoids, tannins, phenolic acids are known to be responsible for free radical scavenging and antioxidant activities in various plant extracts.4 Indeed, it has been suggested by various reports that polyphenolic compounds possess scavenging potential which may justify their inhibitory effect on oxidative stress, mutagenesis, and carcinogenesis.

Applications to other cancers or conditions In this chapter, a description of processes and circumstances generating free radicals in human cells is presented, and it was found that the accumulation of free radicals was one of the mechanisms that initiate or activate cancer progression in humans. This concept applies to all cancers since free radicals are able to modulate various signaling pathways controlling the transcription of genes which play an important role in diverse chronic disorders such as stroke, neurodegenerative diseases, diabetes, and cardiovascular diseases. However, the human body can be protected from the harmful effect of free radicals through its antioxidant defense system constituted of enzymes such as superoxide dismutase, glutathione peroxidase, catalase and biomolecules such as glutathione, vitamins C and E. In addition, external supplements such as plants, fruits, and vegetables known as potential sources of polyphenols can strengthen the antioxidant power of the body to prevent or treat oxidative stress-related diseases. In fact, polyphenolic compounds, characterized by the presence of hydroxyl groups and aromatic rings are able to accept or donate an electron to a free radical in order to neutralize its capacity to cause damage to biological macromolecules such as proteins, lipids, and DNA. It was found in many reports that the antioxidant potency of crude plants correlates well with their phenolic or flavonoid contents. Therefore, the capacity of antioxidants to scavenge free radicals is a common mechanism by which polyphenols are involved in the prevention of oxidative stress-related diseases including cancer.

Oxidative stress resulting from the overproduction of free radicals Free radicals are derived from normal metabolic processes in the body such as phagocytosis, aerobic metabolism, the synthesis of prostaglandins (PGs), or certain external factors such as ionizing radiation (IR), xenobiotics, and pollutants. It is well known that during invasion of pathogens, the organism reacts by activating the macrophages which are the first line of defense, thereby leading to the production of free radicals. An overproduction of these free radicals may result in acute or chronic inflammation which generates oxidative stress. Another process that generates free radicals in the body is known as oxidation which is an essential process in the metabolism of aerobic organisms in which the transfer of electrons from one atom to another occurs. In this system where ATP is produced, oxygen plays the role of ultimate electron acceptor. It may therefore be deduced that uncoupled or unpaired single electron transfer generates oxygen-derived free radicals, resulting in damage to cell tissues. On the other hand, PGs, thromboxane, and leukotrienes collectively termed as eicosanoids are obtained from arachidonic acid (AA) by cyclooxygenase (COX) and lipoxygenase (LOX) pathways. This process will only produce picomolar-to-nanomolar concentrations of PGs which are physiologically not harmful to the body.5 However, in several pathological conditions, the synthesis of PGs is highly increased, and it was confirmed from a report that PGs represent the most potent inducers of intracellular oxidative stress and that the production of ROS in the cells is closely associated with the excitotoxic effect of PGs.6 Ionizing radiation (IR) composed of high-energy photon, proton, and neutron

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FIG. 1 Protective mechanism of antioxidants against oxidative stress-related diseases. Free radicals are generated by internal and external factors which can lead either to oxidative stress-related diseases via the oxidative damage and accumulation of proteins, lipids, and DNA or modify the expression of several genes involved in different signaling pathways. Antioxidants may prevent the occurrence of oxidative stress by interacting and neutralizing free radicals.

current is another factor which triggers the formation of free radicals in living organisms. The free radicals produced by IR can upregulate several enzymes, including nicotinamide adenine dinucleotide phosphate oxidase, LOX, nitric oxide synthase, and COX.7 Xenobiotics and environmental pollutants can produce mutagenic metabolites that lead to the continuous release of superoxide, which finally induce to the formation of DNA adducts thereby activating the superoxidedependent pathways to carcinogenesis.8 All these factors involved in the production of free radicals will activate different pathways to induce oxidative stress. In general, the overproduction of free radicals appears to be one of the fundamental mechanisms explaining the initiation and progression of cancer. In fact, free radicals can bind through electron pairing with biological macromolecules such as proteins, lipids, and DNA in healthy human cells and cause protein and DNA damage along with lipid peroxidation.9 The oxidative DNA damage is a major cause of mutation, and the relevance of DNA damage in the progression of cancer is quite established. The accumulation of DNA damage and denaturation of other macromolecules in normal cell lines initiates a progressively dysplastic cellular appearance, which lead to deregulated cell growth, and finally carcinoma.10 Antioxidants have the capacity to scavenge free radicals to prevent the denaturation of biomacromolecules (Fig. 1). In the absence of an appropriate antioxidant system, oxidative stress results as an imbalance between the generation of free radicals and the availability of antioxidants to scavenge these free radicals favoring an excess production of oxidants.11 The antioxidant defense mechanism of the organism is constituted of molecules such as tocopherol, ascorbic acid, and glutathione or enzymes involved in oxygen radical scavenging.

Free radicals and their implication in oxidative stress-related diseases There are different categories of free radicals which include reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS). ROS contain highly reactive forms of oxygen such as hydroxyl radical (OH%), perhydroxyl radical (HO2%), hypochlorous acid (HOCl), superoxide anion radical (O2%), hydrogen peroxide (H2O2), singlet oxygen (1O2), nitric oxide radical (NO%), hypochlorite radical (OCl%), and peroxynitrite (ONOO).12 RNS are derived from the reaction of nitric oxide (NO) with the superoxide anion (O2%), which lead to the formation of peroxynitrite (ONOO) produced via the enzymatic activity of inducible nitric oxide synthase (iNOS) and other enzymes such as NADPH oxidase and xanthine oxidase.13 On the contrary, RSS are produced from the oxidation of thiols and disulfides with ROS yielding persulfides, polysulfides, and thiosulfate with higher oxidation states.14 These different free radicals generated either by endogenous or exogenous sources are generally eliminated from the body by a combination of antioxidant enzymes and antioxidants provided by the diet (Fig. 1). Oxidative stress results as an imbalance between the antioxidant defense systems and the generation of free radicals which cause damage to major biomolecules such as proteins, DNA, and lipids. A breakdown of the antioxidant defense systems has been associated with a number of chronic diseases. The structural damage-based hypothesis that age-associated functional losses are due to the accumulation of damage caused by free radicals to macromolecules (lipids, DNA, and proteins) leading to oxidative stress has been the basis of the free radical theory of aging, later termed as oxidative stress theory of aging.15 In fact, oxidative stress has been recognized in the pathophysiology of many diseases, and its mechanisms have been clearly described for several diseases including cancer. In fact, the accumulation of free radicals in the organism can activate various

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transcription factors, including nuclear factor-kB (NF-kB), activator protein-1 (AP-1), tumor suppressor protein 53 (p53), hypoxia-inducible factor-1a (HIF-1a), peroxisome proliferator-activated receptor-g (PPAR-g), b-catenin/Wnt, and nuclear factor E2-related factor 2 (Nrf2).16 The modulation of these signaling pathways can lead to the expression of various genes, including genes that regulate growth factors, pro- and anti-inflammatory cytokines, chemokines, and cell cycle regulator molecules.17 Therefore, it is observed via this mechanism that oxidative stress, chronic inflammation, and cancer are closely related. In addition, ROS and RNS are thought to play important roles in diverse nervous system disorders such as stroke, neurodegenerative diseases, and other diseases such as diabetes, cardiovascular diseases, etc. These diseases are clearly correlated to chronic oxidative stress as most of them share the same signaling pathways.

Antioxidant mechanisms of free radical scavengers The antioxidant protection system plays an important role in scavenging free radicals and preventing cell injury in the organism, and this system is constituted of enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and nonenzymes protection (glutathione, vitamin C, and vitamin E). However, the antioxidant system can be strengthened with supplement constituents such as polyphenolic compounds provided by vegetables, fruits, and diet. Free radicals are characterized by the existence of an unpaired electron in an atomic orbital which makes them unstable and highly reactive. As such, they can either donate an electron to or accept an electron from other molecules, thereby behaving as oxidants or reductants.18 On the contrary, antioxidant is a stable molecule which can donate an electron to a free radical in order to neutralize its capacity to cause cellular damage. Antioxidant compounds exhibit their scavenging potential on free radicals through several chemical mechanisms which include the quenching of singlet oxygen, the hydrogen or electron transfer, the breaking of free radical chain reactions, or the chelation of transition metals.19 The chemical structure of the antioxidant compounds characterized by the presence of hydroxyl groups and aromatic rings allows them to interact with electron, hydrogen atom, or transition metals. The most effective antioxidant mechanism is the breaking of the free radical chain reaction. In this mode of action, the radical character of the oxidants (R%) is transferred to the antioxidant compounds (AH) by either accepting or donating electrons to form antioxidant-derived radicals (A%) which are less active and less dangerous than initial oxidants that they have neutralized. These radical intermediates are stable and may be neutralized by other antioxidant compounds or other mechanisms to terminate their radical status.20 The chelation of transition metal ions, such as Fe2+ in particular, can effectively reduce oxidation status due to the fact that these metals via the Fenton reactions are able to catalyze oxidative processes leading to the formation of hydroxyl radicals or hydroperoxides. Another important antioxidant mechanism is the regulation between prooxidant and antioxidant enzymes. NAD(P)H oxidase and xanthine oxidase (XO) are free radical generating enzymes which donates electrons to molecular oxygen, % and/or H2O2 while superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase thereby producing O 2 (GPX) belong to the antioxidant defense system which counteract or neutralize free radicals.21, 22 In fact, SOD can convert % two O 2 into an H2O2 and an oxygen, and CAT and/or GPX are able to eliminate H2O2 in animal cells. Based on this principle, the presence of antioxidants may provide protection against free radicals either by inhibiting the expression of NAD(P)H oxidase and XO or by activating the expression of SOD, CAT, and GPX.

Methods used for the evaluation of antiradical activity Different methods used to evaluate the antiradical activity are based on different chemical reaction mechanisms which include either inhibition or reduction assays. The inhibition assays rely on antioxidants’ potential to interact with or neutralize free radicals generated in the assay system while the reduction assays evaluate the ability of antioxidants to reduce an oxidant which also functions as a probe that changes color when it is reduced.23 The ferric reducing ability of plasma (FRAP), oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ABTS (2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assays are among the commonly used techniques for the evaluation of the antiradical activity due to their simple, rapid, sensitive, and reproducible procedures. Other techniques include superoxide anion and hydroxyl radical scavenging assays which are also used as tools for the evaluation of antioxidant activity.

Ferric reducing ability of plasma (FRAP) assay The FRAP assay is used to measure the antioxidant power based on the reduction at low pH of ferric-tripyridyltriazine (Fe3+-TPTZ) to an intense blue color ferrous-tripyridyltriazine complex (Fe2+-TPTZ) with an absorption maximum at 593 nm.24 In this assay, trolox is mostly used as positive control, and results can be expressed as mmol/mL trolox

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equivalents or as mmol/mL Fe2+ calculated from a standard curve. This assay has been proven to measure the antioxidant capacity of foods and seeds from legumes which is closely related to their polyphenol contents.25

ABTS (2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay The ABTS radical cation (ABTS%+) is produced from the reaction between ABTS solution and sodium persulfate, and the assay is based on the decoloration of the blue-green radical cation (ABTS%+) which has absorption maxima at wavelengths 645, 734, and 815 nm.26 The radical cation ABTS%+ is able to react with most antioxidants including phenolic compounds, and being converted back to its colorless neutral form (ABTS). Moreover, ABTS%+ is not affected by ionic strength and can be used to determine both hydrophilic and hydrophobic antioxidants and the color change can be monitored spectrophotometrically. Trolox, a water-soluble analog of vitamin E, is used as control and the scavenging strength of the tested samples are measured in units referred to as trolox equivalent per milligram.

DPPH (2,2-diphenyl-1-picrylhydrazyl) assay The DPPH radical (DPPH%) is a stable molecule soluble in methanol characterized by its deep-violet color with an absorption maximum at 515 nm. Antioxidants (AH) or other radical species (R%) are able to react with this stable radical (DPPH%) by providing an electron or hydrogen atom, thus reducing it to 2,2-diphenyl-1-hydrazine (DPPH-H) or a substituted analogous hydrazine (DPPH-R) characterized by colorless or pale-yellow color which could be easily monitored with a spectrophotometer.27 This assay is widely used to determine antioxidant activity of crude extracts or purified compounds from plants.28–30

ORAC (oxygen radical absorbance capacity) assay This assay measures the radical chain breaking ability of antioxidants by monitoring the inhibition of peroxyl-free radicals. Based on the fact that peroxyl radicals are the predominant free radicals found in lipid oxidation in foods and biological systems, the ORAC assay is considered as a preferable method due to its biological relevance to the in vivo antioxidant efficacy. Peroxyl-free radicals are generated via thermal decomposition of 2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH) yielding C% centered free radicals which in the presence of oxygen result in the formation of peroxyl-free (COO%) radicals.31 In this assay, b-phycoerythrin (b-PE), a hydrosoluble protein, is used as a fluorescent probe or indicator protein, with an excitation wavelength at 540 nm and an emission at 565 nm, and trolox, a water-soluble vitamin E analog, is used as a control standard.32 Peroxyl-free radicals are able to interact with fluorescent probe, yielding an increase in the fluorescence decay. The presence of antioxidants results in an inhibition of the interaction between the peroxyl-free radicals and the fluorescent probe which is observed by a preservation of the fluorescent signal. Thus, by monitoring the kinetics of the decrease in fluorescence emission for each experimental sample compared to a blank versus time, the resultant difference will be considered as the antiradical protection conferred by the tested sample.

Superoxide anion scavenging assay Superoxide radical (O2%) can be generated by four-electron reduction of molecular oxygen into water or it can be also produced in aerobic cells due to electron leakage from the electron transport chain.33 In addition, during an infection, the activation of phagocytes (monocytes, macrophages, eosinophils, and neutrophils) will result in the production of superoxide 34 radicals (O. 2 ), which play an important role in the killing of microorganisms. In the in vitro experiments, superoxide anion can be generated either enzymatically in a hypoxanthine-xanthine oxidase system or nonenzymatically in a phenazine methosulfate-NADH system by the oxidation of NADH.35 This radical can be monitored spectrophotometrically by measuring the absorbance of the product from the reduction of nitro blue tetrazolium at 560 nm against blank which contained no phenazine methosulfate (PMS). In the nonenzymatically system, PMS acts as an electron carrier while in the enzymatic system, superoxide anions can be estimated either by reduction of cytochrome c or with superoxide dismutase.36

Hydroxyl radical scavenging assay Hydroxyl radicals (OH) is generated from the Fenton reaction on deoxyribose which produces malondialdehyde (MDA), and the thiobarbituric acid reactive substances (TBARS) assay can be used for their measurement.37 On the other hand, hydroxyl radicals can be generated in the ascorbate system where ferric ion (Fe3+) is reduced by ascorbate to the ferrous

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state (Fe2+) which can react with H2O2 yielding OH radicals.38, 39 The hydroxyl radicals are able attack either salicylate used as a probe or hydroxyl radical scavengers (probably in tested samples), and this competition for the OH produced will be used to monitor the chromophore formation from Cu(II)-neocuproine which has a maximum absorption at 450 nm.39 Therefore, the hydroxyl radical scavenging capacity is determined by following this chromophore overtime and by comparing the absorbance in the presence or absence of the tested samples.

Free radical scavenger potency versus polyphenolic contents of plants Crude plant extracts usually contain complex mixture of different bioactive constituents among which secondary metabolites are widely used for the preparation of herbal medicines and their derivative products. It is assumed that the combination of secondary metabolites found in plants may improve health by inhibiting the formation of free radicals responsible for early stage development of some chronic diseases such as cancer. Polyphenols are naturally occurring metabolites broadly distributed throughout the plant kingdom, and largely found in the fruits, vegetables, cereals, and beverages. The production of polyphenols varies widely within different plants and factors such as environmental stress, growth conditions, and plant species modify polyphenol structure and content.40 In fact, plants are able to absorb the sun light and produce via the photosynthesis high levels of oxygen and primary metabolites which are later converted into secondary metabolites as a result of interaction with their environment or in response to stress. These secondary metabolites including alkaloids, glycosides, flavonoids, terpenoids, phenolics, and saponins are stored in the leaves and other parts of the plants. Polyphenols are characterized by the presence of one or more hydroxyl groups binding to one or more aromatic rings. It is known that oxidation is an essential process in the metabolism of aerobic organisms involving transfer of electrons from one atom to another. By acting as metal chelators, reducing agents, hydrogen donors, as well as radical scavengers, polyphenols exhibit their antioxidant potential by scavenging or stabilizing free radicals involved in oxidative processes, or via hydrogenation with oxidizing species.41 In fact, hydroxyl groups and aromatic rings of polyphenols play the role of ultimate electron acceptor which can couple with free radicals yielding to the reduction in free radicals in the organism. Through this mechanism, polyphenols are able to protect the organism from oxidative damage by fixing and scavenging free radicals, which considerably reduces the risk of oxidative stress. Several plant-derived compounds have played an important role as antioxidant agents, and most of these compounds are polyphenols which can be classified into two major groups: flavonoids (flavanols, flavonols, anthocyanidins, flavones, flavanones, and chalcones) and non-flavonoids (stilbene, phenolic acids, saponin, and tannins).4 These compounds are known to exhibit high levels of antioxidant potential which has been linked to their preventive or curative effect in reducing the risk of developing chronic diseases such as cancer, diabetes, obesity, and cardiovascular diseases. Different reports have confirmed the strong correlation values between antiradical activity and total polyphenol and flavonoid contents of plants or seeds.28, 42–45 A great number of natural compounds have been found to possess promising antiradical activity and they are key sources of modern innovative drugs. However, these natural compounds with various molecular structures usually exert their antioxidant potential by diverse mechanisms. The antioxidant activity of phenolic compounds in plants is mainly attributed to their redox properties and chemical structure, which are involved in scavenging free radicals, chelating transitional metals, and quenching singlet and triplet oxygen. Flavonoids are hydroxylated phenolic substances which act as an antioxidant defense system in plant tissues exposed to different abiotic and biotic stresses. A diet containing fruits and vegetables is a great source of flavonoids for humans, and several flavonoids have been reported with antioxidant property and free radical scavenging capacity. Most importantly, flavonoids are known to have potential health benefits in the prevention of many diseases including inflammation, cancer, and coronary heart disease. The basic structure of flavonoids is constituted of a 15-carbon skeleton containing 2 benzene rings linked via a heterocyclic pyrane ring (Fig. 2), and can be divided into different classes such as flavones (e.g., flavone, apigenin, and luteolin), flavonols (e.g., quercetin, kaempferol, myricetin, and fisetin), and flavanones (e.g., flavanone, hesperetin, and naringenin).46 The antioxidant activity of flavonoids depends on their configuration, substitution, and total number of hydroxyl groups which may significantly influence their mode of action against free radicals. In fact, the benzene ring configuration is most relevant for their scavenging activity since they can easily donate hydrogen and an electron to free radicals such as hydroxyl, peroxyl, and peroxynitrite for their stabilization.47, 48 Flavonoids act also by inhibiting the activity of several enzymes such as xanthine oxidase and protein kinase C (responsible for superoxide anion production), and cyclooxygenase, lipoxygenase, microsomal monooxygenase, glutathione-S-transferase, mitochondrial succinoxidase, and NADH oxidase which are reactive oxygen species producers.48 Nonflavonoids include phenolic acids, tannins, stilbenes, and lignans. Phenolic acids are a major class of polyphenols widely distributed in the human diet, particularly in fruits, vegetables, and beverages. Phenolic acids are constituted of free hydroxyl groups and are functional derivatives of benzoic acid and cinnamic acid. The presence of hydroxyl groups and

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FIG. 2 Basic structures of flavonoids and non-flavonoids compounds with antioxidant activity.

aromatic ring justifies their antioxidant activity which is significantly reduced by glucuronidation and sulfation of hydroxyl groups of the phenolic structure.49 Tannins are naturally synthesized and accumulated by higher plants as secondary metabolites, and are structurally presented as phenol derivatives with some common features, which can be classified in two main groups: hydrolysable tannins and condensed tannins.50 Lignans are a subgroup of nonflavonoid polyphenols made up of two units of a phenylpropene derivative joined by C3 side chains or between the aromatic ring and C3 ring while stilbenes are characterized by the presence of a 1,2-diphenylethylene nucleus. Stilbenes have been reported to exhibit an extraordinary potential in the prevention and treatment of different diseases including cancer, due to their antioxidant property associated with low toxicity.51 The antioxidant mechanism of non-flavonoids is quite similar with those previously described since they function both as primary antioxidants (i.e., by donating hydrogen atom or electrons), and as secondary antioxidants (i.e., by chelating metal ions or by interfering with steps of the Fenton reaction). Nonflavonoids such as tannins may also act by inhibiting enzymes such as cyclooxygenase to stop lipid peroxidation.52

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This chapter focuses on the implication of free radicals in the oxidative stress and its prevention with polyphenolic compounds. Free radicals are generated either by normal metabolic processes such as phagocytosis and aerobic metabolism or exogenous factors such as ionizing radiation, xenobiotics, and pollutants. Oxidative stress occurs due to an imbalance between the generation of free radicals and the antioxidant defense system of the body. Free radicals are involved in the modulation of various signaling pathways shared by oxidative stress-related diseases including cancer. The antioxidant defense system of the body is made of enzymes and biomolecules which prevent the accumulation of free radicals. Plants, fruits, and vegetables contain polyphenols which correlate well with their antioxidant capacity. Polyphenols, via their structure, are able to accept or donate electron to free radicals to neutralize them.

References 1. Sloan FA, Gelband H, editors. Cancer control opportunities in low- and middle-income countries. Washington, DC: National Academies Press; 2007. 2. Siddiqui IA, Sanna V, Ahmad N, Sechi M, Mukhtar H. Resveratrol nanoformulation for cancer prevention and therapy. Ann N Y Acad Sci 2015; 1348(1):20–31. 3. IARC/WHO. World cancer report 2014. International Agency for Research on Cancer/World Health Organization; 2014 ISBN: 978-92-832-0429-9 (Edited by Bernard W. Stewart and Christopher P. Wild). 4. Stagos D. Antioxidant activity of polyphenolic plant extracts. Antioxidants (Basel) 2019;9(1). 5. Khanapure SP, Garvey DS, Janero DR, Letts LG. Eicosanoids in inflammation: biosynthesis, pharmacology, and therapeutic frontiers. Curr Top Med Chem 2007;7(3):311–40. 6. Kondo M, Oya-Ito T, Kumagai T, Osawa T, Uchida K. Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress. J Biol Chem 2001;276(15):12076–83.

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7. Wei J, Wang B, Wang H, et al. Radiation-induced normal tissue damage: oxidative stress and epigenetic mechanisms. Oxid Med Cell Longev 2019;2019:3010342. 8. Henkler F, Brinkmann J, Luch A. The role of oxidative stress in carcinogenesis induced by metals and xenobiotics. Cancers (Basel) 2010;2(2):376–96. 9. Islam S, Samima N, Muhammad AK, et al. Evaluation of antioxidant and anticancer properties of the seed extracts of Syzygium fruticosum Roxb. growing in Rajshahi, Bangladesh. BMC Complement Altern Med 2013;13. 10. Acheampong F, Larbie C, Appiah-Opong R, Fareed KNA, Tuffour I. In vitro antioxidant and anticancer properties of hydroethanolic extracts and fractions of Ageratum conyzoides. Eur J Med Plant 2015;7(4):205–14. 11. Chatterjee S. Oxidative stress, inflammation, and disease (Chapter 2). In: Dziubla T, Butterfield DA, editors. Oxidative stress and biomaterials. Elsevier Inc.; 2016. p. 35–58. 12. Aziz AM, Diab AS, Mohammed AA. Antioxidant categories and mode of action. IntechOpen; 2019. Open Access Books. 20 p. 13. Matsubara K, Higaki T, Matsubara Y, Nawa A. Nitric oxide and reactive oxygen species in the pathogenesis of preeclampsia. Int J Mol Sci 2015; 16(3):4600–14. 14. Giles GI, Jacob C. Reactive sulfur species: an emerging concept in oxidative stress. Biol Chem 2002;383(3-4):375–88. 15. Beckman KB, Ames BN. The free radical theory of aging matures. Physiol Rev 1998;78(2):547–81. 16. Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med 2010; 49(11):1603–16. 17. Dasgupta A, Klein K. Oxidative stress and cancer (Chapter 8). In: Dasgupta A, Klein K, editors. Antioxidants in food, vitamins and supplements. Elsevier Inc.; 2014. p. 129–50. 18. Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: impact on human health. Pharmacogn Rev 2010;4(8):118–26. 19. Brewer MS. Natural antioxidants: sources, compounds, mechanisms of action, and potential applications. Compr Rev Food Sci Food Saf 2011;10:221–47. 20. Lu JM, Lin PH, Yao Q, Chen C. Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems. J Cell Mol Med 2010;14(4):840–60. 21. Rojas J, Buitrago A. Antioxidant activity of phenolic compounds biosynthesized by plants and its relationship with prevention of neurodegenerative diseases (Chapter 1). In: Campos MRS, editor. Bioactive compounds: health benefits and potential application. Elsevier Inc.; 2019. p. 3–31. 22. Bevilacqua E, Gomes SZ, Lorenzon AR, Hoshida MS, Amarante-Paffaro AM. NADPH oxidase as an important source of reactive oxygen species at the mouse maternal-fetal interface: putative biological roles. Reprod Biomed Online 2012;25(1):31–43. 23. Carlsen MH, Halvorsen BL, Blomhoff R. Antioxidants in nuts and seeds (Chapter 6). In: Preedy VR, Watson RR, Patel VB, editors. Nuts and seeds in health and disease prevention. Elsevier Inc.; 2011. p. 55–64. 24. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay. Anal Biochem 1996; 239(1):70–6. 25. Galili S, Hovav R. Determination of polyphenols, flavonoids, and antioxidant capacity in dry seeds (Chapter 16). In: Watson RR, editor. Polyphenols in plants: isolation, purification and extract preparation. Elsevier Inc.; 2014. p. 305–23. 26. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolourization assay. Free Radic Biol Med 1999;28:1057–60. 27. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. Lebensm Wiss Technol 1995;28(1):25–30. 28. Mfotie Njoya E, Munvera AM, Mkounga P, Nkengfack AE, McGaw LJ. Phytochemical analysis with free radical scavenging, nitric oxide inhibition and antiproliferative activity of Sarcocephalus pobeguinii extracts. BMC Complement Altern Med 2017;17(1):199. 29. Ondua M, Njoya EM, Abdalla MA, McGaw LJ. Anti-inflammatory and antioxidant properties of leaf extracts of eleven South African medicinal plants used traditionally to treat inflammation. J Ethnopharmacol 2019;234:27–35. 30. Ghuman S, Ncube B, Finnie JF, et al. Antioxidant, anti-inflammatory and wound healing properties of medicinal plant extracts used to treat wounds and dermatological disorders. S Afr J Bot 2019;126:232–40. 31. Litescu SC, Eremia SAV, Tache A, Vasilescu I, Radu G-L. The use of oxygen radical absorbance capacity (ORAC) and trolox equivalent antioxidant capacity (TEAC) assays in the assessment of beverages’ antioxidant properties (Chapter 25). In: Preedy V, editor. Processing and impact on antioxidants in beverages. Elsevier Inc; 2014. 32. Cao G, Alessio HM, Cutler RG. Oxygen-radical absorbance capacity assay for antioxidants. Free Radic Biol Med 1993;14(3):303–11. 33. Wettasinghe M, Shahid F. Scavenging of reactive-oxygen species and DPPH free radicals by extracts of borage and evening primrose meals. Food Chem 2000;20:17–26. 34. Babior BM. Phagocytes and oxidative stress. Am J Med 2000;109(1):33–44. 35. Robak J, Gryglewski RJ. Flavonoids are scavengers of superoxide anions. Biochem Pharmacol 1988;37(5):837–41. 36. Nishikimi M, Appaji N, Yagi K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Commun 1972;46(2):849–54. 37. Bektasoglu B, Esin Celik S, Ozyurek M, Guclu K, Apak R. Novel hydroxyl radical scavenging antioxidant activity assay for water-soluble antioxidants using a modified CUPRAC method. Biochem Biophys Res Commun 2006;345(3):1194–200. 38. Klein SM, Cohen G, Cederbaum AI. Production of formaldehyde during metabolism of dimethyl sulfoxide by hydroxyl radical generating systems. Biochemistry 1981;20(21):6006–12. 39. Ozyurek M, Bektasoglu B, Guclu K, Apak R. Hydroxyl radical scavenging assay of phenolics and flavonoids with a modified cupric reducing antioxidant capacity (CUPRAC) method using catalase for hydrogen peroxide degradation. Anal Chim Acta 2008;616(2):196–206.

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40. Cheynier V, Tomas-Barberan FA, Yoshida K. Polyphenols: from plants to a variety of food and nonfood uses. J Agric Food Chem 2015; 63(35):7589–94. 41. Seyoum A, Asres K, El-Fiky FK. Structure-radical scavenging activity relationships of flavonoids. Phytochemistry 2006;67(18):2058–70. 42. Hu X, Chen L, Shi S, Cai P, Liang X, Zhang S. Antioxidant capacity and phenolic compounds of Lonicerae macranthoides by HPLC-DAD-QTOF-MS/ MS. J Pharm Biomed Anal 2016;124:254–60. 43. Abuashwashi MA, Palomino OM, Gomez-Serranillos MP. Geographic origin influences the phenolic composition and antioxidant potential of wild crataegus monogyna from Spain. Pharm Biol 2016;1–6. 44. Wan Yahaya WA, Abu Yazid N, Mohd Azman NA, Almajano MP. Antioxidant activities and total phenolic content of malaysian herbs as components of active packaging film in beef patties. Antioxidants (Basel) 2019;8(7). 45. Shi P, Du W, Wang Y, Teng X, Chen X, Ye L. Total phenolic, flavonoid content, and antioxidant activity of bulbs, leaves, and flowers made from Eleutherine bulbosa (Mill.) Urb. Food Sci Nutr 2019;7(1):148–54. 46. Kumar S, Pandey AK. Chemistry and biological activities of flavonoids: an overview. Scientific World Journal 2013;2013:162750. 47. Cao G, Sofic E, Prior RL. Antioxidant and prooxidant behavior of flavonoids: structure-activity relationships. Free Radic Biol Med 1997; 22(5):749–60. 48. Pietta PG. Flavonoids as antioxidants. J Nat Prod 2000;63(7):1035–42. 49. Piazzon A, Vrhovsek U, Masuero D, Mattivi F, Mandoj F, Nardini M. Antioxidant activity of phenolic acids and their metabolites: synthesis and antioxidant properties of the sulfate derivatives of ferulic and caffeic acids and of the acyl glucuronide of ferulic acid. J Agric Food Chem 2012;60(50):12312–23. 50. Khanbabaee K, van Ree T. Tannins: classification and definition. Nat Prod Rep 2001;18(6):641–9. 51. Sirerol JA, Rodriguez ML, Mena S, Asensi MA, Estrela JM, Ortega AL. Role of natural stilbenes in the prevention of cancer. Oxid Med Cell Longev 2016;2016:3128951. 52. Zhang Y, DeWitt DL, Murugesan S, Nair MG. Novel lipid-peroxidation- and cyclooxygenase-inhibitory tannins from Picrorhiza kurroa seeds. Chem Biodivers 2004;1(3):426–41.

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Chapter 32

Curcumin, oxidative stress, and breast cancer Gloria M. Calaf Instituto de Alta Investigacio´n, Universidad de Tarapaca, Arica, Chile, Columbia University Medical Center, New York, NY, United States

List of abbreviations 8-isoPGF2a 8-OHdG COX-2 EGFR EMT ER Estrogen GDP GRF GTP HE LET MnSOD or SOD2 NAD(P)H NFkB PARP PGE2 RNS ROS aE2 aEE bE2

8-isoprostaglandin F2a 8-hydroxy-20-deoxyguanosine cyclooxygenase-2 epidermal growth factor receptor epithelial-mesenchymal transition estrogen receptor 17b-estradiol guanidine diphosphate growth-regulating factor guanidine triphosphate hematoxylin and eosin linear energy transfer superoxide dismutase nicotinamide adenine dinucleotide phosphate nuclear factor kB poly (ADP-ribose) polymerases prostaglandin E2 reactive nitrogen species reactive oxygen species 17a-estradiol 17a-ethinylestradiol 17b-estradiol

Introduction Breast cancer, the most frequently diagnosed spontaneous malignancy in women in the western world, is a classical model of hormone-dependent malignancy. There is substantial evidence that breast cancer risk is associated with prolonged exposure to female hormones since the onset of menarche, late menopause, hormone replacement therapy are associated with greater cancer incidence.1 Breast cancer progression follows a complex multistep process that depends on various exogenous (diet, breast irradiation) and endogenous (age, hormonal imbalances, proliferative lesions, and family history of breast cancer) factors.2 Such cancer may have its genesis and cell growth influenced by hormonal factors since about one-third of breast cancers is responsive to endocrine therapies. Estrogen has generally been considered beneficial, based on a variety of hormonal effects; however, in the past 15–20 years, epidemiological studies have increasingly pointed to an increased breast cancer risk associated with them. The potential carcinogenic activity of estrogen-containing medications in humans has not been recognized for many years. Authors1–5 have shown that estrogen administration, a risk factor for humans, increases the risk of breast cancer with continuous doses of estrogen and with the length of treatment. Slightly elevated levels of circulating estrogen are also a risk factor for breast cancer. Several studies have demonstrated strong

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relationships between endogenous estrogen levels and breast cancer risk.6–9 This role of endogenous estrogen in human breast carcinogenesis is supported by risk factors of breast cancer such as high serum or urine estrogen levels.6 Estrogen is associated with carcinogenesis in humans and animals as described in Refs. 10–15 and the exact effect of estrogen in breast cancer remains unclear at this time. Estrogen has been implicated in a variety of cancers. Since that time, many more reports on tumor induction by estrogen have been published, and many rodent tumor models have been reviewed in Ref. 9. The evidence for the carcinogenic activity of estrogen in animals has been reported by those groups. This conclusion was based on numerous experiments related to the administration to rodents of oral or subcutaneous estrogen which resulted in increased the incidence of mammary tumors.16, 17

Estrogens (17b-estradiol) and oxidative stress Oxidative stress plays a crucial role in estrogen-induced carcinogenesis.16, 17 It has been suggested that oxidative stress resulting from metabolic activation of carcinogenic estrogen plays a critical role in estrogen-induced carcinogenesis. In hamsters, a high incidence of malignant kidney tumors occurred in intact and castrated males and in ovariectomized females.16 Hamsters were implanted with 17b-estradiol (bE2), 17a-estradiol (aE2), 17a-ethinylestradiol (aEE), menadione, a combination of aE2 and aEE, or a combination of aEE and menadione for 7 months. The bE2-treated group developed kidney tumors and showed more than twofold increase in 8-isoPGF2a levels compared with controls. Kidneys of hamsters treated with a combination of menadione and aEE showed increased 8-isoPGF2a levels compared with control. This study indicated that chemical known to produce oxidative stress or a potent estrogen with poor ability produced oxidative stress were nontumorigenic in hamsters when given as single agents; however, when given together induced renal tumors.

Oxidative stress Free radical production is ubiquitous in all organisms and is enhanced in many diseases due to carcinogen exposure, such as under conditions of stress, which seems to contribute widely to cancer development.13, 15, 18 A free radical is any chemical species capable of independent existence, possessing one or more unpaired electrons. Chemical reactions seem to occur in every cell including oxidation and reduction of molecules. These reactions can lead to the production of free radicals that react with organic substrates such as lipids, proteins, and DNA. Through oxidation, free radicals cause damage to these molecules, disturbing their normal function and, therefore, may contribute to a variety of diseases. Oxidative stress occurs under pathologic circumstances and leads to an overproduction of highly reactive oxygen species (ROS), which can induce chemical changes in DNA, proteins, and lipids. An antioxidation system, which consists of enzymatic antioxidants and nonenzymatic antioxidants, defends against oxidative stress. Biological free radicals are thus highly unstable molecules that have electrons available to react with various organic substrates. The presence of free radicals and nonradical reactive molecules derived from free radicals at high concentrations are dangerous to living organisms, because of their ability to damage cell organelles. Nitrogen monoxide, superoxide anions, and related ROS and reactive nitrogen species (RNS) also play important modulating roles in certain signal transduction pathways.18 Several ROS-mediated reactions protect the cell from oxidative stress and serve to stabilize redox homeostasis. Chemical compounds capable of generating potential toxic oxygen species are referred to as prooxidants. In a normal cell, there is an appropriate prooxidant–antioxidant balance. However, this balance can be shifted toward the prooxidants when production of oxygen species is increased greatly such as following ingestion of certain chemicals or drugs, or with diminished levels of antioxidants.13 Authors13 have introduced the concept of oxidative stress, that is, the dissolution of the prooxidant–antioxidant equilibrium. Oxidative stress is basically caused by two mechanisms: (i) reduced concentration of antioxidants (e.g., due to mutated antioxidant enzymes, toxins, or the reduced intake of natural antioxidants) and (ii) increased of the number of oxygen-/nitrogen-/carbon-based reactive species. Following the oxidative stress, the activity of a given signal transduction molecule is either reduced or increased; and additionally, the function of the molecule may also change. Large amounts of ROS may be generated in one of the two ways, that is, by a significant stimulation of NAD(P)H oxidases or from the mitochondrial respiratory chain. In the mitochondria, ROS are the unwanted by-products of oxidative metabolism. The severity of the stress seems to affect the cell through the amount of ROS produced and biochemical status of the cell such as the activity of antioxidative and other enzymes, antioxidant content, pH, the integrity of membranes, redox characteristics, and others. Clinical, epidemiological, and experimental findings have provided evidence of a role of free radicals in the etiology of cancer.14 Furthermore, the generation of hydrogen peroxide by oxidative metabolism of estrogens has been documented. Thus an increase in hydroxyl free radical damage to DNA has been observed in human mammary tissue of breast cancer patients compared with controls,15 which are induced by estrogen. It has been reported that the superoxide generated by this

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redox system could be converted to hydrogen peroxide and subsequently to hydroxyl radical, which can cause oxidative damage in DNA.16 It has been generally accepted that active oxygen produced under stress is a detrimental factor, which causes lipid peroxidation and most interestingly oxidative damage to DNA. Both an imbalance of nutrients and ROS generation can alter the antioxidant activity of cells and apoptosis.17 Oxidative stress can be induced by decreasing the ability of a cell to scavenge reactive ROS or by a shortage of antioxidants. Cells utilize enzymatic and nonenzymatic compounds—the so-called antioxidants to defend themselves against oxidative stress. The term antioxidant can serve as a label for any substance whose presence, even at low concentrations, delays or inhibits the oxidation of a substrate.18 There are several molecules that play a role in antioxidant defense; these are either endogenous (internally synthesized) or exogenous (nutritional substances). Antioxidants can be divided into two groups depending on their mechanism of action. They can be either chain-breaking antioxidants or preventive antioxidants. Correlations between the extents of oxidative DNA damage in different tissues are important indicators of the individual oxidative stress levels in different physiological systems. Specific biomarkers show that oxidative damage can characterize cell damage in vitro. 8-Hydroxy-20-deoxyguanosine (8-OHdG) is characteristics for DNA damage.14 Multiple environmental mutagens and carcinogens are known to react with components of DNA with or without metabolic activation and form adducts with adenine, guanine, cytosine, and thymine. Guanine is known to be one of the most important among these targets. Even though background levels of oxidative damage to DNA exist, oxidative stress can lead to an increase in such damage, which has been described in various pathological conditions, such as carcinogenesis. 8-OHdG is one of the most frequent lesions among over 20 known base modifications caused by oxygen radicals.18 It is possible that the formation of 8-OHdG is proportional to that of other DNA modifications, regardless of organisms, type of ROS, and conditions of exposure. Therefore, measurement of 8-OHdG should be very useful in estimating oxygen. Elevated levels of 8-OHdG, a marker of hydroxyl radical-induced damage in DNA, have been detected in DNA from the kidney of hamsters chronically administered with diethylstilbestrol.11 It has been shown that there is concurrent damage not only to lipids but also to DNA during lipid oxidation.

Curcumin as an antioxidant Oxidative stress is one of the important pathogenic factors for cancer development. Among the antioxidants, curcumin (1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione; diferuloylmethane) is a well-known major dietary natural yellow pigment derived from the rhizome of the herb Curcuma longa (Zingiberaceae). It is also named turmeric and is a perennial herb belonging to the ginger family, native to India and Southeast Asia. It measures up to 1 m high with a short stem and tufted leaves. Curcumin is present in extracts of the plant. Curcuminoids are responsible for the yellow color of turmeric and curry powder. It is a pigment of turmeric that is used for imparting color and flavor to foods. This nonnutritive phytochemical is pharmacologically safe, considering that it has been consumed as a dietary spice, at doses up to 100 mg/day, for centuries.19 In the United States, curcumin is used as a coloring agent in cheeses, spices, mustard, cereals, pickles, potato flakes, soups, ice creams, and yogurts. The most active component is curcumin which makes up 2%–5% of the total spice in turmeric; it has been shown to be a potent anti-inflammatory, antioxidant, anticarcinogenic, and chemopreventive agent. This phytochemical has also been shown to suppress the proliferation of numerous types of tumor cells by downregulating c-myc,20 cyclin D1,21 activator protein-1 (AP-1),22 phosphatidylinositol-3-kinase/AKT signaling,23 and epidermal growth factor receptor (EGFR) signaling.24 An imbalance of nutrients and generation of ROS can alter the antioxidant activity of cells and induce apoptosis.17 Curcumin has been previously shown to prevent the formation of many chemically induced cancers including breast cancer in mice25, 26 and rats.27, 28 It has been shown20–30 to prevent cancer in the colon, skin, stomach, and duodenum following oral administration. Curcumin has shown chemopreventive and chemotherapeutic effects by blocking tumor initiation induced by benzo[a]pyrene and 7,12 dimethylbenz[a]anthracene25, 26, 28; it suppressed phorbolester-induced tumor promotion25, 26, suppressed carcinogenesis of the skin,25, 26, 31, 32 the forestomach26, and the colon29, 33 in mice. Curcumin and its analogues are known to protect against peroxidation damage. In the present study, the effect of curcumin as an antioxidant was analyzed by measuring several parameters related to oxidative stress. To gain an insight into the effects of curcumin on oxidative stress, an established in vitro experimental breast cancer model (Alpha-model) was used.34–37 Such a model was developed with the immortalized human breast epithelial cell line, MCF-10F,37 that was exposed to low doses of high LET (linear energy transfer) a particles (150 keV/mm) of radiation, values comparable to a particles emitted by radon progeny, and subsequently cultured in the presence or absence of 17b-estradiol (estrogen). This model consisted of human breast epithelial cells in different stages of transformation: (i) a control cell line, called MCF-10F, (ii) an estrogen-treated cell line, called Estrogen, (iii) a malignant cell line, called Alpha3, (iv) a malignant and tumorigenic cell line, called Alpha5, and (v) Tumor2 cell line derived from cells originating

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from a tumor after injection of Alpha5 cell line into the nude mice. The present study evaluates whether curcumin had any effect on oxidative stress in human breast epithelial cells transformed by the effect of radiation in the presence of estrogen. The Alpha Model gives an opportunity to study phenotypic and molecular alterations induced by an increased amount of oxidative stress.36, 37

Curcumin and a multifunctional nuclear transcription factor and the enzyme manganese superoxide dismutase protein expression It is known that curcumin interferes with the transcription activation induced by transcription factors, such as nuclear factor-kB (NFkB) resulting in the negative regulation of various cell cycle control genes and oncogenes.38–40 The nuclear NFkB complex containing p65 (Rel A) and p50 consists of closely related proteins that act as a multifunctional nuclear transcription factor.41 Such complex acts as a transcription factor that regulates the expression of multiple genes that promote carcinogenesis. Upstream activators of nuclear NFkB include various cellular stressors such as ROS, carcinogens, tumor promoters, apoptosis inducers, and cytokines. Curcumin may inhibit NFkB in experimental conditions and may also regulate DNA binding in pancreatic cancer cells, inducing apoptosis, thereby decreasing cell survival.42, 43 Activation of NFkB has been implicated in the resistance of cancer cells to radiotherapy and chemotherapy.38 It has also been implicated in growth control and G0/G1 to S-phase transition. These studies showed that NFkB protein expression was altered by curcumin since this compound is directly related to specific oxidative stress pathway. Fig. 1A and B shows the effect of curcumin (30 mM) on NFkB (50 kDa subunit) protein expression in the Alpha model. Results indicate that curcumin decreased NFkB protein expression of MCF-10F, Alpha3, Alpha5, and Tumor 2 cell lines in comparison to their counterparts without curcumin. The graph corresponds to the relative grade of luminescence of the protein expression level.

FIG. 1 Curcumin and NFkB. (A) Western blot analysis of MCF-10F, Alpha3, Alpha5, and Tumor2 cell lines treated with 30 mM of curcumin on NFkB protein expression. b-actin was used as a loading control. The graph represents (B) the relative grade of luminescence of the protein expression level. (C) NFkB gene expression of MCF-10F, and Tumor2 cell lines treated with 30 mM of curcumin (*P < .05). (D) Case #1: Cross section of breast specimen with noninvasive ductal carcinoma. Hematoxylin and eosin (HE) staining (left side). Protein expression of isolated cells from such tissue after three passages of estrogen receptor alpha (right side). Middle and bottom left side: nontreated and treated cells with 30 mM curcumin for 2 days in culture and stained with NFkB (E), and COX-2 (F).

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As shown in Fig. 1C, NFkB gene expression is decreased by the effect of curcumin in Tumor2. Fig. 1D corresponds to the cross section of breast specimen with noninvasive ductal carcinoma (Case #1) (HE) and isolated cells positive for ER alpha that were originated from such tissue (right side). Cells were in passage 3 after being cultured with 30 mM curcumin for 2 days. These studies indicated decreased NFkB and COX-2 protein expression by the effect of curcumin (left side). Under oxidative stress conditions, superoxide anions are produced which are converted to hydrogen peroxide through a specific antioxidant system, and then to water to complete the detoxification pathway.44 Abnormal levels of manganese superoxide dismutase (MnSOD) in cancer have been documented to play a critical role in the survival of cells. MnSOD is a nuclear-encoded mitochondrial antioxidant enzyme that catalyzes the conversion of superoxide radicals into molecular oxygen and hydrogen peroxide, which are further reduced into water by peroxide metabolizing enzyme systems, mainly catalase, an endogenous antioxidant that neutralizes hydrogen peroxide by converting it into water and oxygen. It has been reported that MnSOD2 expression was upregulated in response to oxidative stress in various types of cells and tissues by toxic stimuli and treatments, such as ionizing radiation and ultraviolet light.45 Fig. 2A and B shows the effect of curcumin on MnSOD (SOD-2) protein expression in the four cell lines under study. The control MCF-10F cell line responded to curcumin effect by decreasing its expression as indicated by Western blot analysis. Curcumin decreased SOD-2 protein expression in the control MCF-10F, Alpha3, and Tumor2 cell lines; however, MnSOD was slightly increased in the Alpha5 cell line. The MCF-10F and Alpha5 cell lines had a higher background of SOD-2 expression than the other two cell lines. On the other hand, Alpha3, Tumor2, and curcumin-treated cell lines had very low SOD-2 protein expression. Fig. 2C and D shows the representative images of the effects of curcumin (15 mM) analyzed either by peroxidase (Fig. 2C) immunofluorescence (Fig. 2D) techniques on SOD-2 protein expression of MCF-10F, Estrogen, Alpha3, Alpha5,

FIG. 2 Curcumin and MnSOD-2. (A and B) effect of curcumin on MnSOD (SOD-2) protein expression in the four cell lines indicated by Western blot analysis. Representative images of the effect of 15 mM curcumin on MnSOD protein expression analyzed by either (C) peroxidase or (D) immunofluorescence techniques in the MCF-10F, Alpha3, Alpha5, and Tumor2 cell lines.

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and Tumor2 cell lines. SOD-2 protein expression can be appreciated in mitochondria of those treated cell lines by peroxidase techniques. A decrease in such expression was observed by the effect of curcumin in all the cell lines. Oxidative damage can be measured by an agent that recognizes cell damage. High levels of hydrogen peroxide favor ROS formation and an increase in these molecules may play an important role in carcinogenesis.46 An observed excess of hydrogen peroxide can be an indication of the imbalance in redox control that causes oxidative cell damage in vitro. Thus, hydrogen peroxide can be useful to recognize oxidative stress in the cells. It has been reported that curcumin is a good scavenger of hydrogen peroxide at high concentrations (over 27 mM), but at low concentrations, it activates the Fenton reaction to increase the production of hydrogen peroxide.47 Hydrogen peroxide levels were measured by the Amplex™ Red Hydrogen Peroxide Assay kit in MCF-10F, Estrogen, Alpha3, Alpha5, and Tumor2 treated and nontreated cell lines with curcumin (15 mM). It was interesting to find that curcumin decreased the formation of hydrogen peroxide in all the cells of this model when compared with their nontreated counterparts (data not shown).35, 36 Curcumin (15 mM) was studied in MCF-10F, Estrogen, Alpha3, Alpha5, and Tumor2 cell lines. MnSOD, the principal defense against intracellular oxidative stress, decreased protein expression by the effect of curcumin.48

Curcumin and lipid peroxidation It has been generally accepted that active oxygen produced under stress is a detrimental factor that causes lipid peroxidation.49 This is a natural metabolic process under normal conditions; however, it is an important factor in the pathophysiological functions of numerous diseases.50 Specific biomarkers can recognize oxidative cell damage in vitro. The best biomarkers of lipid peroxidation are the isoprostanes such as the 8-isoprostaglandin F2a (8-isoPGF2a), malondialdehyde that shows lipid damage.51–53 It is a good marker to determine the effects of an antioxidant, such as curcumin. Authors have shown that lipid peroxidation is known to be a free radical-mediated reaction leading to cell membrane damage, and the inhibition of peroxidation by curcuminoids is mainly attributed to the scavenging of the reactive free radicals involved in such a process.54 It is one of the major isoprostanes formed in vivo and are initially generated in situ from esterified arachidonic acid in phospholipids and are released in the free form into the circulation. One of the most useful aspects of measuring 8-isoPGF2a to assess oxidative stress is the fact that they can be accurately and specifically measured since they were present in all biological tissues or fluids studied. In animals, the levels of 8-isoPGF2a are increased in oxidative stress models and reduced by dietary antioxidant supplementation. The DNA damage caused by peroxidation of lipids may be implicated in tumorigenesis. Such studies demonstrated that curcumin has a diverse range of molecular targets, confirming the concept that it acts upon numerous biochemical and molecular cascades. Evidence is increasing that isoprostanes, a novel class of prostaglandin-like compounds produced upon peroxidation of lipoproteins, play a causative role in carcinogenesis. Measurement of isoprostane concentrations is likely to have an important diagnostic potential to assess oxidative stress in several disorders, such as carcinogenesis. Studies of the modulation of isoprostanes by antioxidant nutrients are becoming available. For example, authors reported that Vitamin C administered to heavy smokers significantly reduced 8-isoPGF2a excretion in urine.25, 26 It has been generally accepted that active oxygen produced under stress is a detrimental factor which causes oxidative damage to DNA.49–57 Authors57 have shown that lifetime administration of curcumin reduced COX-2 expression in murine intestinal adenomas by decreasing the oxidative DNA adducts. Several studies have indicated that such substance may exert its effect by specifically inhibiting COX-2 enzyme activity and preventing the conversion of arachidonic acid to prostaglandin during prostaglandin synthesis. Therefore, the suppression of prostaglandin synthesis through selective inhibition of COX-2 has been suggested as a strategy applicable to the development of chemopreventive substances. In normal cells, COX-2 gene is highly inducible by signals that activate the NFkB pathway. In contrast, many types of cancer cells possess high basal levels of COX-2, due to permanent activation of NFkB in these cells followed by the expression of the COX-2 gene.57 The downstream product of COX-2 enzymatic activity is prostaglandin E2 (PGE2), which serves as an important stimulus for the induction of several cell signaling pathways, including the NFkB pathway that subsequently regulates cell proliferation and motility.35, 36

Curcumin and epithelial-mesenchymal transition Evidence strongly implicates that epithelial-mesenchymal transition (EMT) is involved in malignant progression affecting numerous genes abnormally expressed in many tumors and favors metastasis. We used MCF-10F and Tumor2 to determine the effect of curcumin (30 mM for 48 h) on EMT, migration, and invasion and found decreased E-cadherin, N-cadherin, b-catenin gene expression in comparison to their own controls all associated with EMT-related genes. Thus, curcumin prevented or delayed cancer progression and since it had an inhibitory effect only on malignant cell lines it can be an

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FIG. 3 Curcumin and epithelial mesenchymal transition. Graphs that represent the effect of curcumin on (A) Axl, (B) Slug, (C) Twist1, (D) N-Cadherin, (E) Caspase 3, and (F) Caspase 8 gene expression in MCF-10F and Tumor2 cell lines (*P < .05).

important substance for the prevention of breast carcinogenesis.58 In other studies we found that curcumin altered not only the gene expression of Axl, Slug, Twist1, and N-cadherin (Fig. 3A–D) but also the gene expression of ZEB2, vimentin, STAT-3, Fibronectin, c-Ha-ras as well as mutant p53, cyclin D1 and caveolin-1 protein expression in Tumor2 cell line when compared to its own controls. These studies were corroborated by apoptotic genes such as caspase 3 (Fig. 3E) and caspase 8 (Fig. 3F). EMT effect was noted in the transcription factor as NFkB.59

Curcumin and genomic instability All the previous changes induced a decrease in migratory and invasive capabilities of such a cell line. Thus, it seems that curcumin influence metastatic properties and apoptosis of malignant cells on antitumor activity in breast cancer cells transformed by low doses of a-particles and estrogen in vitro. Ras proteins must first release bound GDP mediated by GRF to reach their active GTP bound state. Curcumin increased GRF1 protein expression of MCF-10F and E cell line with or without curcumin preventing the binding. The cell lines Alpha3, Alpha5, and Tumor2 had increased protein expression.35, 36 However, curcumin decreased such expression increasing the binding. Results also showed that Rho-A protein expression decreased in the malignant cell lines Alpha3, Alpha5, and Tumor2 with 15 mM curcumin but not in MCF-10F and Estrogen. Previously, our laboratory demonstrated37 that the parental MCF-10F cell line exposed to double doses of alpha-particle radiation and treated with estrogen showed a more complex pattern of allelic imbalance when compared to cell lines treated with a single dose of radiation without estrogen. Progressive changes were observed due to allelic alterations induced by irradiation with either a single or double dose of alpha particles in the presence or absence of estrogen that were expressed either in the form of loss of

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heterozygosity/microsatellite instability and by parallel phenotypic changes, such as anchorage independence or invasive capabilities.34 Consequently, the doses of radiation of the cell lines after irradiation directly influenced these alterations and this genetic effect was more deleterious when given in combination with estrogen. The c-Ha-ras oncogene, mapped to 11p15.5, acquired transforming capacity either by a single-point mutation(s) in codon 12 or 61, resulting in the expression of an aberrant gene product. Then, in order to determine the effect of curcumin on c-Ha-ras, Rho-A, and SOD-2 protein expression in tissues of breast cancer specimens, we analyzed first hormone receptor status and Her 2 neu (antibodies against HER-2) by immunocytochemistry. Representative images of studies done in numerous breast specimens are shown in Fig. 4. Isolated cells derived from breast specimen with ductal carcinoma indicated positive c-Ha-ras and Rho-A protein expression by the effect of 30 mM of curcumin after three passages and 2 days in culture as shown in Fig. 4 (Case #1). Cross section of breast specimen with invasive ductal carcinoma, grade II can be observed in Fig. 4 (Case #2). Isolated cells from such tissue were considered a triple-negative breast cancer patient since cells were negative for ER alpha, ER beta, and ErbB-2 protein expression. Such cells were cultured for 10 passages and then cultured in the presence of 30 mM curcumin for 2 days. Those isolated cells showed decreased protein expression of Rho-A after 30 mM curcumin after 10 passages and 2 days in culture. Another group of isolated cells derived from invasive ductal carcinoma, grade II as shown in Fig. 4 (Case #2) showed decreased protein expression of SOD-2 and Rho-A after 30 mM curcumin, 10 passages, and 2 days in culture. Cross section of breast specimen with lobular carcinoma, grade III can be seen in Fig. 4 (Case #3). Isolated cells from such tissue were positive for ER alpha, ER beta, and ErbB-2 protein expression after 10 passages. Then cells were cultured in the presence of 30 mM curcumin for 2 days. Those isolated cells derived from lobular carcinoma, grade III as shown in Fig. 4 (Case #3) show decreased protein expression of c-Ha-ras and Rho-A in isolated cells derived from such tissue after three passages, and 2 days in culture. These results indicated that 30 mM curcumin induced apoptosis and decreased in cell number. On the other hand, other studies demonstrated that curcumin had a significant inhibitory effect not only on cell growth but also on colony formation in breast carcinogenesis by activating DNA damage. That curcumin seems to inhibit cell proliferation, induce apoptosis, and promote the accumulation of cells in the G2/M phase of the cell cycle has been previously demonstrated.35 Interestingly, curcumin has been found to inhibit proliferation of normal, nonselectively, as well as malignant cells, although its apoptotic effect is more profound in malignant cells.

Curcumin and specific biomarkers for cancer There are specific biomarkers that can recognize oxidative cell damage in vitro such as isoprostanes. Levels of 8-isoprostaglandin F2a (8-isoPGF2a) can indicate lipid damage and it is an end product that provides a useful tool to monitor oxidative stress in human organisms. Enzyme levels of 8-isoPGF2a of benign and malignant breast lesions derived from biopsy specimens is shown in Fig. 5A. Malignant tissues had significantly greater 8-isoPGF2a (pg/mL) than the normal counterpart tissue from the same patients. The DNA damage caused by peroxidation of lipids may be implicated in tumorigenesis58 and it can be suggested as an excellent marker for malignant progression in breast cancer. On the other hand, human breast cell lines have been characterized based on the expression of the cell surface markers CD44 and CD24. CD44 is a type I transmembrane glycoprotein that regulates cell adhesion and cell–cell as well as cellextracellular matrix interactions. CD24 is expressed in benign and malignant solid tumors and is also involved in cell adhesion and metastasis. Thus, we propose the surface expression of CD44 and CD24 as specific marker for breast cancer demonstrated by curcumin. Previous results revealed that curcumin decreased CD44 and CD24 gene and protein expression levels in MCF-10F (normal), Alpha5 (premalignant), and Tumor2 (malignant) cell lines compared with the levels in their counterpart control cells.60 Flow cytometry studies on cell surface can be used as a marker for cancer. As shown in Fig. 5B the CD44 +/CD24 + cell subpopulation was greater than the CD44 +/CD24 subpopulation in these three cell lines by the effect of curcumin. In the normal cell, MCF-10F had no significant effect but on Tumor2 cells decreased the subpopulation CD44 +/CD24 compared with the corresponding control cells. We also reported that in breast cancer specimens from patients, normal tissues were negative for CD44 and CD24 expression, while benign lesions were positive for both markers, and malignant tissues were found to be negative for CD44 and positive for CD24 in most cases. Such results indicated that curcumin improved the proportion of cells with CD44 +/CD24 + cells and decreased the proportion of CD44 +/CD24 type of cells considered malignant, suggesting that curcumin decreased cancerous types of breast cells. It can be suggested as a good marker to be used in the future. In conclusion, curcumin acted upon genomic instability in human breast epithelial cells transformed by the effect of radiation in the presence of estrogen since curcumin had an inhibitory effect only on malignant cell lines. Thus, curcumin could be used as an important substance for the prevention of breast carcinogenesis and since curcumin affected metastatic genes it can be used in breast cancer patients with an advanced disease without side effects commonly observed with

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FIG. 4 Case #1: Cells isolated from a breast specimen with noninvasive ductal carcinoma. Protein expression from such tissue after three passages of nontreated and treated cells with 30 mM curcumin for 2 days in culture and stained with c-Ha-ras and Rho-A. Case #2: Cross section of breast specimen with invasive ductal carcinoma, grade II. Protein expression of positive ER alpha, ER beta, and ErbB-2 of isolated cells derived from such tissue after 10 passages and 2 days in culture in the presence of curcumin. Isolated cells derived from such breast specimen. Effect of SOD-2 and Rho-A protein expression on nontreated and treated cells cultured in 30 mM curcumin after 2 days (10 passages). Case #3: Cross section of breast specimen with lobular carcinoma, grade III. Protein expression of negative ER alpha, positive ER beta, and ErbB-2 of isolated cells derived from such tissue after 10 passages and 2 days in culture in the presence of curcumin. Protein expression of c-Ha-ras and Rho-A in nontreated and treated cells with curcumin (30 mM) in isolated cells derived from the same tissue.

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FIG. 5 Curcumin and biomarkers of genomic instability. (A) 8-Iso-PGF2a enzyme levels (pg/mL) present in benign and malignant breast specimens and measured by an immunoassay kit (Assay Designs, Ann Arbor, MI). (B) Effect of curcumin on CD44+/CD24+ cell and CD44+/CD24 subpopulations by flow cytometry.

therapeutic drugs. On the other hand, this breast cancer model can be considered as an important tool for monitoring the effects of natural dietary compounds on multiple protein expressions that could be key factors in signaling pathways in carcinogenesis.

Summary points l l l l l

l l l

l l

Oxidant stress plays a crucial role in estrogen-induced carcinogenesis. Free radicals play a crucial role in the etiology of cancer. Specific biomarkers of oxidative damage can characterize cell damage in vitro as DNA damage. Oxidative stress is an important pathogenic factor of cancer development. Among antioxidants, curcumin (1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione; diferuloylmethane) is a major dietary natural yellow pigment derived from the rhizome of the herb C. longa (Zingiberaceae). Curcumin and its analogues are known to protect against peroxidation damage. Curcumin as an antioxidant is measured by several parameters related to oxidative stress. To gain an insight into the effects of curcumin on oxidative stress an established in vitro experimental breast cancer model was used. Specific biomarker of lipid peroxidation is the isoprostanes such as the 8-isoprostaglandin F2a. Curcumin as an important substance for the prevention of breast carcinogenesis.

Acknowledgments The financial support received from grant FONDECYT #1080482, #1120006, and #1200656; and MINEDUC-UTA (GMC) is greatly appreciated. I am sincerely thankful for the technical assistance I received from Guiliana Rojas Ordo´n˜ez, Richard Ponce Cusi, Georgina Vargas Marchant, and Leodan A. Crispin.

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References 1. Henderson BE, Pike MC, Ross RK. Epidemiology and risk factors. In: Bonadonna GJ, editor. Breast cancer: diagnosis and management. New York: John Willey and Sons Ltd.; 1984. 2. Krieger N. Exposure, susceptibility and breast cancer risk: a hypothesis regarding exogenous carcinogens, breast tissue development, and social gradients, including black/white differences, in breast cancer incidence. Breast Cancer Res Treat 1989;13(3):205–23. 3. Bernstein L. The epidemiology of breast cancer. LOWAC J 1998;1:7–13. 4. Feigelson HS, Henderson BE. Estrogens and breast cancer. Carcinogenesis 1996;17:2279–84. 5. Bernstein L, Ross RK, Pike MC, Brown JB, Henderson BE. Hormone levels in older women: a study of post-menopausal breast cancer patients and healthy population controls. Br J Cancer 1990;61:298–302. 6. Toniolo PG, Levitz M, Zeleniuch-Jacquotte A, Banerjee S, Koenig KL, Shore RE, et al. A prospective study of endogenous estrogens and breast cancer in post-menopausal women. J Natl Cancer Inst 1995;86:1076–82. 7. Berrino F, Muti P, Micheli A, Bolelli G, Krogh V, Sciajno R, et al. Serum sex hormone levels after menopause and subsequent breast cancer. J Natl Cancer Inst 1996;88:291–6. 8. Li J, Li SA, Klicka JK, Parsons JA, Lam LKT. Relative carcinogenic activity of various synthetic and natural estrogens in the Syrian hamster kidney. Cancer Res 1983;43:5200–4. 9. International Agency for Research on Cancer. Monographs on the evolution of carcinogenic risks to humans: hormonal contraception and postmenopausal hormone therapy. vol. 72. Lyon, France: IARC; 1999. 10. Cavalieri EL, Stack DE, Devanesan PD, Todorovic R, Dwivedy I, Higginbotham S, et al. Molecular origin of cancer: catechol estrogen-3,4-quinones as endogenous tumor initiators. Proc Natl Acad Sci U S A 1997;99:10937–42. 11. Highman B, Greenman DL, Norvell MJ, Farmer J, Shellenberger TE. Neoplastic and preneoplastic lesions induced in female C3H mice by diets containing diethylstilbestrol or 17b estradiol. J Environ Pathol Toxicol 1980;4:81–5. 12. Shull DJ, Spady TJ, Snyder MC, Johansson S, Pennington KL. Ovary intact, but not ovariectomized female ACI rats treated with 17b estradiol rapidly develop mammary carcinoma. Carcinogenesis 1997;18:1595–601. 13. Sies H. Oxidative stress: oxidants and antioxidants. Exp Physiol 1997;82:291–5. 14. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci U S A 1993;90(17):7915–22. 15. Malins DC, Holmes EH, Polissar NL, Gunselman SJ. The etiology of breast cancer. Characteristic alteration in hydroxyl radical-induced DNA base lesions during oncogenesis with potential for evaluating incidence risk. Cancer 1993;71(10):3036–43. 16. Li JJ, Li SA. Estrogen-induced tumorigenesis in hamsters: roles for hormonal and carcinogenic activities. Arch Toxicol 1984;55:110–8. 17. Bhat HK, Calaf GM, Hei TK, Loya T, Vadgama JV. Critical role of oxidative stress in estrogen-induced carcinogenesis. Proc Natl Acad Sci U S A 2003;100(7):3913–8. 18. Halliwell B. Oxygen and nitrogen are pro-carcinogens. Damage to DNA by reactive oxygen, chlorine and nitrogen species: measurement, mechanism and the effects of nutrition. Mutat Res 1999;443:37–52. 19. Ammon HP, Wahl MA. Pharmacology of curcuma longa. Planta Med 1991;57(1):1–7. 20. Aggarwal BB, Kumar A, Bharti AC. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res 2003;23:363–98. 21. Mukhopadhyay A, Banerjee S, Stafford LJ, Xia C, Liu M, Aggarwal BB. Curcumin-induced suppression of cell proliferation correlates with downregulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phodphorilation. Oncogene 1994;21:8852–61. 22. Surh YJ, Han SS, Keum YS, Seo HJ, Lee SS. Inhibitory effects of curcumin and capsaicin on phorbol ester-induced activation of eukaryotic transcription factors, NF-kappaB and AP-1. Biofactors 2000;12:107–12. 23. Korutla L, Kumar R. Inhibitory effect of curcumin on epidermal growth factor receptor kinase activity in A431 cells. Biochim Biophys Acta 1994;1224:597–600. 24. Hussain AR, Al-Rasheed M, Manogaran PS, Al-Hussein KA, Platanias LCA, Kuraya K, et al. Curcumin induces apoptosis via inhibition of PI3’-kinase/AKT pathway in acute T cell leukemias. Apoptosis 2006;11:245–54. 25. Huang MT, Newmark HL, Frenkel K. Inhibitory effects of curcumin on tumorigenesis in mice. J Cell Biochem 1997;27:26–34. 26. Huang MT, Lou YR, Ma W, Newmark HL, Reuhl KR, Conney AH. Inhibitory effects of dietary curcumin on forestomach, duodenal, and colon carcinogenesis in mice. Cancer Res 1994;54(22):5841–7. 27. Pereira MA, Grubbs B, Barnes LH. Effects of phytochemicals, curcumin and quercetin upon azoxymethane-induced colon cancer and 7, 12-dimethylbenz[a]anthracene-induced mammary cancer in rats. Carcinogenesis 1996;17:1305–11. 28. Singletary K, MacDonald C, Wallig M, Fisher C. Inhibition of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis and DMBA-DNA adduct formation by curcumin. Cancer Lett 1996;103(2):137–41. 29. Rao CV, Rivenson A, Simi B, Reddy BS. Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound. Cancer Res 1995;55(2):259–66. 30. Kawamori T, Lubet R, Steele VE, Kelloff GJ, Kaskey RB, Rao CV, et al. Chemopreventive effect of curcumin, a naturally occurring antiinflammatory agent, during the promotion/progression stages of colon cancer. Cancer Res 1999;59(3):597–601. 31. Limtrakul P, Lipigorngoson S, Namwong O, Apisariyakul A, Dunn FW. Inhibitory effect of dietary curcumin on tumorigenesis in mice. J Biochem Suppl 1997;27:26–34. 32. Lu YP, Chang RL, Lou YR, Huang MT, Newmark HL, Reuhl KR, et al. Effect of curcumin on 12-O-tetradecanoylphorphol-13-acetate-and ultraviolet B light-induced expression of c-jun and c-fos in JB6 cells and in mouse epidermis. Carcinogenesis 1994;15(10):2363–70.

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33. Kim JM, Araki DJ, Park CB, Takasuka N, Baba-Toriyama H, Ota T, et al. Chemopreventive effect of carotenoids and curcumins on mouse colon carcinogenesis after 1,2 dimethylhydrazine initiation. Carcinogenesis 1998;19(1):81–5. 34. Calaf GM, Hei TK. Establishment of a radiation- and estrogen induced breast cancer model. Carcinogenesis 2000;21:769–76. 35. Calaf GM, Echiburu´-Chau C, Wen G, Balajee AS, Roy D. Effect of curcumin on irradiated and estrogen-transformed human breast cell lines. Int J Oncol 2012;40:436–42. 36. Calaf GM, Echiburu´-Chau C, Roy D, Chai Y, Wen G, and. Balajee, A. S. Protective role of curcumin in oxidative stress of breast cells. Oncol Rep 2011;26:1029–35. 37. Roy D, Calaf GM, Hei TK. Allelic imbalance at 11p15.5-15.4 correlated with c-Ha-Ras mutation during radiation-induced neoplastic transformation of human breast epithelial cells. Int J Cancer 2003;103:730–7. 38. Aggarwal BB, Surth YH, Shishodia S. The molecular targets and therapeutics of curcumin in health and disease. Advances in experimental biology, vol. 995. Springer; 2007. 39. Aggarwal BB, Shishodia S, Takada Y, Banerjee S, Newman RA, Bueso-Ramos CE, et al. Curcumin suppresses the paclitaxel-induced nuclear factorkappa B pathway in breast cancer cells and inhibits lung metastasis of human breast cancer in nude mice. Clin Cancer Res 2005;11:7490–8. 40. Baeuerle PA. The inducible transcription activator NF-kappa B: regulation by distinct protein subunits. Biochim Biophys Acta 1991;1072:63–80. 41. Brennan P, O’Neill LA. Inhibition of nuclear factor kappaB by direct modification in whole cells—mechanism of action of nordihydroguaiaritic acid, curcumin and thiol modifiers. Biochem Pharmacol 1998;55:965–73. 42. Siebenlist U, Franzoso G, Brown K. Structure, regulation and function of NF-kappa B. Annu Rev Cell Biol 1994;10:405–55. 43. Anand P, Sundaram C, Jhurani S, Kunnumakkara AB, Aggarwal BB. Curcumin and cancer: an ‘old-age’ disease with an age-old’ solution. Cancer Lett 2008;267:133–64. 44. Basile V, Ferrari E, Lazzari S, Belluti S, Pignedoli F, Imbriano C. Curcumin derivatives: molecular basis of their anti-cancer activity. Biochem Pharmacol 2009;78:1305–15. 45. Wispe JR, Clark JC, Burhans MS, Kropp KE, Korfhagen TR, Whitsett JA. Synthesis and processing of the precursor for human mangano-superoxide dismutase. Biochim Biophys Acta 1989;994:30–6. 46. Klaunig JE, Kamendulis LM. The role of oxidative stress in carcinogenesis. Annu Rev Pharmacol Toxicol 2004;44:239–67. 47. Kunchandy E, Rao MN. Effect of curcumin on hydroxyl radical generation through Fenton reaction. Int J Pharm 1989;57:173–6. 48. Ahn J, Gammon MD, Santella RM, Gaudet MM, Britton JA, Teitelbaum SL, et al. Associations between breast cancer risk and the catalase genotype, fruit and vegetable consumption, and supplement use. Am J Epidemiol 2005;162:943–52. 49. Urata Y, Yoshito I, Hiroaki M, Shinji G, Takehiko K, Junji Y, et al. 17b estradiol protects against oxidative stress-induced cell death through the glutathione/glutaredoxin-dependent redox regulation akt in myocardiac H9c2 cells. J Biol Chem 2006;281(19):13092–102. 50. Roberts II LJ, Morrow JD. Measurement of F2-isoprostanes as an index of oxidative stress in vivo. Free Radic Biol Med 2000;28:505–13. 51. Morrow JD, Minton TA, Badr KF, Roberts II LJ. Evidence that the F2-isoprostane, 8-epi-prostaglandin F2a, is formed in vivo. Biochim Biophys Acta 1994;1210:244–8. 52. Sodergren E, Cederberg J, Basu S, Vessby B. Vitamin E supplementation decreases basal levels of F(2)-isoprostanes and prostaglandin (2alpha) in rats. J Nutr 2000;130:10–4. 53. Wong YT, Ruan R, Tay FE. Relationship between levels of oxidative DNA damage, lipid peroxidation and mitochondrial membrane potential in young and old F344 rats. Free Radic Res 2006;40:393–402. 54. Priyadarsini I. Free radical reactions of curcumin in membrane models. Free Radic Biol Med 1997;23(6):838–43. 55. Valko M, Izakovic M, Mazur M, Rhodes CJ, Telser J. Role of oxygen radicals in DNA damage and cancer incidence. Mol Cell Biochem 2004;266:37–56. 56. Marnett LJ. Oxyradicals and DNA damage. Carcinogenesis 2000;21:361–70. 57. Williams CS, Mann M, Dubois RN. The role of cyclooxygenases in inflammation cancer and development. Oncogene 1999;18:7908–16. 58. Gallardo M, Calaf GM. Curcumin inhibits invasive capabilities through epithelial-mesenchymal transition in breast cancer cell lines. Int J Oncol 2016;49(3):1019–27. 59. Gallardo M, Calaf GM. Curcumin and epithelial-mesenchymal transition in breast cancer cells transformed by low doses of radiation and estrogen. Int J Oncol 2016;48(6):2534–42. 60. Calaf GM, Ponce-Cusi R, Abarca-Quinones J. Effect of curcumin on the cell surface markers CD44 and CD24 in breast cancer. Oncol Rep 2018;39 (6):2741–8.

Chapter 33

Curcumin analogs, oxidative stress, and prostate cancer Marco Bisoffi and Justin M. O’Neill Chemistry and Biochemistry, Schmid College of Science and Technology, Chapman University, Orange, CA, United States

List of abbreviations AhR AIF ANT AP-1 AR ARE ARNT CRPC CYP DHT Endo G ERK GCL GP GR GSH GS-SG GST H2O2 HO% HO-1 IM Keap1 mPTPC NAD(P)H NCBI NCI NFkB NQO1 Nrf2 O2%2 OH OM PIN PKC ROS SAR SH SOD

arylhydrocarbon receptor apoptosis inducing factor adenine nucleotide translocase activated protein 1 androgen receptor antioxidant response element AhR nuclear translocator castration resistant prostate cancer cytochrome P450 dihydrotestosterone endonuclease G extracellular regulated kinase glutamate cysteine ligase glutathione peroxidase glutathione reductase reduced glutathione oxidized glutathione glutathione S-transferases hydrogen peroxide hydroxyl radical heme oxygenase-1 mitochondrial inner membrane Kelch-like ECH-associated protein 1 mitochondrial permeability transition pore complex nicotinamide adenine dinucleotide (phosphate) H National Center for Biotechnology Information National Cancer Institute nuclear factor kappa B NAD(P)H:quinone oxidoreductase-1 nuclear factor erythroid 2 related factor 2 superoxide radical hydroxyl mitochondrial outer membrane prostatic intraepithelial neoplasia protein kinase C reactive oxygen species structure activity relationship sulfhydryl superoxide dismutase

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00033-X © 2021 Elsevier Inc. All rights reserved.

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2,3,7,8-tetrachlorodibenzo-p-dioxin thioredoxin reductase transgenic adenocarcinoma of the mouse prostate voltage-dependent ion channel mitochondrial membrane potential

Introduction Within the sections of this chapter three main subjects will be covered: l

l

l

The plant natural product curcumin (diferuloylmethane) and its naturally occurring curcuminoids are presented as major polyphenolic constituents in the popular spice turmeric, which is gained from the roots of the plant Curcuma longa (Linn.) and other species that are members of the ginger family Zingiberaceae, a native plant throughout South-East Asia.1 In this chapter, we discuss curcumin, curcuminoids, and chemical analogs of curcumin, which were synthesized by combinatorial chemistry based on the structural components of the original chemical structure, featuring a “combichemical” design.2, 3. Throughout this chapter, the word “analog” will be used instead of “derivative”. Although the two are largely overlapping in meaning, “analog” better accommodates the de novo synthesis of new structures, as opposed to the chemical modifications of existing molecules. The effect of natural curcuminoids and chemical analogs of curcumin on the redox status of human cells, taking into consideration the dual capacity to act as both anti- and prooxidant agents, depending on the chemical nature, dose, and the cellular and molecular context in which they are investigated. Such a dual role often also depends on the concentration, which is typical for hormetic compounds, including curcumin. In fact, there is now sufficient evidence to list this polyphenolic natural product and its derivatives in this category of molecules.4 The role of oxidative stress in prostate cancer as it pertains to its contribution to the malignant process underlying the transformation of normal epithelial cells into the premalignant precursor of prostate intraepithelial neoplasia (PIN) and the further progression into invasive and metastatic cells. This chapter will focus on reactive oxygen species as a supporter of prostate cancer development.5

The individual subjects listed above represent rather large bodies of scientific research reflected in a high number of published studies. Their full coverage would thus exceed the scope of this chapter and the reader is referred to excellent previously published reports reviewing these topics.1, 3–5 However, there remains substantial room to combine these main subjects specifically for prostate cancer and to study the effect of naturally occurring and synthetic agents with curcuminoid structure on the redox status and on the cellular response in cells of prostatic origin. In fact, much of the knowledge about how curcumin and its analogs influence the redox balance in prostate cancer cells is implied, often from data generated in other cell systems, rather than proven. The scope of this chapter is to explore future avenues and possibilities of utilizing the oxidant properties of curcuminoid agents in the prevention of and fight against a major contributor to mortality from a malignancy in men worldwide, that is, prostate cancer.6, 7 This is achieved by exploring the available published information, specific for prostate cancer, on the effects of curcuminoids on components of the cellular machinery responsible for the balanced redox status. To achieve this, the chapter will: l

l

l

Introduce the subject of oxidative stress, in particular, its factors and pathways as they pertain to prostate cancer and review the chemistry of curcumin and its analogs, as well as explore the mechanisms of action underlying their anti- and prooxidant activities. Discuss the current knowledge of the effect of curcuminoid agents in prostate cancer cells relative to the mechanisms of action. Summarize the current status of knowledge of curcuminoid agents and their ability to influence the redox status in prostate cancer cells, and their possible clinical use.

Prostate cancer and oxidative stress Prostate cancer: A brief introduction Prostate cancer or prostatic adenocarcinoma is a hormonally driven and dependent malignancy of the glandular structures primarily within the peripheral zone of the prostate, which is an organ that contributes to the optimal composition of the

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ejaculate fluid. Prostate cancer can develop through well-defined precursors including PIN to become a highly invasive and metastatic disease. In addition, prostate cancer cells initially thrive on the relatively high concentrations of androgen (testosterone, dihydrotestosterone [DHT]) produced by the testicles and adrenal glands. This constitutes the basis of androgen ablation therapy, which aims at reducing the androgen concentration systemically and locally and/or at blocking the androgen receptor (AR); the latter is a proproliferative signaling factor in prostate cancer cells and its blockage leads to cell death.8 Unfortunately, the progression of prostate cancer cells to a more aggressive and metastatic phenotype is possible through a number of pathways, including the androgen-independent activation of the AR through gene amplification and mutation as well as intra-tumoral synthesis of testosterone.9 This constitutes castration-resistant prostate cancer (CRPC) which often displays simultaneous metastatic capabilities with a propensity for the bone microenvironment causing much morbidity.10 At this stage, therapeutic options are limited, and the disease has become incurable. Of the approximately 900,000 new cases diagnosed annually worldwide, about a third develop this advanced phenotype and results in the estimated annual mortality of over a quarter-million men, making this malignancy one of the most common causes of death from cancer in men.7

Prostate cancer and oxidative stress: Possible factors Denham Harman introduced his “free radical theory of aging” in the 1950s, according to which the aging process is based on the cumulative production of free radicals over time that affects cellular lifespan by damaging essential biomolecules including DNA, lipids, and proteins.11 Since that time, the theory was expanded to age-related diseases, including prostate cancer, which tends to develop primarily in men over the age of 55.5 Based on epidemiological and experimental data using cell models and human tissues, it is now widely accepted that prostatic tissue, as it ages, changes from a neutral oxidant to a prooxidant state, and that components of the diet, including those that affect cell oxidant state play an important role in influencing this process (Fig. 1). Because of the effect of free radicals on pathways that are essential for cellular transformation, it is conceivable that the change toward a prooxidant state is linked to the onset of cancer. Many etiologic factors potentially responsible for increased oxidative stress in prostatic tissues have long been recognized, including diet, obesity, hormone imbalance, and chronic inflammation.12, 13 Once the cellular transformation has been initiated, persistent oxidative stress continues to support cancer progression through acquired genomic mutations that lead to the failure of antioxidant defense mechanisms, thereby constituting a vicious cycle of prooxidant activity5 (Fig. 1).

Reactive oxygen species: A paradox in (prostate) cancer Aerobic cells have been referred to as “The antioxidant machine” in Nick Lane’s book “Oxygen, the Molecule that made the World”. Although oxygen can inflict potential harm on cells, it is absolutely necessary for aerobic life. Oxidative stress is induced by deviations from the tightly controlled redox balance, as defined by the levels of reactive oxygen species (ROS) and enzymatic antioxidant activities.5, 11, 12 These players will be introduced in the next sections in the context of curcuminoids and prostate cancer. Diet, obesity, hormone imbalance

ROS

Age Redox balance

Redox imbalance Impaired antioxidation

Oxidative stress Genomic instability

FIG. 1 Oxidative stress in prostate cancer. The level of reactive oxygen species (ROS) and inflammation increase with age, which is influenced by diet, obesity, and hormone imbalance. Redox balance changes to redox imbalance, which induces a vicious cycle of oxidative stress leading to genomic instability, impaired antioxidant mechanisms, and further redox imbalance.

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Normal cells exert redox balance

Required to induce cell death

Level of ROS

Required for normal funcon

Cancer cells are inhibited

Level of ROS

Anoxidant intervenon

Normal cells

Cancer cells

Normal cells exert redox balance

Prooxidant intervenon

Cancer cells are killed Level of ROS

Normal cells

Cancer cells

Normal cells

Cancer cells

FIG. 2 The “ROS threshold concept” for the anti- and prooxidant intervention by redox-active agents, including curcuminoids. Bars represent the levels of ROS in normal (white) and cancer (black) cells. The light and dark shadowed areas represent ROS levels required for normal function and for the induction of cell death, respectively. Antioxidant intervention would inhibit cancer cells, while prooxidant intervention would kill cancer cells. Normal cells would compensate both situations and maintain redox balance.

While research on cellular redox balance was initiated with a focus on the deleterious effects of ROS on cellular components and ultimately cell viability, subsequent studies have revealed that ROS are important second messengers that support signal transduction pathways implicated in normal cell function. ROS have thus been called “a double-edged sword”.14, 15 This concept of a double edge sword is of particular interest when considering a change in redox balance as a therapeutic intervention against cancer, and leading some authors to include Shakespearean innuendos in the title of their reports, such as: “Cancer cell killing via ROS—To increase or decrease, that is the question.”15 This question is justified and applies perfectly to the subject of this chapter, that is, the use of curcuminoids against prostate cancer because curcumin and its analogs can be potent anti-, as well as prooxidant agents. How does this dual capacity affect their use as potential anticancer agents? To answer this question, the widely accepted “ROS threshold concept” (Fig. 2) should be considered.14, 15 As outlined earlier, prostate cancer cells are characterized by higher ROS levels when compared to their normal counterparts.5 According to the ROS threshold concept, cancer cells are adapted to a higher level of ROS which they exploit for elevated survival and proliferation. However, cancer cells are also more sensitive to any further elevation in ROS levels and because of it, they are more prone to cell death. It is conceivable then that antioxidant intervention, for example, by curcuminoid agents would decrease ROS levels in normal cells, which would compensate by downregulating scavenging agents such as glutathione, thereby maintaining redox balance, while cancer cells would be inhibited by the diminished ROS levels. In contrast, normal cells would compensate a prooxidant intervention and increased ROS levels by upregulating the scavenging defense system to maintain redox balance, while cancer cells would be pushed toward cell death due to the overwhelming of the antioxidant system (Fig. 2). Whether this is a viable antiprostate cancer scheme for the use of bifunctional oxidants such as the xenobiotic curcuminoid agents, remains to be shown. The next section will review the dual function of curcumin and its analogs with respect to the chemistry that underlies their anti- and prooxidant activities.

Curcumin, curcuminoids, and curcumin analogs Chemistry and biochemistry of curcumin, curcuminoids, and curcumin analogs Curcumin (1E,6E)-(1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), or diferuloylmethane, is the principal curcuminoid component of the widely used spice turmeric, which is isolated from the roots of the plant Curcuma longa,

Curcumin analogs, oxidative stress, and prostate cancer Chapter

Curcumin

OCH3

H3CO Enone

HO Oxy

Ene

OH

1,3-Ketoenolyl Aryl

C7-Alkenyl

O OH

O OCH3

H3CO HO

OH

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FIG. 3 The chemical structure and individual moieties of the natural product curcumin. All moieties are targets of combinatorial chemistry, including the methoxy, oxy, aryl, enone, ene, 1,3-ketoenolyl, and the C7-alkenyl groups. Some of the naturally occurring curcuminoids are isolated from the popular spice turmeric from the roots of the plant Curcuma longa (Linn.) and other species that are members of the ginger family Zingiberaceae are shown.

H O O

OMe

33

O

(H3CO) HO

Hexahydrocurcumin

OH (Bis)demethoxycurcumin

OH OH

O OCH3

H3CO

OCH3

H3CO

HO

OH HO

OH

Hexahydrocurcuminol Cyclocurcumin

a member of the ginger family Zingiberaceae, native throughout South-East Asia.1 The curcuminoids are natural phenols which exist in several tautomeric forms, including a keto and an energetically more stable enol form1, 3, 16 (Fig. 3). Curcumin incorporates several functional groups: The aromatic phenol ring systems are connected by two a,b-unsaturated carbonyl (1,3-ketoenolyl) groups. These groups are good Michael acceptors and readily undergo nucleophilic addition by forming irreversible adducts with the sulfhydryl (SH) group of multiple target molecules. The diketones form stable enols and are readily deprotonated to form enolates.1, 3, 16 The term “curcuminoid” relates to organic compounds with chemical moieties that are either identical or similar to those found in curcumin itself. However, “curcuminoid” is not a very well-defined term, making it difficult to identify the exact rules for inclusion or exclusion of compounds into this group. In taking a less stringent view, this group includes other components of turmeric, such as the di-, tetra-, and hexahydrocurcumins, the demethoxy- and bisdemethoxycurcumins, the curcuminols, and cyclocurcumin (Fig. 3), and also gingerol, capsaicin, and caffeic acid.2 The chemical structure of curcumin is being extensively exploited by combinatorial chemistry and structure–activity relationship (SAR) studies due to its relative simplicity, its well-known bioactivity, and its safe pharmacological profile, including prostate cancer model systems.2, 3, 16 A major driver of these efforts is its long-known limited bioavailability, which has barred its rapid development into a clinically used preventive or therapeutic agent.17 Curcumin is a hydrophobic compound and although its physicochemical parameters are not unfavorable for drug development purposes, it has been shown to be poorly absorbed. Oral and intraperitoneal administration indicates that serum concentrations are particularly low and often do not reach more than 0.1% of intake, followed by rapid clearance in the feces and in bile/urine,17 and metabolization to the less bioactive curcumin glucuronide, curcumin sulfate, and ferulic acid.2, 17 To date, there have been many reports on the generation of chemical libraries using curcumin as a starting structure.2, 3, 16 Most often, detailed information on their bioavailability remains scarce, but whenever a more potent effect is observed, it could be due to enhanced absorption and distribution, longer retention, and/or decreased systemic secretion. Human data pertaining to the bioavailability of curcumin analogs is currently unavailable since these analogs are not yet in clinical trials. This is in contrast to curcumin itself, which in its many different formulations to increase bioavailability and thus, bioefficacy, is now featured in over 200 clinical trials.18 To be able to understand the oxidant actions of agents with curcuminoid character with respect to prostate cancer, it is necessary to explore the several mechanisms of action by which curcuminoids can affect the cellular redox status. Interestingly, curcuminoids exert both anti- and prooxidant effects. This is not entirely surprising, as an initial prooxidant effect by curcumin can lead to a secondary antioxidant response. Hence, curcumin, as well as its many derivatives are now being viewed as hormetic compounds.4 While these effects have not all been shown in prostate cancer cells, they can be reasonably implied by extension, as the targets described in other systems are also present in cells of prostatic origin.

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Antioxidant versus prooxidant activities of curcumin, curcuminoids, and curcumin analogs Antioxidant activities: Mechanisms Curcumin, curcuminoids, and the chemical structures described in its various analogs have been recognized as antioxidant agents. This capability is thought to be at the very heart of the chemopreventive activity of curcumin, as postulated from epidemiological data in population studies.1 In this capacity, it is assumed that long-term exposure to dietary curcumin would counteract the age-dependent increase of oxidative stress in prostate tissue (Fig. 1). Different mechanisms of action responsible for the antioxidant activities of curcumin and agents with curcuminoid structure have been identified and further investigated. There are two major mechanisms, that is, nonenzymatic (direct) and enzymatic (indirect) (Fig. 4). The nonenzymatic mechanisms are due to the scavenging capability of these molecules. Accordingly, curcuminoids have been shown to scavenge free radicals and ROS, including hydroxyl radicals (HO%), superoxide radicals (O2%
), singlet oxygen, and peroxyl radicals. This reaction can occur after the following scheme: (i) R-OO% + curcumin > ROOH + curcumin% (reversible step) (ii) Curcumin% + X% ! non-radical curcuminoid products (irreversible step) where R-OO% is radical, ROOH is the oxidized reactant, and X% is another radical species. General conclusions from SAR studies can be made for the structural requirements of curcumin to act as a direct antioxidant. The hydrogen bonding interaction between the phenolic hydroxyl and the ortho-positioned methoxy groups in curcumin markedly influences the OdH bond energy and H-atom abstraction by free radicals. Changes in the composition and distribution of these moieties affect scavenging potential, as shown for bisdemethoxycurcumin and demethoxycurcumin.19 The phenolic hydroxyl groups are needed for antioxidant activity and the presence of more than one of these groups, for example in the curcumin derivative bis(3,4-dihydroxycinnamoyl)-methane, confers enhanced activity.20 The position of the hydroxyl groups also affects the antioxidant potential, with position 2 (ortho) yielding enhanced antioxidant activity (e.g., in bis(2-hydroxycinnamoyl)-methane), and similar findings were reported for curcumin analogs with shortened (C-5) carbon linkers, that is, diarylpentanoids or mono-carbonyl/mono-ketone compounds, with additional hydroxyl substituents on the phenyl rings, and in structures carrying 3-alkoxy-4-hydroxyphenyl units.21 The b-diketone moiety, which is part of the Michael acceptor reactivity of curcuminoids (see below), was also shown to be involved in the antioxidant effect (e.g., in dimethyltetrahydrocurcumin).22 In addition, the presence of an alkoxy group in the ortho position relative to the hydroxyl group has been reported to potentiate the antioxidant activity.23 Curcumin and analogs with curcuminoid character may also act as antioxidants by enzymatic (indirect) mechanisms. The best-studied is the effect on the nuclear factor erythroid 2 related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/ Keap1) pathway, which regulates the expression of downstream phase 2 cytoprotective (detoxification) proteins by binding to antioxidant response elements (ARE) in their promoters (Fig. 4). In this scheme, Keap1 acts as a major sensor for the redox status within the cell through its cysteine residues. When these residues are in their reduced form, Keap1 is bound to the transcription factor Nrf2 in the cytoplasm, thereby sequestering it and inhibiting its nuclear translocation. When the cellular redox balance shifts toward a more prooxidative state, Keap1 cysteine residues are oxidized which results in the dissociation of the two proteins, allowing Nrf2 to translocate into the nucleus. Upon dimerization with a number of different co-transcription factors, including Maf proteins, the resultant Nrf2 heterodimers bind to the ARE regulatory region of phase 2 cytoprotective genes, including glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase 1 (HO-1), glutamate-cysteine ligase (GCL), glutathione peroxidase (GP), and superoxide dismutase (SOD)24 (Fig. 4). A xenobiotic agent with a curcuminoid structure could induce the Nrf2/Keap1 pathway via both its anti-, as well as its prooxidant capability. The antioxidant mechanism is based on the Michael acceptor capability, as shown in excellent works by Albena Dinkova-Kostova and Paul Talalay.25 Accordingly, the a,b-unsaturated 1,3-diketone moiety of curcumin is a very attractive site for nucleophilic attacks by sulfhydryl groups. Keap1 carries a number of cysteine residues that are prone to be directly oxidized by ROS or by xenobiotics with Michael acceptor capability. It is thus conceivable to assume that the a,b-unsaturated 1,3-diketone moiety of curcumin and its analogs are able to oxidize Keap1, disrupt its interaction with Nrf2, and trigger its transcriptional activity for phase 2 antioxidant genes (Fig. 4). Validity for this concept was provided by a comprehensive SAR study of curcuminoids, as shown by the induction of the activities of the Nrf2 downstream targets NQO1 and GST.26 This study indicated that hydroxyl groups at position 2 (ortho) in the phenolic rings greatly enhanced the capability of curcuminoid structures containing a,b-unsaturated carbonyl groups with sulfhydryl groups to induce phase 2 cytoprotective proteins.26 Nrf2 activation also involves its phosphorylation by several different kinases, for example, protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) (Fig. 4). These kinases have been shown to be redox-sensitive and directly activated by high levels of ROS.27, 28

FIG. 4 Mechanisms of antioxidant activities of curcuminoids. Left: nonenzymatic (direct) mechanisms: scavenging abilities of free radicals, for example, superoxide radical O2%
and the hydroxyl radical HO%. R-OO%, the radical; ROOH, the oxidized reactant; and X%, another radical species. The responsible structural moieties (hydroxyl and a,b-unsaturated carbonyl [1,3-ketoenolyl] groups) are shaded. Right: enzymatic (indirect) mechanisms: induction of phase 2 cytoprotective enzymes, for example, glutathione transferases (GST), NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), glutamate-cysteine ligase (GCL), glutathione peroxidase (GP), and superoxide dismutase (SOD) via Michael acceptor (1,3-ketoenolyl) activation and nuclear translocation of the redox sensor nuclear factor erythroid 2 related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/Keap1). Bolting flash indicates point of attacks by curcuminoids. SH and S-S, reduced and oxidized sulfhydryl groups, respectively; ARE, antioxidant response elements; PKC, protein kinase C; ERK, extracellular regulated kinase.

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Prooxidant activities: Mechanisms The same redox capacity of curcumin and agents with curcuminoid moieties discussed above under the antioxidant effects can be responsible for strong prooxidant activities (Fig. 5). Agents with curcuminoid character that features a,b-unsaturated 1,3-diketone moieties can be strong Michael acceptors. They react with nucleophilic centers, which are characteristic for reduced glutathione and thioredoxin, two of the most prominent members of the cellular reservoir of natural thiols. It is thus conceivable that curcumin and agents with curcumin character rapidly deplete these thiols and shift the redox balance toward a prooxidant state. An additional effect of agents able to bind glutathione is its cellular extrusion without affecting the redox status of the cell. This has been termed “nonoxidative loss of glutathione” and was shown for several cytotoxic and chemotherapeutic agents.29 An example of the diverse and often contradictory actions of curcumin and its analogs, is the inhibitory actions on antioxidant phase 2 cytoprotective enzymes that are often induced by these agents (see above). This is the case for the thioredoxin system, such as the inhibition of thioredoxin reductase. This enzyme is crucial in the replenishing of reduced thioredoxin as an important cellular thiol buffer. Accordingly, curcumin and several curcumin analogs featuring furan moieties have been shown to convert thioredoxin reductase from an antioxidant to a prooxidant enzyme, changing it to a NADPH oxidase that transfers electrons from NADPH to oxygen, by alkylating cysteines and selenocysteines in the catalytically active sites of the enzyme.30 Independent of these actions of curcuminoids on thioredoxin reductase activity, curcumin can also significantly downregulate thioredoxin reductase expression by as yet unknown mechanisms.31 Similarly, curcumin and several structurally different curcumin analogs are able to inhibit GST expression in the low micromolar range, which is one of the key enzymes in the replenishing function of glutathione as an important cellular thiol buffer. These studies were conducted in different cell systems and indicated that GST inhibition may involve the upstream inhibition of the binding of transcription factors activated protein 1 (AP-1) and nuclear factor kappa B (NFkB) to recognition sites located on the GSTP1-1 gene promoter.32 Curcumin and several of its analogs can exert their cell death via mitochondrial processes in conjunction with ROS production.19, 33 This is not surprising, as the mitochondria are the primary source of cellular ROS production. However, the exact mechanism of action of xenobiotic agents with curcuminoid character within the mitochondria remains largely unexplained and may differ greatly in different cell systems. An intriguing possibility is that they affect the mitochondrial permeability transition pore complex (mPTPC). The mPTPC is a multicomponent channel spanning the inner and the outer mitochondrial membrane. Essential components are the adenine nucleotide translocase (ANT) located in the inner membrane and the voltage-dependent ion channel (VDAC) located in the outer membrane.34 The mPTPC is responsible for the mitochondrial ADP import and ATP export, and for the exchange of metabolites between the mitochondrial matrix and the cytosol. The mPTPC component ANT is also a sensor for ROS. It carries redox-sensitive cysteine residues that are normally reduced. In this state, ANT binds nucleotides and function as an ATP/ADP translocase. When these cysteine residues are oxidized, ANT undergoes a conformational change resulting in its inability to bind nucleotides and allowing calcium (Ca2+) to flux into the mitochondria where it induces additional pore opening.34 This mitochondrial permeability transition leads to changes in the mitochondrial membrane potential (Dcm), outer membrane permeabilization, the release of apoptotic proteins, including cytochrome C, apoptosis-inducing factor (AIF), and endonuclease G (Endo G), and ultimately cell death. Intramitochondrial ROS levels are kept in balance by the cellular redox systems. Accordingly, the superoxide radical O2%
produced by complex III of the electron transport chain is converted to hydrogen peroxide (H2O2) by SOD and further neutralized by catalase or GP to water thereby oxidizing glutathione. The cysteine residues of ANT are kept reduced by the thioredoxin/thioredoxin reductase and/or the glutathione/glutathione reductase systems. Several mechanisms by which curcumin and curcumin analogs could interfere with the normal function of the mPTPC can be envisioned (Fig. 5). First, the Michael acceptor reactivity could deplete the natural thiol pool such as glutathione and thioredoxin, leading to elevated ROS, oxidized ANT, pore opening, changes in the membrane potential Dcm, and membrane permeabilization. Second, it is not inconceivable that curcuminoids could directly interfere with ANT to oxidize the redox-sensitive cysteine residues. This is in agreement with earlier studies that suggested curcumin affects the mPTPC by the involvement of “oxidation of membrane thiol functions”.35 Third, curcumin and fluorinated derivatives have been shown to reduce iron (Fe3+) to Fe2+, which converts H2O2 to hydroxyl anion OH
and hydroxyl radical HO% in a Fenton reaction, leading to the oxidation of ANT.36 Remarkably, in this prooxidant scheme, curcumin acts as an antioxidant which represents a perfect example of the intricate interdependence of the two systems. Most of the above anti- and prooxidant mechanisms were not elucidated in prostate cancer cells. However, it is conceivable that they would be at work in cells of prostatic or uroepithelial provenance. The next section will elucidate the current knowledge on the oxidant actions of curcuminoid agents in cells of prostatic origin.

Prooxidant

Depleon of thiol buffers:

Inhibion of phase 2 cytoprotecve enzymes: GST, TR

Oxidaon of the mitochondrial permeability transion pore complex:

GSH  GS-SG and Conversion of enzyme funcons: TR  NADPH oxidase

FIG. 5 Mechanisms of prooxidant activities of curcuminoids. Left: depletion of thiol buffers, for example, oxidation of reduced glutathione (GSH) to oxidized glutathione (GS-SG) via Michael acceptor (1,3-ketoenolyl) action. Middle: inhibition of phase 2 cytoprotective enzymes, for example, glutathione transferase (GST) and thioredoxin reductase (TR) via unknown mechanisms; and conversion of TR to NADPH oxidase by alkylation of TR cysteines and selenocysteines. Right: oxidation of the mitochondrial permeability transition pore complex. Bolting flash indicates the point of attacks by curcuminoids. ANT, adenine nucleotide translocase; VDAC, voltage-dependent ion channel; OM and IM, mitochondrial outer and inner membrane, respectively; Compl III, complex III; SH and S-S, reduced and oxidized sulfhydryl groups, respectively.

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The potential of curcumin, curcuminoids, and curcumin analogs as oxidant agents in prostate cancer Molecular targets of curcumin, curcuminoids, and curcumin analogs in prostate cancer Prostate cancer is characterized by a myriad of molecular changes, some or even the majority of which may well be just consequential bystanders.37 However, despite this heterogeneous background of deregulated molecular pathways, some of them have been reported to be strongly causative for the development and progression of the disease, driving the development from normal epithelial cells to the well-characterized precursor PIN, and on to invasive and metastasizing carcinoma. In fact, some pathways have been recognized and proven to be necessary for the survival of prostate cancer cells; these constitute “oncogenic addiction.”8, 38 An imbalance in the pathways responsible for the maintenance of the cellular redox status may fuel the development of such oncogenic addiction. Backed by extensive epidemiological and population data, curcumin is widely accepted to be a dietary supplement with chemopreventive efficacy against many cancers, including prostate adenocarcinoma.6, 39 In a substantial number of in vitro and in vivo experimental studies, curcumin and its various analogs have proven to be strongly pleiotropic agents with a plethora of reported targets of importance to the biology of prostate cancer cells.6, 39 In fact, these targets include oncogenic pathways known to be essential for cell survival, proliferation, and resistance to therapeutic intervention. Interference with these pathways has been shown to lead to cell death of prostate cancer cells in vitro and to the inhibition of tumor formation in vivo.33 Furthermore, there is direct evidence that curcumin affects the transcriptional regulation of genes involved in oxidative stress in prostate cancer cells.40 However, although curcumin and agents with curcuminoid character are proven effectors of the cellular redox status, this particular aspect of their bioactivity remains to date remarkably unexplored in the specific context of prostate cancer. The following sections review the current knowledge of the anti- and prooxidant actions of curcuminoids and curcumin analogs with a focus on cells of prostatic origin. In the cases where the exact mechanisms are not known, it is reasonable to imply the mechanisms outlined in the previous section. Although the number of published reports has increased in recent years on compounds with curcuminoid character that act through their effect on the cellular redox system, either in an antior prooxidant manner, it remains rather small overall, especially with respect to their mechanism in cells of the prostate.

Curcumin, curcuminoids, and curcumin analogs as antioxidants in prostate cancer The published literature is rich in reports on the use of natural products with curcuminoid character with efficacies against prostate cancer cells (Refs. 6, 39 and references therein). It is also well established that curcumin and by extrapolation or experimentation, curcumin analogs, can be potent antioxidants (Ref. 41 and references therein). However, the term “antioxidant” is often used as an accompanying adjective when the effect of curcumin and curcuminoids are experimentally investigated. In quite a few of these studies the antioxidant mechanisms are assumed as opposed to proven by experimentation, although H-atom and electron transfer mechanisms seem to be the main scavenging activities at work.41 Of course, curcumin itself is established as an antioxidant product in several cellular backgrounds, for example in hepatic cells, where it was shown to protect diabetic pathophysiology by counteracting ROS and inhibiting the activation of the tumor suppressor p53 and stress response pathways.42 In another example, the antioxidant activity of cyclodextrin-complexed curcumin was shown to improve the success of kidney transplantation in a preclinical pig model.43 Surprisingly, there are still relatively few studies specific for prostate cancer that include analyses of the antioxidant capabilities of curcuminoid compounds and to the components of the cellular redox machinery. In addition, clinical trials using the pharmacologically safe natural product curcumin are typically not designed to specifically address its antioxidant capacities, as they tend to focus on more clinical end-points.18, 44 However, the antioxidant mechanisms outlined in the previous section (Fig. 4) can be reasonably implied in cells of prostatic origin. An example of this is the ability of curcumin to inhibit the proproliferative effects of H2O2 in LNCaP human prostate cancer cells due to its free radical scavenging activity.45 Another example is given by the complexation of curcumin and its derivative diacetylcurcumin with gallium, which has been shown to affect the structure and function of peroxidases in LNCaP cells, thereby enhancing their stability and antioxidant activity.46 One prominent mono-carbonyl analog of curcumin with enhanced antioxidant activity in a multitude of biochemical assays and in the PC-3 prostate cancer cells was simply called compound 14 (Fig. 6).41 One of the strongest effects of curcumin and its analogs with respect to their antioxidant capability in prostate cells is the induction of phase 2 cytoprotective genes (see earlier). In this context, curcumin was shown to reactivate the master regulator of cellular antioxidant defense systems Nrf2 by restoring its expression via demethylation of CpG islands in its promoter, therefore acting as DNA hypomethylation agent. Nrf2 was previously shown to be epigenetically silenced during the progression of prostate tumorigenesis in

Curcumin analogs, oxidative stress, and prostate cancer Chapter

O O OCH3

H3CO

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F

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OH Compound 14

Antioxidant N

HO-3867

Prooxidant F

OH

33

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FIG. 6 Synthetic curcumin analogs that exert their effects in prostate cancer cells through either an antioxidant or a prooxidant mechanism of action. HO-3867, HO-4200, and compound 14 are examples of synthetic curcuminoids/curcumin analogs that exert cytotoxic effects in prostate cancer cells via antioxidation.41,47 EF24, ca27, WZ35, and P1 are examples of synthetic curcuminoids that exert cytotoxic effects in prostate cancer cells via prooxidation.48–52

F

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transgenic adenocarcinoma of the mouse prostate (TRAMP) mice.53 In addition, two mono-carbonyl curcumin analogs, HO-3867 and HO-4200 (Fig. 6), which are difluoro-diarylidenylpiperidones and feature an additional substituted N-hydroxypyrroline (NOH nitroxide precursor), were reported to act as enhanced radical-scavenging compounds in several different types of cells, including PC-3 human prostate cancer cells. Of note, this activity was observed to be specific for cancerous cells and remarkably diminished in normal cells with noncancerous character.47

Curcumin, curcuminoids, and curcumin analogs as prooxidants in prostate cancer It is remarkable that many, if not most, scientific reports on the oxidant effect of curcumin and its various analogs in prostate cancer cells relate to their prooxidant, as opposed to their antioxidant capacity. This is surprising in the light of the fact that this polyphenol natural product has been and continues to be listed as a parade example for antioxidant activity,1, 6, 41 although it has been known for 20 years that curcumin can have prooxidant activity affecting DNA integrity.19 In fact, curcumin and its naturally occurring and synthetic analogs are being increasingly recognized as prooxidant agents based on the mechanisms discussed in the previous section, as well as new ones. The capability of curcumin to act as a prooxidant agent in prostate cancer cells has been confirmed in different studies utilizing different cell models and schemes. For example, ROS induction by curcumin followed by changes in the mitochondrial membrane potential and cell death was shown in the widely-used human prostate cancer cell line LNCaP.54 Formulations of curcumin complexations with palladium and bipyridines induced growth inhibition and cell death in various prostate cancer cells, including LNCaP, DU145, and PC-3, through a ROS-dependent mechanism involving complex 1 and mitochondrial membrane depolarization.55 This study also revealed another potential mechanism of curcuminoids, that is, the downregulation of the important phase 2 cytoprotective enzyme GSTp1,55 which lead to a shift toward a prooxidant cellular status. An important consequence of ROS induction by curcumin has been shown to be the selective downregulation of proteins that are implicated in prostate tumorigenesis and cancer progression. A prominent example of this action was shown in the context of the induction of cytochrome P450 enzymes by the toxic environmental contaminant 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). TCDD mediated P450 induction occurs through the activation of the arylhydrocarbon receptor (AhR) in the cytoplasm, which translocates to the nucleus, dimerizes with the AhR nuclear translocator (ARNT) and drives the expression of CYP1A1 and 1B1. TCDD is known to mediate cell transformation and carcinogenesis via the generation of genotoxic metabolites. Accordingly, curcumin was shown to downregulate nuclear AhR and ARNT in normal human embryonic kidney cells and normal prostate cells and to inhibit malignant cell transformation. Of interest to this chapter, these actions were dependent on the concomitant induction of ROS leading to oxidative stress.56 In fact, in the setting of prostate cells, curcumin now holds a firm role as a prooxidant molecule. In a number of human prostate cancer cell models, curcumin induced nonautophagic vacuolation death through the induction of ROS-mediated endoplasmic reticulum stress.57 The induction of endoplasmic reticulum stress by curcumin, triggering multiple proapoptotic signaling pathways in prostate cancer cells was corroborated in another independent study.58 Furthermore, endoplasmic reticulum

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stress is accompanied by the inhibition of the signal transducer and activator of transcription 3 (STAT3) protein when alkylated mono-carbonyl analogs of curcumin are engaged in cancer cell treatment schemes.59 In addition, curcumin was shown to promote the oxidation of thioredoxin through the induction of ROS and to alter its subcellular localization.60 Regarding synthetic analogs of curcumin that act through prooxidant mechanisms, differently structured compounds have been tested in a variety of cellular backgrounds, including pulmonary, pancreatic, mammary, ovarian, hepatic, cervical, gastrointestinal, colorectal, glial, and endothelial (in NCBI PubMed library). In contrast, the number of reports on compounds with curcuminoid character, that is, curcumin analogs, that have been shown to exert prooxidant effects in cells specifically of prostatic origin, remains relatively small. The following paragraphs cover these compounds, as published in approximate chronological order. Adams and coworkers characterized a synthetic fluorinated curcumin analog, 3,5-bis-(2-fluorobenzylidene)-4piperidone (termed EF24; Fig. 6) with respect to its capability to induce cell cycle arrest and apoptosis by means of a redox-dependent mechanism in DU145 human prostate cancer cells.48 EF24 was previously identified as a curcumin analog with higher antitumorigenic potential in the 60 cancer cell line test panel provided by the National Cancer Institute (NCI).61 EF24 caused G2/M arrest followed by the induction of apoptosis, as shown by caspase-3 activation, phosphatidylserine externalization, and an increased number of cells with a sub-G1 DNA fraction. In addition, EF24 induced depolarization of the mitochondrial membrane potential, indicating the involvement of mitochondrial deregulation and subsequent activation of apoptotic pathways. EF24, as well as an additional derivative (termed EF31) featuring nitrogen substitutions for the ortho-positioned fluorine groups, was later shown to act similarly in breast cancer cells when pre-combined with glutathione.61 With respect to the mechanism(s) of action of these curcumin analogs in relation to oxidative stress, they were shown to induce ROS by depleting the pool of natural thiol buffers, that is, reduced glutathione (GSH) and thioredoxin via their Michael acceptor reactivity toward the sulfhydryl groups of GSH and thioredoxin.48 Since EF24 was reported for the first time, this remarkable curcumin analog has shown to be active in several different types of cancer cells, including mammary, pancreatic, and leukemic origin. The broad bioactivity of EF24 was recently reviewed.62 Another protein target of importance to prostate cancer development and progression is the androgen receptor (AR), which is the major target of the mainstay therapeutic intervention of androgen ablation.8, 38 The expression of the AR is retained and often increased during cancer progression. Therefore, AR protein downregulation could be an effective therapeutic approach against prostate cancer. Our own work has shown that the curcumin analog 5-bis(2-hydroxyphenyl)-1,4pentadien-3-one (termed ca27; Fig. 6) downregulates AR expression at low micromolar concentrations in several prostate cancer cell lines including the androgen-dependent LNCaP and LAPC4 and the androgen ablation resistant C4-2 cells.49 Pertinent to the link to oxidative stress, ca27-mediated AR downregulation was rapidly induced via the generation of ROS, as the antioxidant N-acetyl cysteine was able to inhibit this effect. Interestingly, the prostate cancer cells exerted an antioxidant response to ca27 by inducing the expression of the cellular redox sensor Nrf2 and the phase 2 cytoprotective enzymes NQO1 and aldoketoreductase 1C1. The latter shows that prooxidant effects and antioxidant responses are intricately linked in the cellular redox status of a cell.49 The effect of curcumin analogs on the expression of the AR has been extensively covered by other authors.63 While these authors presented a number of curcumin analogs with this interesting effect, the role of oxidative stress was not specifically addressed. The downregulation/induction of AR degradation is also interesting in the context of the aforementioned work by Dinkova-Kostova and colleagues.26 These authors had suggested that the powerful induction of protective phase 2 proteins by the benzylidenealkanones with curcuminoid character under investigation was due to the ortho-positioned hydroxyl groups on the aryl rings in combination with the closely positioned a,b-unsaturated carbonyl (1,3-ketoenolyl) group on the carbon chain. In particular, it was hypothesized that the conformational vicinity of these two groups leads to an enhanced Michael acceptor reactivity by lowering the pKa of sulfhydryl groups on target proteins through inductive hydrogen bonding of the neighboring phenolic hydroxyl groups (Fig. 7). To test this hypothesis, we are currently conducting SAR studies on ca27 that focus on the position of the hydroxyl groups on the aryl ring. We thereby hypothesize a gradual loss in the potency to induce ROS with increasing distance of the hydroxyl group from the a,b-unsaturated carbonyl group (Michael acceptor), as well as a marked loss of prooxidant activity upon removal of the hydroxyl groups and/or saturation of the a,b-carbonyl group (Fig. 7). The following compounds complete the list of additional curcuminoids and curcumin analogs that have been shown to either affect the phenotypic behavior or distinctive molecular pathways in prostate cancer cells since the first edition of this book. The compounds fall into two categories with respect to the knowledge of whether they act through oxidant mechanisms or not. The latter is not always proven, often simply because the study design did not include experimental methods to investigate this possibility. However, based on reports documenting several other molecules and their prooxidant mechanisms of action, it is reasonable to assume that the cellular redox status and the molecular targets covered in the previous section are affected. In this category, we find the naturally occurring curcuminoid demethoxycurcumin (Fig. 3), which inhibits cell proliferation, migration, and invasion of PC-3 prostate cancer cells.64 While the oxidant activity was not

Curcumin analogs, oxidative stress, and prostate cancer Chapter

H

R

R –O:

S

OH S

OH

O

33

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R O–

OH S

OH

OH

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H+ OH

O

OH

OH

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ortho OH

No OH

O HO

α,β-saturated no OH

OH

OH

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α,β-saturated

meta OH para OH

ROS

O HO

para OH

OH

ortho OH

meta OH

FIG. 7 Representative structure activity relationship (SAR) studies of oxidative stress induction by a diarylpentanoid/mono-carbonyl curcumin analog. Top: molecular redox mechanism (after Ref. 26) by which an ortho-positioned hydroxyl (OH) group can enhance the Michael acceptor reactivity of the a,b-unsaturated carbonyl (1,3-ketoenolyl) moiety of the diarylpentanoid curcumin analog ca27.49 The nucleophilic attack by a putative protein bearing a sulfhydryl group (italic and underlined) is shown leading to its covalent bonding to the diarylpentanoid. Bottom: structural analogs of ca27 proposed to be tested for their induction of reactive oxygen species (ROS). The compounds differ with respect to the position of the OH group and the presence/absence of the Michael acceptors (a,b-unsaturated carbonyl). The projected relative capacity of the compounds to induce ROS is shown in the sun chart.

specifically shown in this study, it can be implied from experiments performed in other cell systems, including of pulmonary origin.65 Similar to EF24, Mapoung and colleagues synthesized two cyclohexanones, 2,6-bis-(4-hydroxy-3methoxy-benzylidene)-cyclohexanone (2A) and 2,6-bis-(3,4-dihydroxy-benzylidene)-cyclohexanone (2F) that inhibited metastatic behavior of castration-resistant prostate cancer cells in vitro and in vivo through inhibiting the production and secretion of matrix metalloproteinases (MMP).66 Compound 2A, in particular, was very potent in inhibiting metastatic tumor colonization in the lungs by inducing G1 phase arrest and apoptosis. Similarly, Wang et al. synthesized a series of 1,7-diarylhepta-1,4,6-trien-3-ones as the major backbone moiety derived from curcumin (featuring an additional unsaturated carbon bond) and tested them successfully against the three major human prostate cancer cell models, LNCaP, PC-3, and DU145.67 Interestingly, however, the most effective compounds with respect to their capacity to inhibit cell growth were the ones with different terminal nitrogen- and partially sulfur-containing heteroaromatic rings, including the most potent structure featured in compound 40. Of note, compounds 2A, 2F, and 40 were not tested for their capability to elicit oxidative stress through the induction of ROS, but one can reasonably assume that they do. This is in contrast to the last two curcumin analogs covered in this chapter, which have been shown to act through prooxidant mechanisms in prostate cancer cells. First, Peng and colleagues introduced a diarylpentanoid mono-ketone, a tropinone curcuminoid with an additional nitrogen-containing ring, termed P150 (Fig. 6). This compound strongly inhibited the NFkB activity through the marked induction of ROS, which was primarily generated in the mitochondria. The latter supports the proposed mechanisms of actin depicted in Fig. 5. Second, Zhang and colleagues, as well as Chen and coworkers tested the asymmetrically structured diarylpentanoid WZ35 in a number of prostate cancer cell models, including PC-3, DU145, and RM-1.51, 52 Not surprisingly, and in agreement with other reported molecules of curcuminoid nature, WZ35 exerted its cancer cell toxicity by inducing ROS-dependent endoplasmic reticulum stress, G2/M cell cycle arrest, intracellular calcium (Ca2+) surge, and induction of mitochondrial apoptosis. Lastly, it must be noted that very few preclinical studies on curcumin analogs have been reported in prostate cancer animal (in vivo) models. The aforementioned compounds EF24, 2A, and WZ35 stand out in this category.51, 66, 68 Such preclinical trials are necessary to assess the bioavailability and bioefficacy of curcumin analogs in comparison to curcumin itself.

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FIG. 8 Open questions and tasks for the future. Open questions, unresolved issues, and tasks related to agents with diarylheptanoid and diarylpentanoid curcuminoid character and their possible clinical implications for prostate cancer.

Diarylheptanoid analogs

Diarylpentanoid analogs

Prostate-specific studies

Paradox of oxidave stress

Structure acvity relaonship

… mechanisms of acon …

“… increase or decrease …”

… bioacvity vs. selecvity …

Preclinical (in vivo) studies

Phase I clinical trials

Summary points l

l

l

l

l

The development and progression of prostate cancer are characterized by increasing oxidative stress due to elevated levels of free radicals and other reactive oxygen species. Both the anti- and the prooxidant capacity of curcumin and its analogs can be exploited as therapeutic strategies against prostate cancer. More studies are necessary to determine the oxidant mechanisms of action of curcuminoid agents specifically in cells of prostate origin, in order to pursue their possible clinical implications for prostate cancer. There is also a need to increase the number of preclinical animal (in vivo) studies (Fig. 8). Structural modification by combinatorial chemistry approaches, as featured in de novo synthesized chemical analogs continues to improve the bioavailability of agents with curcuminoid character (Fig. 8). Structure analysis relationship studies of curcumin analogs are necessary to identify agents with a pharmacologically safe profile that discriminate between prostate cancer and normal cells, in order to facilitate the development of phase I clinical trials (Fig. 8).

References 1. Gupta SC, Patchva S, Koh W, Aggarwal BB. Discovery of curcumin, a component of Golden spice, and its miraculous biological activities. Clin Exp Pharmacol Physiol 2012;39(3):283–99. 2. Anand P, Thomas SG, Kunnumakkara AB, et al. nBiological activities of curcumin and its analogues (congeners) made by man and mother ature. Biochem Pharmacol 2008;76(11):1590–611. 3. Rodrigues FC, Anil Kumar NV, Thakur G. Developments in the anticancer activity of structurally modified curcumin: an up-to-date review. Eur J Med Chem 2019;177:76–104. 4. Moghaddam NSA, Oskouie MN, Butler AE, Petit PX, Barreto GE, Sahebkar A. Hormetic effects of curcumin: what is the evidence? J Cell Physiol 2019;234(7):10060–71. 5. Roumeguere T, Sfeir J, El Rassy E, et al. Oxidative stress and prostatic diseases. Mol Clin Oncol 2017;7(5):723–8. 6. Aggarwal BB. Prostate cancer and curcumin: add spice to your life. Cancer Biol Ther 2008;7(9):1436–40. 7. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68(6):394–424. 8. Coutinho I, Day TK, Tilley WD, Selth LA. Androgen receptor signaling in castration-resistant prostate cancer: a lesson in persistence. Endocr Relat Cancer 2016;23(12):T179–97. 9. Mansinho A, Macedo D, Fernandes I, Costa L. Castration-resistant prostate cancer: mechanisms, targets and treatment. Adv Exp Med Biol 2018;1096:117–33. 10. Jin JK, Dayyani F, Gallick GE. Steps in prostate cancer progression that Lead to bone metastasis. Int J Cancer 2011;128(11):2545–61. 11. Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol 1956;11(3):298–300. 12. Fleshner NE, Klotz LH. Diet, androgens, oxidative stress and prostate cancer susceptibility. Cancer Metastasis Rev 1998;17(4):325–30.

Curcumin analogs, oxidative stress, and prostate cancer Chapter

33

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13. Omabe M, Ezeani M. Infection, inflammation and prostate carcinogenesis. Infect Genet Evol 2011;11(6):1195–8. 14. Acharya A, Das I, Chandhok D, Saha T. Redox regulation in cancer: a double-edged sword with therapeutic potential. Oxid Med Cell Longev 2010; 3(1):23–34. 15. Wang J, Yi J. Cancer cell killing via ROS: to increase or decrease, that is the question. Cancer Biol Ther 2008;7(12):1875–84. 16. Agrawal DK, Mishra PK. Curcumin and its analogues: potential anticancer agents. Med Res Rev 2010;30(5):818–60. 17. Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. Bioavailability of curcumin: problems and promises. Mol Pharm 2007;4(6):807–18. 18. Kunnumakkara AB, Harsha C, Banik K, et al. Is curcumin bioavailability a problem in humans: lessons from clinical trials. Expert Opin Drug Metab Toxicol 2019;15:705–33. 19. Ahsan H, Parveen N, Khan NU, Hadi SM. Pro-oxidant, anti-oxidant and cleavage activities on DNA of curcumin and its derivatives demethoxycurcumin and bisdemethoxycurcumin. Chem Biol Interact 1999;121(2):161–75. 20. Priyadarsini KI, Maity DK, Naik GH, et al. Role of phenolic O-H and methylene hydrogen on the free radical reactions and antioxidant activity of curcumin. Free Radic Biol Med 2003;35(5):475–84. 21. Weber WM, Hunsaker LA, Abcouwer SF, Deck LM, Vander Jagt DL. Anti-oxidant activities of curcumin and related enones. Bioorg Med Chem 2005;13(11):3811–20. 22. Sugiyama Y, Kawakishi S, Osawa T. Involvement of the beta-diketone moiety in the antioxidative mechanism of tetrahydrocurcumin. Biochem Pharmacol 1996;52(4):519–25. 23. Venkatesan P, Rao MN. Structure-activity relationships for the inhibition of lipid peroxidation and the scavenging of free radicals by synthetic symmetrical curcumin analogues. J Pharm Pharmacol 2000;52(9):1123–8. 24. Surh YJ, Kundu JK, Na HK. Nrf2 as a master redox switch in turning on the cellular signaling involved in the induction of Cytoprotective genes by some Chemopreventive phytochemicals. Planta Med 2008;74(13):1526–39. 25. Dinkova-Kostova A, Talalay P. Direct and indirect antioxidant properties of inducers of vytoprotective proteins. Mol Nutr Food Res 2008;52(Suppl 1): S128–38. 26. Dinkova-Kostova AT, Massiah MA, Bozak RE, Hicks RJ, Talalay P. Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups. Proc Natl Acad Sci U S A 2001;98(6):3404–9. 27. Chen CA, Chen TS, Chen HC. Extracellular signal-regulated kinase plays a proapoptotic role in podocytes after reactive oxygen species treatment and inhibition of integrin-extracellular matrix interaction. Exp Biol Med (Maywood) 2012;237(7):777–83. 28. Konishi H, Tanaka M, Takemura Y, et al. Activation of protein kinase C by tyrosine phosphorylation in response to H2O2. Proc Natl Acad Sci U S A 1997;94(21):11233–7. 29. Ghibelli L, Coppola S, Rotilio G, Lafavia E, Maresca V, Ciriolo MR. Non-oxidative loss of glutathione in apoptosis via GSH extrusion. Biochem Biophys Res Commun 1995;216(1):313–20. 30. Qiu X, Liu Z, Shao WY, et al. Synthesis and evaluation of curcumin analogues as potential thioredoxin reductase inhibitors. Bioorg Med Chem 2008;16(17):8035–41. 31. Cai W, Zhang B, Duan D, Wu J, Fang J. Curcumin targeting the thioredoxin system elevates oxidative stress in HeLa cells. Toxicol Appl Pharmacol 2012;262(3):341–8. 32. Appiah-Opong R, Commandeur JN, Istyastono E, Bogaards JJ, Vermeulen NP. Inhibition of human glutathione S-transferases by curcumin and analogues. Xenobiotica 2009;39(4):302–11. 33. Khan N, Adhami VM, Mukhtar H. Apoptosis by dietary agents for prevention and treatment of prostate cancer. Endocr Relat Cancer 2010; 17(1):R39–52. 34. Fruehauf JP, Meyskens Jr. FL. Reactive oxygen species: a breath of life or death? Clin Cancer Res 2007;13(3):789–94. 35. Morin D, Barthelemy S, Zini R, Labidalle S, Tillement JP. Curcumin induces the mitochondrial permeability transition pore mediated by membrane protein thiol oxidation. FEBS Lett 2001;495(1–2):131–6. 36. Ligeret H, Barthelemy S, Zini R, Tillement JP, Labidalle S, Morin D. Effects of curcumin and curcumin derivatives on mitochondrial permeability transition pore. Free Radic Biol Med 2004;36(7):919–29. 37. Wang G, Zhao D, Spring DJ, Depinho RA. Genetics and biology of prostate cancer. Genes Dev 2018;32(17–18):1105–40. 38. Knudsen KE, Scher HI. Starving the addiction: new opportunities for durable suppression of Ar signaling in prostate cancer. Clin Cancer Res 2009; 15(15):4792–8. 39. Jordan BC, Mock CD, Thilagavathi R, Selvam C. Molecular mechanisms of curcumin and its semisynthetic analogues in prostate cancer prevention and treatment. Life Sci 2016;152:135–44. 40. Thangapazham RL, Shaheduzzaman S, Kim KH, et al. Androgen responsive and refractory prostate cancer cells exhibit distinct curcumin regulated transcriptome. Cancer Biol Ther 2008;7(9):1427–35. 41. Li Q, Chen J, Luo S, Xu J, Huang Q, Liu T. Synthesis and assessment of the antioxidant and antitumor properties of asymmetric curcumin analogues. Eur J Med Chem 2015;93:461–9. 42. Ghosh S, Bhattacharyya S, Rashid K, Sil PC. Curcumin protects rat liver from streptozotocin-induced diabetic pathophysiology by counteracting reactive oxygen species and inhibiting the activation of p53 and MAPKs mediated stress response pathways. Toxicol Rep 2015;2:365–76. 43. Thuillier R, Allain G, Giraud S, et al. Cyclodextrin curcumin formulation improves outcome in a preclinical pig model of marginal kidney transplantation. Am J Transplant 2014;14(5):1073–83. 44. Gupta SC, Patchva S, Aggarwal BB. Therapeutic roles of curcumin: lessons learned from clinical trials. AAPS J 2013;15(1):195–218.

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45. Polytarchou C, Hatziapostolou M, Papadimitriou E. Hydrogen peroxide stimulates proliferation and migration of human prostate cancer cells through activation of activator protein-1 and up-regulation of the heparin affin regulatory peptide gene. J Biol Chem 2005;280(49):40428–35. 46. Jahangoshaei P, Hassani L, Mohammadi F, Hamidi A, Mohammadi K. Investigating the effect of gallium curcumin and gallium diacetylcurcumin complexes on the structure, function and oxidative stability of the peroxidase enzyme and their anticancer and antibacterial activities. J Biol Inorg Chem 2015;20(7):1135–46. 47. Selvendiran K, Ahmed S, Dayton A, et al. Safe and targeted anticancer efficacy of a novel class of antioxidant-conjugated difluorodiarylidenyl piperidones: differential cytotoxicity in healthy and cancer cells. Free Radic Biol Med 2010;48(9):1228–35. 48. Adams BK, Cai J, Armstrong J, et al. EF24, a novel synthetic curcumin analog, induces apoptosis in cancer cells via a redox-dependent mechanism. Anticancer Drugs 2005;16(3):263–75. 49. Fajardo AM, Mackenzie DA, Ji M, et al. The curcumin analog Ca27 down-regulates androgen receptor through an oxidative stress mediated mechanism in human prostate cancer cells. Prostate 2012;72(6):612–25. 50. Peng YM, Zheng JB, Zhou YB, Li J. Characterization of a novel curcumin analog P1 as potent inhibitor of the NF-Kappab signaling pathway with distinct mechanisms. Acta Pharmacol Sin 2013;34(7):939–50. 51. Chen M, Zhou B, Zhong P, et al. Increased intracellular reactive oxygen species mediates the anti-cancer effects of WZ35 via activating mitochondrial apoptosis pathway in prostate cancer cells. Prostate 2017;77(5):489–504. 52. Zhang X, Chen M, Zou P, et al. Curcumin analog WZ35 induced cell death via Ros-dependent Er stress and G2/M cell cycle arrest in human prostate cancer cells. BMC Cancer 2015;15:866. 53. Li W, Su ZY, Guo Y, et al. Curcumin derivative epigenetically reactivates Nrf2 antioxidative stress signaling in mouse prostate cancer TRAMP C1 cells. Chem Res Toxicol 2018;31(2):88–96. 54. Shankar S, Srivastava RK. Involvement of Bcl-2 family members, phosphatidylinositol 3’-kinase/AKT and mitochondrial P53 in curcumin (Diferulolylmethane)-induced apoptosis in prostate cancer. Int J Oncol 2007;30(4):905–18. 55. Valentini A, Conforti F, Crispini A, et al. Synthesis, oxidant properties, and antitumoral effects of a heteroleptic palladium(Ii) complex of curcumin on human prostate cancer cells. J Med Chem 2009;52(2):484–91. 56. Choi H, Chun YS, Shin YJ, Ye SK, Kim MS, Park JW. Curcumin attenuates cytochrome P450 induction in response to 2,3,7,8-tetrachlorodibenzoP-dioxin by ROS-dependently degrading AhR and ARNT. Cancer Sci 2008;99(12):2518–24. 57. Lee WJ, Chien MH, Chow JM, et al. Nonautophagic cytoplasmic vacuolation death induction in human Pc-3m prostate cancer by curcumin through reactive oxygen species-mediated endoplasmic reticulum stress. Sci Rep 2015;5:10420. 58. Rivera M, Ramos Y, Rodriguez-Valentin M, et al. Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells. PLoS One 2017;12(6): E0179587. 59. Rajamanickam V, Zhu H, Feng C, et al. Novel allylated monocarbonyl analogs of curcumin induce mitotic arrest and apoptosis by reactive oxygen species-mediated endoplasmic reticulum stress and inhibition of Stat3. Oncotarget 2017;8(60):101112–29. 60. Rodriguez-Garcia A, Hevia D, Mayo JC, et al. Thioredoxin 1 modulates apoptosis induced by bioactive compounds in prostate cancer cells. Redox Biol 2017;12:634–47. 61. Sun A, Lu YJ, Hu H, Shoji M, Liotta DC, Snyder JP. Curcumin analog cytotoxicity against breast cancer cells: exploitation of a redox-dependent mechanism. Bioorg Med Chem Lett 2009;19(23):6627–31. 62. He Y, Li W, Hu G, Sun H, Kong Q. Bioactivities of EF24, A Novel Curcumin Analog: A Review. Front Oncol 2018;8:614. 63. Shi Q, Shih CC, Lee KH. Novel anti-prostate cancer curcumin analogues that enhance androgen receptor degradation activity. Anticancer Agents Med Chem 2009;9(8):904–12. 64. Ni X, Zhang A, Zhao Z, Shen Y, Wang S. Demethoxycurcumin inhibits cell proliferation, migration and invasion in prostate cancer cells. Oncol Rep 2012;28(1):85–90. 65. Ko YC, Lien JC, Liu HC, et al. Demethoxycurcumin induces the apoptosis of human lung cancer NCI-H460 cells through the mitochondrial-dependent pathway. Oncol Rep 2015;33(5):2429–37. 66. Mapoung S, Suzuki S, Fuji S, et al. Cyclohexanone curcumin analogs inhibit the progression of castration-resistant prostate cancer in vitro and in vivo. Cancer Sci 2019;110(2):596–607. 67. Wang R, Zhang X, Chen C, et al. Structure-activity relationship studies of 1,7-diheteroarylhepta-1,4,6-trien-3-ones with two different terminal rings in prostate epithelial cell models. Eur J Med Chem 2017;133:208–26. 68. Yang CH, Yue J, Sims M, Pfeffer LM. The curcumin analog EF24 targets NF-Kappab and miRNA-21, and Has potent anticancer activity in vitro and in vivo. PLoS One 2013;8(8): E71130.

Chapter 34

Fern extract, oxidative stress, and skin cancer Concepcio´n Parradoa, Yolanda Gilaberteb, Neena Philipsc, Angeles Juarranzd, and Salvador Gonzaleze,f a

Department of Histology and Pathology, Faculty of Medicine, University of Ma´laga, Ma´laga, Spain, b Dermatology Service, Miguel Servet Hospital,

Zaragoza, Spain, c School of Natural Sciences, University College, Fairleigh Dickinson University, Teaneck, NJ, United States, d Biology Department, Sciences School, Universidad Auto´noma de Madrid, Madrid, Spain, e Dermatology Service, Memorial Sloan-Kettering Cancer Center, New York, NY, United States, f Medicine and Medical Specialties Department, Alcala University, Madrid, Spain

List of abbreviations 8-OH-dG AP-1 CD CIE COLIa1 CPDs CTSK DC ECM eLC ELN FBN GP GST HPLC IL iNOS IR MMP NB-UVB NF-kB NIEHS NO NOS OX-2 PG PL PMLE PUVA ROS SOD

8-hydroxy-20 -deoxyguanosine activator protein 1 common deletion C Commission Internationale de l’Eclairage (CIE) collagen type I cyclobutane pyrimidine dimers cathepsin K dendritic cells extracellular matrix. epidermal Langerhans cells elastin fibrillin glutathione peroxidase glutathione S-transferase high-performance liquid chromatography interleukine inducible nitric oxide synthase infrared matrix metalloprotease narrow-band UVB nuclear factor kappa beta national Institute of Environmental Health Sciences nitric oxide nitric oxide synthase cyclooxygenase-2 prostaglandin Polypodium leucotomos polymorphic light eruption psoralens + UVA reactive oxygen species superoxide dismutase

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00034-1 © 2021 Elsevier Inc. All rights reserved.

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SSR TGF- b Th1 TIMP TNF-a UCA UV VIS

B Antioxidants and cancer

simulator simulating radiation transforming growth factor beta T helper 1 lymphocyte inhibitor of metalloproteinase tumor necrosis factor-alpha urocanic acid ultraviolet light visible light

Introduction Ultraviolet radiation and oxidative stress Skin cancer accounts for at least 40% of all human malignancies. Solar radiation is a potent environmental human carcinogen.1 It is widely accepted that overexposure to sunlight is the cause of the harmful effects on the skin. Four out of five skin cancers could be prevented since excessive exposure to sunlight can be avoided.2 Ultraviolet (UV) radiation (UVR) is a major causal agent in most skin cancers.3 UVR is a part of the electromagnetic spectrum emitted by the sun. Most of the UVA radiation (315–400 nm) and approximately 10% of the UVB rays (280–315 nm) reach the superficies of the Earth. UVA and UVB rays are of great importance for human health. UVC (100–280 nm) is absorbed by atmospheric ozone. UVR generates reactive oxygen species (ROS) involved in the skin photodamage. It is also well documented that ROS can induce photoaging and photocarcinogenesis.3 UVR damages DNA through generation ROS.3 UVR also alters DNA directly by the formation of pyrimidine-pyrimidine dimers. The dimers creations are the basis of mutations in specific genes, including the tumor suppressor gene P53. Broad-spectrum UVR also causes suppressing the immune system response and inflammation.1 UVR induces oxidation of the membrane lipids and ROS productions as a result of oxidation of arachidonic acid (AA). Cyclooxygenase enzymes (COX) convert AA into prostaglandin (PG), amplifying the recruitment of inflammatory cells. ROS also induce the activation of tumor necrosis factor-a (TNF-a) and the nuclear factor-kB (NF-kB) expression. NF-kB causes the appearance of the various proinflammatory gene.3 Also, UVR induces peroxidation, which causes the synthesis of inducible nitric oxide synthase (iNOS). iNOS mediates angiogenesis and hyperpermeability.4 The excess of ROS produced by the action of UVR also activates the mitogen-activated protein kinases (MAPKs) and increases the transcription of the matrix metalloproteinases (MMPs) and dysregulation of the extracellular matrix ECM homeostasis.3 UVR also induces immunosuppression and immunological tolerance. UVR decreases the numbers of epidermal Langerhans cells and induces isomerization of the urocanic acid [3-(1H-imidazole-4-yl)-2-propenoic acid; UCA]. The isomerization of trans-UCA to the cis-isomer represents a signal of immune suppression.4

Infrared radiation (IR) and visible light (VIS) and oxidative stress Not only UVR produces oxidative stress and photodamage but also infrared radiation (IR) has a deleterious impact on the skin.5 IR-A produces the generation of mitochondrial ROS, followed by the activation of the MAPK and increases in MMP-1. Finally, IR-A decreases collagen synthesis and increases angiogenesis and the number of mast cells. In the same way, the first works in 1962 by Pathak6 and those of Kollias and Baqer in 19847 hypothesized that VIS could contribute to photodamage. After the exposition of the skin to VIS induced hyperpigmentation. Recently has been confirmed that exposure of human skin to different doses of VIS (40–180 J/cm2) induces ROS production. VIS also through ROS induction produces DNA damage.8 Also in human skin, VIS plus IR increases MMP expression, decreases procollagen levels, and recruits macrophages.8 Due to the multiple harmful effects of UVR, VIS, and IR radiation in the skin, including photoaging and photocarcinogenesis, prevention measures and treatment to avoid the skin sun damage are increasing. In addition to the avoidance of solar overexposure, the use of topical and systemic photoprotection is a topic of great interest in health, since numerous cutaneous pathologies can be avoided. Topical and systemic photoprotective agents, including antioxidants, provide additional protection against solar radiation.9

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In this context, a hydrophilic extract obtained from the aerial parts of the fern Polypodium leucotomos (PL, Fernblock) exhibits strong photoprotective properties following its topical or oral administration.10–16 In vivo and in vitro studies have reported the antioxidant properties of PL.12, 13, 15, 17–24 In this chapter, we will address the composition of PL extract and its antioxidant and anticarcinogenic properties. We address the molecular basis of the effect of oral PL in preventing the adverse effects of UV, VIS, and IR radiation on the skin.

Fernblock, oxidative stress, and photoprotection Photoprotective agents Sunscreens are defined as substances that protect the skin from the harmful effects of solar UV radiation by absorbing, reflecting, scattering, or otherwise deflecting UV photons, avoiding their absorption by the components of the skin. Sunscreens must be either prophylactic or therapeutic against skin cancer.9, 17 These substances are usually antioxidants.17 Many botanical and phytochemical extracts have antioxidant properties and decrease systemic oxidative stress caused by UV radiation. PL, which has been widely studied and proven useful as a photoprotector and antioxidant, both orally and topically.15, 17 A standardized aqueous extract of PL (Fernblock) was marketed in Europe in 2000, both topically and orally. In 2006 the FDA approved Fernblock as an oral dietary supplement, and now PL extracts are available in more than 26 countries, including the USA.25 Its success in clinical trials has placed PL as an interesting photoprotective and antioxidant option.16 The NOAEL (no observed adverse effect level) from the 90-day study was determined to be 1200 mg/kg bw/day, the highest dose tested.26

Polypodium leucotomos. Origen and composition General features of the Polypodium genus and P. leucotomos species The fern genus Polypodium (Greek poly ¼ many, pod ¼ feet, due to the rhizomes shaped like small feet) is also known by the name of Phlebodium. This genus belongs to the family of Polypodiaceae.27 To survive in adverse conditions, these plants have developed a diversity of antioxidants that remove ROS. A major component of PL is polyphenol.27 Polyphenols comprise the primary antioxidant moiety of extracts made of these ferns. PL is the most common species used to prepare extracts. PL has also been described as P. aureum, “calaguala” and “anapsos.”17 The first clue of the medicinal properties of the ferns of the family Polypodiaceae goes back to the classic Greek botanist and pharmacist Dioscorides.15, 28, 29

Composition Recent studies have addressed the molecular composition of the PL extract that is commercially available for oral administration. These studies determined that the total phenols content is 250 mg/L.29 Different phenolic compounds were detected according to their retention time.30 Phenolic compounds have been identified in the aqueous extract Fernblock.30 The most abundant were phenolic acids, specifically caffeic acid, chlorogenic acid, and ferulic acid.31 These phenolic compounds displayed excellent absorption through an epithelial monolayer in vitro, ranging between 70% and 100%. Absorption of these compounds seemed to depend on a concentration-dependent, saturable active transport mechanism, as absorption was more effective at a dose of 50 mM than 200 mM.31 Importantly, three of the major phenolics of the PL extract (coumaric, ferulic, and vanillic acids) were catabolized by phase I and II enzymes, which resulted in the extended persistence of the compounds; conversely, chlorogenic, and caffeic acids were unstable and disappeared rapidly.31 Nonphenolic compounds were also found, for example, adenosine, which inhibits the protease elastase, that is released by neutrophils as part of the inflammatory cascade.28

Molecular, cellular, and clinical evidence of the photoprotective properties of Fernblock Fernblock in DNA photodamage and repair UVR damages DNA through ROS. These may promote the generation of 8-hydroxy-20 -deoxyguanosine (8-OH-dG),15 which is a marker of DNA oxidative damage and is mutagenic, favoring GC ! TA mutations.15 PL, orally administered, inhibited DNA damage and mutagenesis induced by UVR in humans and mice.10, 24, 32, 33

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PL exerts its effect through a double mechanism by decreasing both the accumulation of CPD and the oxidative damage, reducing 8-OH-dG.32 Even before UVR, oral PL decreased levels of 8-OH-dG in a mouse model of xeroderma pigmentosum (Xpc ), suggesting that oral PL decreases the damage of constitutive oxidative DNA.34 In this model, PL inhibits COX2 expression and accelerates the elimination of CPD.32 These data indicate that PL increases DNA repair capacity instead of preventing damage. Recently in a model of skin reconstructed human epidermis (RHE) (Episkin), a suitable model for the evaluation of acute UV-induced cell damage, Torricelli reported a decrease of CPD in PL treated samples vs. no treated (0% vs. 20% positive cells) after UVB irradiation.11 In human in a small-sample clinical study of healthy human volunteers also PL decreases CPD.10, 15, 24, 26 Finally, another small-sample clinical study has revealed that PL decreases UVA-dependent mitochondrial DNA damage as evidenced by decreased common deletions (CD),35 which are mitochondrial markers of chronic UVA radiation in fibroblasts and keratinocytes (Table 1).

Fernblock effect on free radicals during inflammation Evidence of the role of PL in preventing UV-mediated inflammation comes from experiments that revealed that PL prevented sunburn and erythema in UV-treated human skin19, 21 and also in PUVA-based therapy,23, 35 which is often used in the treatment of psoriasis, vitiligo, and other inflammatory skin conditions15.

TABLE 1 Fernblock, UV radiation, DNA photodamage, and inflammation. Studies

Molecular mechanism/cellular target

References

DNA In vitro Skin reconstructed human epidermis (RHE) (Episkin)

Inhibit accumulation of CPD

11

Hairless Xpc() mice

Inhibition of 8-hydroxy-20 -deoxyguanosine

32

Hairless Xpc() mice

Inhibition of mutation of DNA

32

Human

Decrease mitochondria DNA mutations

35

Human

Inhibit accumulation of CPD

10, 24

In vivo

Inflammation In vitro Human keratinocytes

Inhibition of increase of TNF-a

33

Inhibition of increase of NO and iNOS Human keratinocytes

Inhibition of activation of NF-kB and AP-1

33

PBMC

Increase of antiinflammatory cytokines IL-10

36

Hairless albino mice (Skh-1)

Reduction mast cell infiltration

36

Hairless Xpc() mice

Inhibition of neutrophil and macrophage infiltration

32

Hairless Xpc() mice

Inhibition of COX-2 expression

32

C57BL/6 mice

Reduction mast cell infiltration

37

Human

Inhibition of vasodilation

23

Human

Reduction mast infiltration

23, 24

Human

Inhibition of COX-2 and PGE2

10

In vivo

Beneficial effects of Fernblock in DNA photodamage and inflammation induced by UV radiation in the human skin, and in normal and hairless albino mice. The beneficial effects have also been tested in vitro in human keratinocytes and skin reconstructed human epidermis (RHE).

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The molecular basis of its antiinflammatory properties can be explained in terms of its ability to suppress the transcription factors NF-kB and AP-1 induced by UV radiation.33 Experiments using solar simulated radiation (SSR) proved this. Pretreatment of human keratinocytes with PL inhibited SSR mediated increase of tumor necrosis factor TNFa-1 and also abrogated nitric oxide (NO) production. Consistent with this, PL blocked the induction of iNOS elicited by SSR radiation.33 The mechanisms by which COX-2 produces cell damage involve the expression of proinflammatory molecules and markers (e.g., TNF-a and inducible NOS (iNOS))33, 36, 38 among others. This is consistent with the ability of PL to block the transcriptional activation of activator protein 1 (AP-1) and NF-kB induced by UV radiation.33 In this regard, as will be discussed further, the antitumoral effect of PL is also due to suppression of the induction of proinflammatory and growth-promoting genes by downregulating the activation of the biosynthesis of PG, which generates free oxygen radicals. Together, these effects account for the decrease of leukocyte extravasations and decrease the presence of mast cells in the irradiated area after PL treatment.23, 24, 32, 37 These data complement the in vitro studies using human PHA-stimulated peripheral blood mononuclear cells (PBMC), which showed that PL induced a decrease in the production of IL-2, IFN-gamma, and TNF-alpha and completely inhibited the expression of the inflammatory cytokine IL-6. In the same experiments, the addition of PL increased the antiinflammatory cytokines IL-10 production.36 PL also inhibited apoptosis and cell death,33, 39 therefore preventing apoptosis/necrosis-triggered inflammation (Table 1).

Fernblock prevents UV radiation-mediated immunosuppression UV radiation induces immunosuppression, anergy, and immunological tolerance. This is mediated by a marked decrease in the numbers of epidermal Langerhans cells (eLCs), which leads to T helper 1 lymphocyte (Th1) clonal anergy.3, 40 PL efficiently blocked eLC depletion upon UV irradiation and prevented the appearance of abnormal dendritic cell (DC) morphologies after irradiation using a solar simulator. In these experiments, PL inhibited DC apoptosis and promoted the secretion of antiinflammatory cytokines and inhibited expression of proinflammatory cytokines (e.g., TNF-a).33 The molecular mechanism of enhanced DC survival seems to implicate the inhibition of trans-urocanic acid (UCA) isomerization12, 15, 34 and blockade of iNOS expression induced by UV radiation41 (Table 2).

Fernblock, an anti-UV-induced tumor progression agent Several studies have documented the antitumor properties of different fern extracts, including PL. In vivo experiment in a hairless albino mice model, PL blocked skin tumor formation and photoaging as a result of exposure to UVB, even after discontinuation of the treatment for 8 weeks.22 The biological effects of UV radiation lead to skin cancer. UVR exposure modulates the expression of genes, activates signal transducer, transcription factor, and molecules that induce carcinogenesis.3, 12, 13 PL induced activation of the tumor suppressor p53.32 PL enhanced both p53 expression and activation in irradiated Xpc  mice.32 In agreement with increased p53 activity, PL also decreased UV radiation-induced cell proliferation.23, TABLE 2 Fernblock, UV radiation and immunosuppression. Studies

Molecular mechanism/cellular target

References

Human fibroblasts

PL interferes with the cis-UCA isomerization

34

Human keratinocytes

Inhibition of TNF-a

33

Immature human DCs

Protects DCs from apoptosis

40

Immature human DCs

Induces DCs production of antiinflammatory cytokines (IL-12)

40

Hairless rats

Reduction of glutathione oxidation in blood and epidermis

41

Human

Inhibition of UVR-mediated Langerhans cell depletion

21, 23, 24

In vitro

In vivo

Beneficial effects of Fernblock® in photoimmunosuppression induced by UV radiation in the human and mice skin. In vitro experiments using immature human dendritic cells and human keratinocytes probed the inhibition of the immunosuppression elicited by Fernblock.

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B Antioxidants and cancer

24

In this line, and recently in an experimental model of human skin (RHE) (Episkin), PL applied after UVB reduced the number of cells expressing p21, and Ki-67,11 decreasing the proliferating rate induced by UVB.11 The activation of p53 has been shown to decrease the expression of COX-2, thereby reducing the inflammatory response.32 Levels of phospho-p53ser15, presumably active form of p53, were increased in the skin of PL-treated mice, which inversely correlated with decreased COX-2 levels, suggesting that orally administered PL reduces UV-induced COX-2 levels in mouse skin, at least in part, by activating tumor suppressor protein p53. Importantly, the antioxidant activity of PL was not lost during digestion but reached the bloodstream. Besides, the observed increase in superoxide dismutase (SOD), glutathione peroxidase (GP), and glutathione transferase (GST) activities cannot be explained in terms of increased expression as erythrocytes are anucleated. Instead, it was postulated that PL might play an allosteric activating role on these enzymes. This is particularly striking for SOD because, in vehicle-treated and irradiated animals, SOD activity decreased, but PL prevented such decrease.39 Regarding the anticarcinogenic effect, PL has been useful in a small group of patients with actinic keratoses (AK).42 It has been reported whether PL could improve the effect of photodynamic therapy (PDT), one of the most effective treatments in AK. One group of patients combined oral therapy with PL 1 week after PDT42 and another group received only PDT. Both therapies were successful at 6 months of treatment and reduced the number of injuries; however, supplementation with PL increased the clearance rate compared with PDT alone. The PL supplement to the PDT seems to be a complementary agent for the treatment of the cancer field.42 Recently the changes that PL produces about the process of carcinogenesis have been verified in humans.11 Oral PL in individuals with Fitzpatrick skin phototype I-III, after radiation with UVB, reduced the levels of cells expressing cyclin D1 and Ki67, thereby decreasing the proliferation induced by UVB. The expression of COX also decreased. PL shows chemoprotective and antiinflammatory properties against UVB-induced damage11 (Table 3).

TABLE 3 Fernblock, UV radiation and photocarcinogenesis. Studies

Molecular mechanism/cellular target

References

Dermal fibroblasts Melanoma cells

Increase the expression of the inhibitor TIMP-1

43

Skin reconstructed human epidermis (RHE) (Episkin)

Reduce number of cells p21, and Ki-67

11

Hairless albino mice

Reduction number of mice showing skin tumors at 8 weeks after the cessation of chronic UVB exposure

22

Hairless albino mice

Inhibits angiogenesis

36

Hairless albino mice

Enhance the antioxidant plasma capacity

39

Hairless albino mice Hairless Xpc() mice

Increase the number of p53(+) cells

3, 32

Hairless Xpc() mice

Inhibition of DNA mutations

32

Hairless Xpc() mice

Inhibition of COX-2 expression

32

Hairless Xpc() mice

Reduce the number of proliferating epidermal cells

32

Human

Reduce number of proliferating epidermal cells

24

Human

Increase the clearance rate of AK

42

Human

Reduce expression of cyclin D and Ki-67

10

Human

Inhibition of COX-2 expression

10

Human

Increased the MED in patients with familial MM

44

In vitro

In vivo

Fernblock, and anti-UV-radiation-induced tumor progression agent. In vitro and in vivo studies in humans and experimental animals that support the power of Fernblock to inhibit the mechanisms involved in photocarcinogenesis.

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Fernblock and malignant melanoma The effect of PL as a systemic photoprotector has been studied in patients with familial and/or multiples MM, patients with sporadic MM, and control without a history of MM. After UVR, PL was administered with a total dose of 1080 mg. PL treatment significantly increased the minimal erythema dose (MED) mean in all patients. However, in patients with familial MM, those with DKN2A mutation and/or polymorphisms in MC1R, genes to confer a known high degree of susceptibility to MM, were those who most beneficiary effect PL. These results seem promising regarding possible PL oral therapy in patients at high risk of developing melanoma.44 PL also complemented the effect of ascorbate in limiting melanoma cell growth and their ability to remodel the extracellular matrix (ECM), among other effects, by increasing the expression of the metalloprotease inhibitor TIMP-145 (Table 3).

Fernblock prevention of matrix remodeling and other cellular effects (Table 4) In vitro and in vivo studies have shown that PL counteracts the ECM alterations due to UVR. PL acts as photoprotective and antioxidant. PL inhibits the overexpression of MMP in keratinocytes and fibroblasts and increases the expression of the endogenous metalloprotease inhibitor, TIMP.20, 43, 47 Besides, in vitro, in melanoma cells, PL decreased MMP-1 expression in an AP-1 dependent manner and stimulated TIMP-2, involved in the modulation of basal membrane homeostasis.43 Transforming growth factor-b (TGF-b) is also essential in the lesions of ECM since it is a primary regulator of ECM.47 UV radiation has been shown to induce decreased procollagen synthesis by the inhibition of TGF-b.48 Besides, AP-1 that is activated after UVR exposure plays a vital role in MMP upregulation and TGF-b expression blockade.48 Concerning a direct effect of PL on collagen synthesis, in UVR irradiated fibroblasts, PL stimulated collagen type I deposition and V and collagen types I, III, and V in fibroblasts unirradiated.43 In contrast, UV radiation decreases the induction of type III collagen synthesis induced by PL. These data imply that PL promotes the synthesis of collagen types I and V in the skin exposed to UV radiation and the assembly of fibrillar collagen in the skin protected by the sun. Stimulation of collagen expression was associated with the increase in TGF-b, promoted by PL. UVB induced the inhibition of collagen synthesis and a decrease in TGF-b expression. However, UV radiation did not decrease the PL stimulation of TGF-b, suggesting that UV and PL rays regulate TGF-b expression by separate but related pathways.43 Specifically, PL promotes the expression of TGF-b in nonirradiated or UV irradiated fibroblasts. This effect may be responsible for the observed inhibition of angiogenesis in vivo.43 In summary, PL protects ECM through two types of actions: one depends on its effect on the decreased expression of ECM proteolytic enzymes and TIMP overexpression, and the second is related to the expression/assembly of structural collagens (types I, III, and V) and the increase

TABLE 4 Fernblock, UV radiation and extracellular matrix. Studies

Molecular mechanism/cellular target

References

Fibroblasts, keratinocytes

Inhibit UVR-induced MMP-1, MMP-2

20, 43

Fibroblasts

Stimulate TIMPs

43

Fibroblasts

Increase the synthesis of types I, III, and V collagen in nonirradiated fibroblasts

43

Fibroblasts

Increase the synthesis of types I and V collagen in UV-radiated fibroblasts

43

Fibroblasts

Stimulatory effects on TGF- b expression in nonirradiated or UV-irradiated

43

Melanoma cells

Inhibit MMP-1 Stimulate TIMP-2 Inhibit TGF- b expression in melanoma cell

43

Improved skin hydration and elasticity Decrease transepidermal water loss (TEWL)

46

In vitro

In vivo Human

Fernblock prevents UV-radiation-induced matrix disturbance in vitro in keratinocytes, fibroblast, and human melanoma cells and in vivo in human.

394 SECTION

B Antioxidants and cancer

TGF-b in fibroblasts. PL exhibits a strong antiaging effect. It prevents the morphological effects associated with increased oxidative stress, which include dramatic disorganization of the microfilaments and loss of cell-matrix and cell-cell anchorage points.49 Recently in a group of 20 women of 37.2  5.5 years. PL has improved the ECM biophysical properties. PL at a dose of 480 mg/day for 3 months increases hydration and elasticity of the skin and decreased transepidermal water loss (TEWL)46 (Table 4).

Fernblock preventions of photodamage induced by visible light and infrared radiation Visible light (VIS) and infrared (IR) radiation may also contribute to the pathogenesis of photoaging and photocarcinogenesis.50 Among other VIS and IR, they produce hyperpigmentation, erythema, genotoxicity, or increased expression of MMPs. Until a few decades ago, as previously mentioned, VIS did not appear to induce harmful effects on the skin.6, 7 VIS also produces indirect DNA damage through the production of ROS.50 IR radiation, similar to UVR, seems to be involved in photoaging and potentially photocarcinogenesis.51 IR exposure induces increased expression of MMPs.52 In human skin, PL acts against the detrimental effects produced by IR (specifically IRA) and VIS radiation in dermal fibroblasts. PL prevents the changes induced by IRA and VIS radiation on the viability and the cell cycle of human fibroblasts, as well as on the expression of ECM components including fibrillins (FBN) 1 and 2, elastin (ELN), MMP-1, and cathepsin K (CTSK).50 The cell mortality rate increased significantly after 247.3 J/cm2 of VIS compared to the control; however, pretreatment with PL before VIS light decreased the cell death rate. Similarly, IRA radiation (1.56 and 1.95 J/cm2) induced a significant increase in cell death rate concerning control cells. However, treatment with PL before irradiation significantly reduced the cell mortality rate.50 MMP-1 was increased after VIS exposure (247.3 J/cm2) in human fibroblasts. Pretreatment with PL significantly decreased MMP-1 expression. In the case of IRA light, it also produced a significant increase in MMP-1 expression after irradiation (1.56 J/cm2). Incubation with PL before irradiation with IRA significantly counteracted this increase. CTSK is involved in ECM remodeling. VIS and IRA light induces in the dermal fibroblast increased the expression of CTSK. PL, therefore, prevents such an increase, indicating that CTSK is a factor present in photoaging.50 Fibrillin (FBN) 1 (FBN1) and 2 (FBN2) are the main components responsible for the biomechanical properties of most tissues. They also intervene in the assembly of elastin (ELN). After treatment with PL, the expression of FBN1 and FBN2 generally increased after VIS or IRA radiation. PL per se stimulated the expression of FBN1 and FBN2.50 When irradiation was preceded by treatment with PL, the ELN increased markedly.50 In general, the results suggest that PL could be considered as a preventive treatment against cellular skin damage caused by VIS and IRA radiation, and therefore could be to prevent photoaging50 (Table 5).

TABLE 5 Fernblock and the effects on the skin of visible light (VIS) and infrared (IR) radiation. Studies

Molecular mechanism/cellular target

References

Human fibroblast

Decreases cell mortality rate induced by VIS or IRA radiation

50

Human fibroblast

Increase the expression of FBN1 and FBN2 after VIS or IRA radiation

50

Human fibroblast

Increase the expression of ELN after VIS or IRA radiation.

50

Human fibroblast

Decrease the expression of MMP-1after VIS or IRA radiation

50

Human fibroblast

Decrease the expression of CTSK after VIS or IRA radiation

50

In vitro

Fernblock, prevent skin photodamage induced by visible light (VIS) and infrared (IR) radiation.

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Potential use of Fernblock in the treatment of other pathological skin conditions Idiopathic photodermatosis These lesions include clinical conditions that emerge from exposure of the skin to normal sunlight. Some examples include polymorphic light eruption (PMLE), solar urticaria, chronic actinic dermatitis, and actinic prurigo.37, 53 Recent studies have addressed the potential of PL to counter the occurrence of PMLE.53 Oral PL treatment produced a significant reduction in positive photoprovocation results and a significant delay in PMLE lesions formation.54 Other studies have reported the beneficial effects of PL in patients affected with PMLE. In those studies, patients exposed to sunlight were also treated with PL orally (480 mg/day). Both studies displayed a significant improvement in subjective symptoms and a reduction of skin reaction.42, 55 All the results suggested that PL has a beneficial effect in the treatment of PMLE, which may be extended to other idiopathic photodermatoses (Table 6).

Pigmentary disorders Vitiligo One of the most efficient methods to treat vitiligo vulgaris is a narrow band (311–312 nm) UVB phototherapy, which stimulates melanocyte reservoirs to counter depigmentation. A double-blind, placebo-controlled study has shown that the conjoined use of PL with narrow-band UVB (NB-UVB) increases the re-pigmentation of the head and neck area of vitiligo patients.56 In a second study, patients with generalized vitiligo receive therapy with both NB-UVB and PL (twice-weekly NB-UVB and oral PL 480 mg daily, 6 months) or NB-UVB phototherapy alone. The combination of PL and NB-UVB improved the re-pigmentation compared with NB-UVB alone.57 Together with its beneficial effect on PUVA therapy, these studies suggest that PL may be a beneficial, general use adjuvant in phototherapy protocols.58 PL effect might be attributable to their immunomodulatory and antioxidant properties. Pathophysiology of vitiligo includes loss of melanocyte adhesion, inflammasome activation, activation of innate immunity, and oxidative stress.13

Melasma Melasma, a common skin disorder in adults, is acquired hyper melanosis in areas exposed to the sun. There are several various treatment methods available; however, the development of new therapies could help to understand the pathogenesis and improve this skin condition, which has significant psychological and social repercussions.59, 60 TABLE 6 The potential use of Fernblock in other skin pathology. Studies

Molecular mechanism/cellular target

References

In vivo Idiopathic photodermatosis Human

Delay in PMLE lesions formation

53

Human

Reduces skin reactions and subjective symptoms

54, 55

Pigmentary disorders Vitiligo Human

PL to the treatment with NB-UVB shows an increased re-pigmentation in the head and neck area

56, 57

Human

PUVA plus PL versus PUVA increase the percentage of patients with more than 50% of re-pigmentation

58

Decreased in mean melasma area and severity index

59, 60

Melasma Human

In vivo human studies have shown the beneficial effect of Fernblock® in the treatment polymorphic light eruption, melasma, and vitiligo.

396 SECTION

B Antioxidants and cancer

In this sense, PL has proven effective in treating patients with melasma. In two clinical studies conducted in the Hispanic59 and Asian population,60 in both cases after 12 weeks of treatment with oral PL, Melasma Area and Severity Index significantly decreased in patients treated with PL vs. placebo59, 60 (Table 6).

Applications to Fernblock to skin cancers or other conditions Fernblock exhibits a wide array of beneficial effects and displays no significant toxicity or allergenic properties. Its dual route of administration—topical and oral—suggests that it prevents UV-induced damage (taken orally before exposure) and is also useful to protect the skin during exposure. It may also contribute to the healing and regeneration of the skin that is required postexposure. These regenerative properties also underlie its potential as an antiaging and anticancer tool. Most of its beneficial effects are related to its antioxidant and ROS-scavenging capability, but its ability to prevent apoptosis and block the improper ECM rearrangements that occur during oxidative damage suggest that its profile may extend beyond skincare and be useful as a systemic antioxidant tool. Recently, evidence suggests that PL reduces IR-VIS damage. Further research will be aimed to study its effect on other parameters related to aging, such as telomere length and telomerase activity.

Summary points Photoprotective and antitumoral effects of oral-systemic administration of an extract of Polypodium leucotomos (Fernblock) include the following: l l l l l l l l l l l l l

Prevents sunburn and erythema mediated by UV radiation Prevents DNA photodamage and favors its repair Blocks t-UCA photoisomerization Blocks lipid peroxidation Enhances natural cutaneous and plasmatic antioxidant systems Blocks photoinduction of TNFa, iNOS, AP-1, and NF-kB Inhibits UV-induced expression of COX-2 Prevents cell death and apoptosis Prevents photoinduced depletion of Langerhans cells and preserves their function Enhances TGF-beta expression Inhibits cytoskeletal disarray and MMP expression and activation Enhances TIMP expression Prevent and reduce VIS and IR radiation photodamage

Acknowledgments Salvador Gonzalez is a consultant for Industrial Farmaceutica Cantabria (IFC). This work has been partially supported by two grants: from the Carlos III Health Institute, Ministry of Science and Innovation, Spain (PS09/01099) and from the Comunidad Auto´noma de Madrid (S2010/ BMD-2359). The authors want to dedicate this chapter to the memory of Dr. Vicente Garcia Villarrubia, a very special scientist and teacher, in recognition of his remarkable contribution in the field.

References 1. NTP (National Toxicology Program) Report on carcinogens, 13th ed. Department of Health and Human Services, Public Health Service; Research Triangle park, NC, USA, 2014. (Accessed 24 March 2016). Available online: http.//ntp.niehs.nih.gov/pubhealth/roc/roc13/ 2. Gies P, van Deventer E, Green A, Sinclair C, Tinker R. Review of the global solar UV index 2015 workshop report. Health Phys 2018;114:84–90. 3. Mullenders LHF. Solar UV damage to cellular DNA, from mechanisms to biological effects. Photochem Photobiol Sci 2018;17:1842–52. 4. Christensen L, Suggs A, Baron E. Ultraviolet photobiology in dermatology. Adv Exp Med Biol 2017;996:89–104. 5. Albrecht S, Jung S, M€uller R, Lademann J, Zuberbier T, Zastrow L, et al. Skin type differences in solar-simulated radiation-induced oxidative stress. Br J Dermatol 2019;180:597–603. 6. Pathak MA, Riley FC, Fitzpatrick TB. Melanogenesis in human skin following exposure to long-wave ultraviolet and visible light. J Investig Dermatol 1962;39:435–43. 7. Kollias N, Baqer A. An experimental study of the changes in pigmentation in human skin in vivo with visible and near-infrared light. Photochem Photobiol 1984;39:651–9.

Fern extract, oxidative stress, and skin cancer Chapter

34

397

8. Piazena H, Meffert H, Uebelhack R. Spectral remittance and transmittance of visible and infrared-a radiation in human skin-comparison between in vivo measurements and model calculations. Photochem Photobiol 2017;93:1449–61. 9. Yeager DG, Lim HW. What’s new in photoprotection, a review of new concepts and controversies. Dermatol Clin 2019;37:149–57. 10. Kholi I, Griffith JL, Isedeh P, Silpa-Archa N, Al-Jamal M, Lim HW, et al. The effect of oral Polypodium leucotomos extract (PLE) on ultravioletinduced changes in the skin. J Am Acad Dermatol 2017;77:33–41. 11. Torricelli P, Fini M, Fanti PA, Dika E, Milani M. Protective effects of Polypodium leucotomos extract against UVB-induced damage in a model of reconstructed human epidermis. Photodermatol Photoimmunol Photomed 2017;33:156–63. 12. Parrado C, Philips N, Gilaberte Y, Juarranz A, Gonza´lez S. Oral photoprotection, effective agents, and potential candidates. Front Med (Lausanne) 2018;26:188. https://doi.org/10.3389/fmed.2018.00188. 13. Parrado C, Mascaraque M, Gilaberte Y, Juarranz A, Gonzalez S. Fernblock (Polypodium leucotomos extract), molecular mechanisms and pleiotropic effects in light-related skin conditions, photoaging and skin cancers, a review. Int J Mol Sci 2016;29(7):17. 14. Berman B, Ellis C, Elmets C. Polypodium leucotomos—an overview of basic investigative findings. J Drugs Dermatol 2016;15:224–8. 15. Gonza´lez S, Gilaberte Y, Juarranz A, Wang SQ. Oral and systemic photoprotection. In: Lim HW, editor. Principles and practice of photoprotection. Switzerland: Springer International Publishing; 2016. p. 387–403. 16. Bhatia N. Polypodium leucotomos, a potential new photoprotective agent. Am J Clin Dermatol 2015;16:73–9. 17. Gonzalez S, Gilaberte Y, Philips N. Mechanistic insights in the use of a Polypodium leucotomos extract as an oral and topical photoprotective agent. Photochem Photobiol Sci 2010;9:559–63. 18. Gomes AJ, Lunardi CN, Gonzalez S, Tedesco AC. The antioxidant action of Polypodium leucotomos extract and kojic acid, reactions with reactive oxygen species. Braz J Med Biol Res 2001;34:1487–94. 19. Gonzalez S, Pathak MA. Inhibition of ultraviolet-induced formation of reactive oxygen species, lipid peroxidation, erythema and skin photosensitization by Polypodium leucotomos. Photodermatol Photoimmunol Photomed 1996;12:45–56. 20. Philips N, Smith J, Keller T, Gonzalez S. Predominant effects of Polypodium leucotomos on membrane integrity, lipid peroxidation, and expression of elastin and matrixmetalloproteinase-1 in ultraviolet radiation exposed fibroblasts, and keratinocytes. J Dermatol Sci 2003;32:1–9. 21. Gonzalez S, Pathak MA, Cuevas J, Villarubia VG, Fitzpatrick TB. Topical or oral administration with an extract of Polypodium leucotomos prevents acute sunburn and psolaren-induced phototoxic reactions as well as depletion of Langerhans cells in human skin. Photodermatol Photoimmunol Photomed 1997;13:50–60. 22. Alcaraz MV, Pathak MA, Rius F, Kollias N, Gonza´lez S. An extract of Polypodium leucotomos appears to minimize certain photoaging changes in a hairless albino mouse animal model. Photodermatol Photoimmunol Photomed 1999;15:120–6. 23. Middelkamp-Hup MA, Pathak MA, Parrado C, Garcia-Caballero T, Rius-Diaz F, Fitzpatrick TB, et al. Orally administered Polypodium leucotomos extract decreases psoralen-UVA-induced phototoxicity, pigmentation, and damage of human skin. J Am Acad Dermatol 2004;50:41–9. 24. Middelkamp-Hup MA, Pathak MA, Parrado C, Goukassian D, Rius-Diaz F, Mihm MC, et al. Oral Polypodium leucotomos extract decreases ultraviolet-induced damage of human skin. J Am Acad Dermatol 2004;51:910–8. 25. Choudhry SZ, Bhatia N, Ceilley R, Hougeir F, Lieberman R, Hamzavi I, et al. Role of oral Polypodium leucotomos extract in dermatologic diseases, a review of the literature. J Drugs Dermatol 2014;13:148–53. 26. Murbach TS, Beres E, Vertesi A, Gla´vits R, Hirka G, Endres JR, et al. A comprehensive toxicological safety assessment of an aqueous extract of Polypodium leucotomos (Fernblock(®)). Food Chem Toxicol 2015;86:328–41. 27. Bagniewska-Zadworna A, Zenkteler E, Karolewski P, Zadworny M. Phenolic compound localization in Polypodium vulgare L. rhizomes after mannitol-induced dehydration and controlled desiccation. Plant Cell Rep 2008;27:1251–9. 28. Vasange-Tuominen M, Perera-Ivarsson P, Shen J, Bohlin L, Rolfsen W. The fern Polypodium decumanum, used in the treatment of psoriasis, and its fatty acid constituents as inhibitors of leukotriene B4 formation, prostaglandins, Leukotrienes Essent. Fatty Acids 1994;50:279–84. 29. Sempere JM, Rodrigo C, Campos A, Villalba JF, Diaz J. Effect of Anapsos (Polypodium leucotomos extract) on in vitro production of cytokines. Br J Clin Pharmacol 1997;43:85–9. 30. Garcia F, Pivel JP, Guerrero A, Brieva A, Martinez-Alcazar MP, Caamano-Somoza M, et al. Phenolic components and antioxidant activity of Fernblock, an aqueous extract of the aerial parts of the fern Polypodium leucotomos. Methods Find Exp Clin Pharmacol 2006;28:157–60. 31. Gombau L, Garcia F, Lahoz A, Fabre M, Roda-Navarro P, Majano P, et al. Polypodium leucotomos extract, antioxidant activity and disposition. Toxicol In Vitro 2006;20:464–71. 32. Zattra E, Coleman C, Arad S, Helms E, Levine D, Bord E, et al. Oral Polypodium leucotomos decreases UV-induced cox-2 expression, inflammation, and enhances DNA repair in Xpc  mice. Am J Pathol 2009;175:1952–61. 33. Janczyk A, Garcia-Lopez MA, Fernandez-Penas P, Alonso-Lebrero JL, Benedicto I, Lopez-Cabrera M, et al. Polypodium leucotomos extract inhibits solar-simulated radiation-induced TNF-alpha and iNOS expression, transcriptional activation and apoptosis. Exp Dermatol 2007;16:823–9. 34. Capote R, Alonso-Lebrero JL, Garcia F, Brieva A, Pivel JP, Gonzalez S. Polypodium leucotomos extract inhibits trans-urocanic acid photoisomerization and photodecomposition. J Photochem Photobiol B 2006;82:173–9. 35. Villa A, Viera MH, Amini S, Huo R, Perez O, Ruiz P, et al. Decrease of ultraviolet a light-induced “common deletion” in healthy volunteers after oral Polypodium leucotomos extract supplement in a randomized clinical trial. J Am Acad Dermatol 2010;62:511–3. 36. Gonzalez S, Alcaraz MV, Cuevas J, Perez M, Jaen P, Alvarez-Mon M, et al. An extract of the fern Polypodium leucotomos (Difur) modulates Th1/Th2 cytokines balance in vitro and appears to exhibit anti-angiogenic activities in vivo, pathogenic relationships and therapeutic implications. Anticancer Res 2000;20:1567–75. 37. Siscovick JR, Zapolanski T, Magro C, Carrington K, Prograis S, Nussbaum M, et al. Polypodium leucotomos inhibits ultraviolet B radiation-induced immunosuppression. Photodermatol Photoimmunol Photomed 2008;24:134–41.

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38. Ikehata H, Mori T, Douki T, Cadet J, Yamamoto M. Quantitative analysis of UV photolesions suggests that cyclobutane pyrimidine dimers produced in mouse skin by UVB are more mutagenic than those produced by UVC. Photochem Photobiol Sci 2018;17:404–13. ´ , Cuevas J, Gonzalez S, Mallol J. Polypodium leucotomos decreases UV-induced epidermal cell proliferation and 39. Rodrı´guez-Yanes E, Juarranz A enhances p53 expression and plasma antioxidant capacity in hairless mice. Exp Dermatol 2012;21:638–40. 40. De la Fuente H, Tejedor R, Garcia-Lopez MA, Mittelbrunn M, Alonso-Lebrero JL, Sanchez-Madrid F, et al. Polypodium leucotomos induces protection of UV-induced apoptosis in human skin cells. J Invest Dermatol 2005;124(A):121. 41. Mulero M, Rodriguez-Yanes E, Nogues MR, Giralt M, Romeu M, Gonzalez S, et al. Polypodium leucotomos extract inhibits glutathione oxidation and prevents Langerhans cell depletion induced by UVB/UVA radiation in a hairless rat model. Exp Dermatol 2008;17:653–8. 42. Auriemma M, Di Nicola M, Gonza´lez S, Piaserico S, Capo A, Amerio P. Polypodium leucotomos supplementation in the treatment of scalp actinic keratosis, could it improve the efficacy of photodynamic therapy? Dermatol Surg 2015;41:898–902. 43. Philips N, Conte J, Chen YJ, Natrajan P, Taw M, Keller T, et al. Beneficial regulation of matrix metalloproteinases and their inhibitors, fibrillar collagens and transforming growth factor-beta by Polypodium leucotomos, directly or in dermal fibroblasts, ultraviolet radiated fibroblasts, and melanoma cells. Arch Dermatol Res 2009;301:487–95. 44. Aguilera P, Carrera C, Puig-Butille JA, Badenas C, Lecha M, Gonza´lez S, et al. Benefits of oral Polypodium leucotomos extract in MM high-risk patients. J Eur Acad Dermatol Venereol 2013;27:1095–100. 45. Philips N, Keller T, Hendrix C, Hamilton S, Arena R, Tuason M, et al. Regulation of the extracellular matrix remodeling by lutein in dermal fibroblasts, melanoma cells, and ultraviolet radiation exposed fibroblasts. Arch Dermatol Res 2007;299:373–9. 46. Emanuele E, Berton M, Biagi M. Comparative effects of a fixed Polypodium leucotomos/pomegranate combination versus Polypodium leucotomos alone on skin biophysical parameters. Neuro Endocrinol Lett 2017;38:38–42. 47. Lesiak A, Rogowski-Tylman M, Danilewicz M, Wozniacka A, Narbutt J. One week’s holiday sun exposure induces expression of photoaging biomarkers. Folia Histochem Cytobiol 2016;54:42–8. 48. Gao W, Lin P, Hwang E, Wang Y, Yan Z, Ngo HTT, et al. Pterocarpus santalinus L. regulated ultraviolet b irradiation-induced Procollagen reduction and matrix metalloproteinases expression through activation of TGF-b/Smad and inhibition of the MAPK/AP-1 pathway in normal human dermal fibroblasts. Photochem Photobiol 2018;94:139–49. 49. Alonso-Lebrero JL, Domı´nguez-Jimenez C, Tejedor R, Brieva A, Pivel JP. Photoprotective properties of a hydrophilic extract of the fern Polypodium leucotomos on human skin cells. J Photochem Photobiol B 2003;70:31–7. ´ . Fernblock prevents dermal cell damage induced by visible and infrared A radiation. Int J Mol Sci 50. Zamarro´n A, Lorrio S, Gonza´lez S, Juarranz A 2018;19(8) pii, E2250. 51. Schroeder P, Haendeler J, Krutmann J. The role of near infrared radiation in photoaging of the skin. Exp Gerontol 2008;43:629–32. 52. Schieke SM, Schroeder P, Krutmann J. Cutaneous effects of infrared radiation, from clinical observations to molecular response mechanisms. Photodermatol Photoimmunol Photomed 2003;19:228–34. 53. Tanew A, Radakovic S, Gonzalez S, Venturini M, Calzavara-Pinton P. Oral administration of a hydrophilic extract of Polypodium leucotomos for the prevention of polymorphic light eruption. J Am Acad Dermatol 2012;66:58–62. 54. Caccialanza M, Recalcati S, Piccinno R. Oral Polypodium leucotomos extract photoprotective activity in 57 patients with idiopathic photodermatoses. G Ital Dermatol Venereol 2011;146:85–7. 55. Caccialanza M, Percivalle S, Piccinno R, Brambilla R. Photoprotective activity of oral Polypodium leucotomos extract in 25 patients with idiopathic photodermatoses. Photodermatol Photoimmunol Photomed 2007;23:46–7. 56. Middelkamp-Hup MA, Bos JD, Rius-Diaz F, Gonzalez S, Westerhof W. Treatment of vitiligo vulgaris with narrow-band UVB and oral Polypodium leucotomos extract, a randomized, double-blind placebo-controlled study. J Eur Acad Dermatol Venereol 2007;21:942–50. 57. Pacifico A, Vidoli P, Leone G, Iacovelli P. Combined treatment of narrowband ultraviolet B light (NBUVB) phototherapy and oral Polypodium leucotomos extract versus NBUVB phototherapy alone in the treatment of patients with vitiligo. J Am Acad Dermatol 2009;60(Suppl. S1)AB154. 58. Reyes E, Jaen P, de las Heras E, Carrion F, Alvarez-Mon M, de Eusebio E, et al. Systemic immunomodulatory effects of Polypodium leucotomos as an adjuvant to PUVA therapy in generalized vitiligo, a pilot study. J Dermatol Sci 2006;41:213–6. 59. Martin LK, Caperton C, Woolery-Lloyd H. A randomized, double-blind placebo-controlled study evaluating the effectiveness and tolerability of oral Polypodium leucotomos in patients with melasma. J Am Acad Dermatol 2012;66(Suppl. S1)AB21. 60. Goh CL, Chuah SY, Tien S, Thing G, Vitale MA, Delgado-Rubin A. Double-blind, placebo-controlled trial to evaluate the effectiveness of Polypodium leucotomos extract in the treatment of Melasma in Asian skin, a pilot study. J Clin Aesthet Dermatol 2018;11:14–9.

Chapter 35

Lycium barbarum (goji berry), human breast cancer, and antioxidant profile Anna Wawruszaka, Marta Halasaa, and Karolina Oklab a

Department of Biochemistry and Molecular Biology, Medical University of Lublin, Lublin, Poland, b The First Department of Gynecological Oncology

and Gynecology, Medical University of Lublin, Lublin, Poland

List of abbreviations BrdU BC Bcl-xl CDK 6 DNA DPPH EEGB ERK HCAAs HER2 IĸB LBB LBF LBPs NF-ĸB NO PI ROS TAA TNBC

bromodeoxyuridine breast cancer B-cell lymphoma-extra large cyclin-dependent kinase 6 deoxyribonucleic acid 2,2-diphenyl-1-picrylhydrazyl goji berry ethanol extract extracellular signal-regulated kinase hydroxycinnamic acid amides human epidermal growth factor receptor 2 inhibitor of kappa B Lycium barbarum bark extract Lycium barbarum berry extract Lycium barbarum polysaccharides nuclear factor kappa-light-chain-enhancer of activated B cells nitric oxide propidium iodide reactive oxygen species total antioxidative activities triple negative breast cancer

Introduction The global cancer statistics are alarming. In 2018, 18 million individuals were diagnosed worldwide and half of them died due to cancer. By 2035, the incidence of cancers may double.1 Additionally, cancer treatment costs are increasing (7%–10% annually), and only half of the patients respond to them, while less than 10% new anticancer drugs in phase I of clinical trials will reach phase III.2, 3 It is estimated that global cancer costs will exceed $150 billion in 2020 which causes a relevant socioeconomic burden to the humankind.2 Breast cancer (BC) is the most common malignancy among women worldwide.4 In 2018, more than 2 million new cases of BC were diagnosed.1 It is estimated that 1/8 to 1/10 women will get BC during their lifetime.5 Although huge advances in targeted therapies of BC has been made, it is still the leading cause of cancer-related deaths in women accounting for 627,000 deaths in 2018.1 One of the reasons of this high mortality is huge heterogeneity of tumors, recurrence of disease, resistance to current therapies, and the progression to metastasis.5–7 There is a pressing need to find novel therapeutic options which would provide oncological patients with the best possible chance of combatting their cancer.

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00035-3 © 2021 Elsevier Inc. All rights reserved.

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Natural compounds in cancer therapy and chemoprevention For centuries, herbs have been used in traditional medicines in the therapy and prevention of different diseases. Tooth plaque analyses of Neanderthals revealed that about 50,000 years ago, they used natural plants to treat their health aliments.8 The traditional Chinese and Indian medicine (Ayurveda) are two remaining living traditions which have provided relevant current knowledge about medicinal plants.9 Today, in highly economically and technology-developed countries, traditional medicines are still popular in many of these countries as basic healthcare. It is estimated that 70%–95% of the population in developing countries continues to use traditional medicines.10 Medicinal plants have aroused scientific interest as complementary or alternative medicines, and for the hunt of bioactive compounds. A lot of anticancer agents derived from plants, such as taxol, vinblastine, podophyllotoxin, and camptothecin are currently used as antitumor therapy.11–13 It is estimated that above 60% of the current anticancer agents are derived from natural sources.14 Natural therapies, such as the use of some plants or plant-derived natural products, are being beneficial to combat cancer in contrast to synthetic drugs which cause relevant harm to the human body and the main disadvantage is the suppression of immune system.15 Furthermore, the advantages of most substances of natural origin compared with synthetic ones include lower costs of extraction as well as widespread accessibility. By state-of-the-art chemical and analytical methods, novel natural compounds from plants can be isolated using fractionation and isolation. Interestingly, only a small part of species of higher plants (5%–15% of 250,000 species) has been screened for natural bioactive compounds. To study novel therapeutic options, researchers used cell lines to investigate new compounds and their potential effects on tumor cells.16 Herbs, fruits, vegetables, and marine-derived compounds are well characterized as possessing a wide spectrum of antitumor properties, for example, induction of apoptosis and autophagy as well as inhibition of cell proliferation. Active compounds such as alkaloids, flavonoids, terpenoids, saponins, and polysaccharides derived from natural products have potent biological activities including analgesia, antiinflammatory, antitumor, immunomodulation, and antiviral properities.9, 17–20 Cancer chemoprevention involves ingestion of dietary or pharmaceutical agents in order to prevent, reduce, or delay the occurrence of neoplastic changes (before invasion). Broad spectrum of potential chemopreventive compounds in both plant extracts as well as purified molecules isolated from herbs, fruits, spices, vegetables, or teas have been explored.9, 21–23 Potential mechanisms of chemoprevention include scavenging of free radicals, antioxidant activity, and induction of deoxyribonucleic acid (DNA) repair mechanisms.24 Moreover, plant-derived compounds can enhance the immunological system, antitumor activity, and inhibit cancer cell proliferation. Goji berries are one of the most prominent chemopreventive agents studied.25

Characteristics of Lycium barbarum (goji berry) Goji berries are the fruits of L. barbarum and Lycium Chinese (Fig. 1). Lycium (named Ningxia gouqi in Chinese and Goji berry (wolfberry) in English) has been used by the population of China, and some other countries of Asia, for more than 2000 years as a traditional natural medical plant as well as food. Goji berries have recently gained growing popularity as a “superfood” because of their highly advantageous antioxidant and nutritive properties as well as potential health-promoting

FIG. 1 Lycium barbarum fruit.

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properties.26 Nowadays, China is the greatest supplier of these fruits worldwide and commercial amounts of goji berry are grown in the Chinese northwest territory: Hebei, Gansu, Ningxia, Inner Mongolia, Qinghai, and Shanxi provinces.27 Lycium is a part of the Solanaceae family which yields abundant foods, for example, some fruits that are yellow to red, including potatoes, tomatoes, or eggplants. Goji berries are 1–2 cm long (ellipsoid) fruits with bright orange-red color and a sweet and tangy flavor.26 After harvesting in late summer/early autumn, berries are sun-dried and, traditionally, the dried fruits are cooked before they are consumed. Goji berries are often used in Chinese herbal tea and soups. Furthermore, goji fruits are used for the production of wine, juice, and tincture.28 The nutrients in berries contain carbohydrate (46%), dietary fiber (16%), protein (13%), and fat (1.5%). Therefore, these fruits can be a great source of macronutrients. High content of micronutrients (minerals and vitamins) can also be found in the berries.26, 29

Anticancer properties of L. barbarum in breast cancer Goji berry shows numerous bioactive activities, including anticancer, antiinflammatory, immunomodulating, antioxidative, radioprotective, antiaging, antidiabetic, antiviral, antifatigue, cardioprotective, neuroprotective, hepatoprotective, hypolipidemic, and antiosteoporosis effects (Fig. 2).30–32 Lycium barbarum polysaccharides (LBPs) have been found to have antiproliferative and proapoptotic activities against a great deal of cancer cells both in vitro and in vivo.30 Goji berry is widely used in Chinese medicine as an adjuvant in the treatment of patients with malignant melanoma, nasopharyngeal, renal cell, lung, or colorectal carcinoma.33–35 Moreover LBPs are able to strengthen the action, and reduce adverse effects, of other forms of anticancer therapies.30 The anticancer activity of goji berry is associated with the modulation of biochemical pathways involved in the induction of apoptosis and cell cycle arrest at the miscellaneous cell cycle checkpoints, inhibition of proliferation, and regulation of signal transduction pathways.33 Current forms of breast cancer therapy often fail due to serious side effects and the occurrence of chemoresistance; therefore, natural medicine is a complementary approach for breast cancer patients’ therapy.30 Ethanol extract isolated from goji berry (EEGB) inhibited the proliferation of T47D cells in a dose- and time-dependent mode. Antiproliferative properties of the extract negatively affected both cancer cells’ DNA synthesis as well as proliferation signaling pathways. T47D breast cancer cells after EEGB treatment revealed a decline in the bromodeoxyuridine (BrdU) incorporation during the process of DNA synthesis. Interestingly, the same extract shows the lack of cytotoxic effects on human normal skin fibroblasts. Moreover, western blot analysis demonstrated a decrease in antiapoptotic proteins and an increase in the expression of proapoptotic proteins in breast cancer cells treated with the EEGB. Treatment of T47D cells with the EEGB caused an increase in p21 and p53 protein expression. Increase in the proapoptotic Bax protein expression and decrease in the expression of antiapoptotic B-cell lymphoma-extralarge (Bcl-xL) protein indicates apoptosis mediated by EEGB via the mitochondrial pathway. Moreover, T47D cells treated with EEBG treatment showed decreased expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) protein, and an increased expression of inhibitor of kappa B (IkB) protein. An analysis of the expression of proteins involved in cell cycle inhibition demonstrated a slight decrease in cyclin D1 and cyclin-dependent kinase 6 (CDK 6) proteins expression after EEBG treatment.25 Pectin-free and polysaccharide fractions of goji berry showed antiproliferative effects on three different types of breast cell lines—MCF-7 (estrogen receptor +, progesterone receptor +, human epidermal growth factor receptor 2

FIG. 2 Biological properties of goji berry (Lycium barbarum).

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(HER2)—cancer line), MDA-MB-231 (triple negative breast cancer line, TNBC), and MCF-10A (nontumorigenic epithelial cell line). The pectin-free fraction further showed dose-dependent growth inhibition of all analyzed cell lines, but the most significant effect was achieved on the two breast cancer lines (MCF-7 and MDA-MB-231). In contrast to the pectin-free fraction, the polysaccharide fraction demonstrated insignificant activity in relation to all analyzed breast cell lines. A combination of polysaccharide and pectin-free fractions did not show a synergistic effect on the MCF7 cell line. Polyphenols contained in goji berry fruit showed selective activity to breast cancer cells (MCF7 and MDA-MB-231) without affecting MCF-10A nontumorigenic breast cells. The pectin-free fraction with the highest content of polyphenols exhibited the highest anticancer potential against breast cancer cells.36 LBPs from goji berry induce growth inhibition in MCF-7 breast cancer cells, cause inhibition of the cell cycle in the S phase as well as induction of apoptosis via the extracellular signal-regulated protein kinase (ERK) signaling pathway. ERK pathway is one of the key factors of LBP anticancer activity. The level of active ERK, phosphorylated ERK (pERK), was decreased after LBP treatment in MCF-7 breast cancer cells. It can be concluded that LBP suppresses the cell cycle depending on the activation of ERK. p53, p-p53, and p21 protein levels increased after LBP treatment in MCF-7 cancer cells. Therefore, p53 can act as one of the upstream regulators of ERK activation for the induction of apoptosis in MCF-7 cells treated with LBP.33 LBPs induce growth inhibition of estrogen receptor-positive MCF-7 cells due to specific alterations of the metabolic pathways of estradiol (E2). LBPs exhibit an increase in estrone (E1), 2-OH-E1, and estriol (E3) formation as well as a decrease in 16a-OH-E1.30 L. barbarum downregulates the growth of MCF7 cells stimulated by estradiol via the creation of antiproliferative 2-OH-E1 and accelerates conversion of mitogenic 16a-OH-E1 to antimitogenic estriol. L. barbarum treatment caused an increase in the 2-OH-E1:16a-OH-E1 ratio because of an increase in 2-OH-E1 formation and a decrease in 16a-OH-E1:estriol ratio due to an increase in estriol formation. L. barbarum changes the cellular metabolism of 17bestradiol in a mode that is favorable to the upregulation of the 2-hydroxylation pathway and to enhance the transformation of 16a-OH-E1 to estriol. Since these two pathways are associated with the output of antiproliferative metabolites of 17bestradiol, it is likely that changes in estradiol biosynthesis represent a mechanism responsible for antiproliferative action of goji berry.37 The total aqueous water extracts from Lycium barbarum bark (LBB) showed about a 20-fold greater potency than berry aqueous water extract (LBF) for growth arrest in MCF-7 cells.36, 38 Growth inhibitory profiles of LBB and LBF can be caused by their different chemical composition and their complementary actions on the metabolism of estrogen.30, 38 LBB and LBF affected distinct pathways of 17b-estradiol metabolism to exert their growth inhibitory effect on MCF-7 cells. LBB treatment mainly affected the C2-hydroxylation pathway, increasing the formation of antiproliferative 2OH-E1. In turn, LBF treatment substantially enhanced the reduction of pro-mitogenic 16a-OH-E1 to comparatively inert estriol. Lycium barbarum extract, used as a single agent, inhibited the growth of MCF-7 cells by accelerating the conversion of 16a-OH-E1 to estriol.38 Nonfractionated aqueous extracts from Lycium barbarum bark inhibited the growth of MCF-7 cells, induced cell cycle progression (G1 or G2/M arrest) and apoptosis, and formed the antiproliferative 17b-estradiol metabolites. Extracts efficiently downmodulated the growth-promoting effects of 17b-estradiol through distinct mechanisms in the isogenic model of cell culture with modulated estrogen receptor function (Table 1).39

Antioxidative properties of L. barbarum in breast cancer Two sources of reactive oxygen species (ROS), endogenous and exogenous, are proceeded from a variety of cellular processes as the byproducts of semireduction of molecular oxygen. The highest concentration of endogenous ROS occurs in mitochondria and peroxisomes The most common sources of exogenous ROS are tobacco smoking, drugs and xenobiotics, global pollutants, and radiation. Overhigh level of ROS has a strong influence on the pathological states of the body, including carcinogenesis through the DNA and protein damage. On the other hand, ROS in low concentrations are highly needed in homeostatic maintenance and intracellular signaling pathway modulation.26, 40, 41 Goji berry shows high levels of polysaccharides, polyphenols, flavonoids, carotenoids, and their derivatives, which play an important role in the quality of human health through free radical neutralization. LBPs’ fraction features stands out from other fractions in terms of quantity and quality, constituting 23% of dry goji berry after extraction.42 Antioxidant enzyme activity is enhanced by the LBPs’ dose-dependent treatment in mice.30 Other compounds found in goji berry— hydroxycinnamic acid amides (HCAAs) play a crucial role in inflammatory phenomena. HCAAs are able to effectively block nitric oxide (NO) generation, thus they are considered as a prominent antiinflammatory effector.39, 43 Polysaccharides and phenolic compounds, which condition the total antioxidative activities (TAA) from goji berry extract, are related with ROS and the quantitative differentiation of these compounds strongly depends on goji berry genotypes.44 Black goji

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TABLE 1 Mechanisms of action of Lycium barbarum in breast cancer. Extract/fraction of extract

Model of breast cancer

Mechanism of action

References

Nonfractionated ethanol extract

T47D cancer cells

-

Inhibition of proliferation Decline of the BrdU incorporation Increase in p21 and p53 proteins expression Increase in Bax and decrease in Bcl-xl proteins expression Increase in IkB and decrease in NF-kB proteins expression Decrease in cyclin D1 and CDK 6 proteins expression

25

Pection-free fraction

MCF7, MDA-MB-231 cancer cells and MCF-10A nontumorigenic cells

- Dose-dependent inhibition of proliferation of all analyzed cell lines

36

Polysaccharide fraction

- Insignificant antiproliferative activity in relation to all analyzed breast cell lines

Polyphenol fraction

- Selective antiproliferative activity to MCF7 and MDAMB-231 without affecting MCF-10A cells

Polysaccharide fraction

MCF-7 cancer cells

- Inhibition of the cell cycle in S phase - Induction of apoptosis via ERK signaling pathway - Increase in p53, p-p53 and p21 proteins expression

33

Polysaccharide fraction

MCF-7 cancer cells

- Increase in estrone (E1), 2-OH-E1 and estriol (E3) as well as decrease in 16a-OH-E1 formation - Increase of the 2-OH-E1:16a-OH-E1 ratio because of the increase in 2-OH-E1 formation and decrease of the 16aOH-E1:estriol ratio due to increase in estriol formation

30, 37

Bark aqueous water extracts

MCF-7 cancer cells

- Increase in the formation of 2-OH-E1

38

Berry aqueous water extract Nonfractionated aqueous extracts

- Reduction of pro-mitogenic 16a-OH-E1 to estriol MCF-7 cancer cells

- Induction of the cell cycle progression (G1 or G2/M arrest) and apoptosis - Formation of the antiproliferative 17b-estradiol metabolites - Downmodulation the growth-promoting effects of 17bestradiol

39

berries exhibit better antioxidant properties than red goji berries due to their high abundance of phenolics and flavonoids. Antioxidant potential of these compounds was checked using colorimetric methods and the absorbance was measured by ultraviolet spectroscopy.45 Goji berry is known for its antioxidative properties in breast cancer cells (MCF7 and MDA-MB231). The goji berry fraction with the highest amount of polyphenols has the strongest antioxidative properties against the breast cancer cells mentioned above.36

Applications to other cancers or conditions In this chapter, we review the antioxidative and anticancer properties of medicinal plant Lycium barbarum (goji berry) in breast cancer. We found a significant association between different fractions isolated from goji berries containing varying abundance of active compounds and their influence on breast cancer cells.25, 33, 36 We described the polysaccharide fraction as one of the most significant factor in cancer cell combat during in vitro and in vivo studies.30 Polysaccharides from goji berries show antiproliferative effects through specific alteration of estradiol cellular metabolism in estrogen receptorpositive breast cancer cells and influence ERK signaling pathways, causing the cell cycle inhibition and induction of

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apoptosis.33, 37 Interestingly, LBPs decrease doxorubicin-induced cardiotoxicity but do not decrease doxorubicin antitumor activity.46 Anticancer properties of goji berries are observed in many different types of cancers, including hepatocellular, gastric, or colorectal cancer.35, 47, 48 Hot-water extract from L. barbarum dose dependently inhibits cell proliferation and is considered as a stimulator of p53-induced apoptosis in hepatocellular carcinoma (HCC) cells.47 Polysaccharides contained in goji berries cause cell cycle arrest in the G0/G1 phase in MGC-803 cell lines, and in the S phase in SGC-7901 human gastric cancer cells.48 In colorectal cancer (SW480 and Caco-2 cell lines), LBPs significantly inhibit cancer cell growth. Cell cycle distribution was enhanced in the G0/G1 phase and decreased in the S phase.35 In addition to the anticancer and antioxidant properties described above, goji berries exhibit a number of other healthpromoting properties, such as antiinflammatory, antiaging, immunomodulating, antiviral, antifatigue, antidiabetic, cardioprotective, hepatoprotective, neuroprotective, hypolipidemic, or antiosteoporosis.49–58

Summary points l l

l

l

l

l

l

l

l

Lycium barbarum (goji berry) is a member of the Solanaceae family exhibiting beneficial effects on human health. The desired properties of L. barbarum for human health result from high concentrations of polysaccharides, flavonoids, polyphenols, carotenoids, and their derivatives. Active compounds contained in goji berries are able to neutralize free radicals, thereby affecting the maintenance of cellular homeostasis and regulation of intracellular signaling pathways. Goji berry shows anticancer, antioxidant, antiinflammatory, antiaging, antifatigue, immunomodulating, antiviral, hypolipidemic, antidiabetic, cardioprotective, neuroprotective, hepatoprotective, or antiosteoporosis activities. Goji berry is widely used in Asian medicine as an adjuvant in the therapy of patients with malignant melanoma, renal cell, nasopharyngeal, colorectal, or lung carcinoma. Extracted fractions from L. barbarum, depending on the content of individual compounds, show antiproliferative properties against many types of cancer cells, including breast carcinoma, while there is a lack of the cytotoxic effect on human normal cells. Anticancer activity of L. barbarum is associated with alteration of biochemical pathways involved in the inhibition of cancer cell proliferation, cell cycle arrest, and induction and regulation of signal transduction pathways. Lycium barbarum polysaccharides (LBPs) are the best described and the most effective faction in the fight against cancer in vitro and in vivo. LBPs regulate the steroid biogenesis, increase the expression level of p53, and activate the extracellular signal-reduced kinase 1/2 (ERK 1/2) pathway.

References 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394–424. 2. Prager GW, Braga S, Bystricky B, Qvortrup C, Criscitiello C, Esin E, et al. Global cancer control: responding to the growing burden, rising costs and inequalities in access. ESMO Open 2018;3:e000285. 3. Davis C, Naci H, Gurpinar E, Poplavska E, Pinto A, Aggarwal A. Availability of evidence of benefits on overall survival and quality of life of cancer drugs approved by European medicines agency: retrospective cohort study of drug approvals 2009–13. BMJ 2017;359:j4530. 4. Sudhakar A. History of cancer, ancient and modern treatment methods. J Cancer Sci Ther 2009;01:1–4. 5. Harbeck N, Gnant M. Breast cancer. Lancet (London, England) 2017;389:1134–50. 6. Woolston C. Breast cancer. Nature 2015;527:S101. 7. Braden A, Stankowski R, Engel J, Onitilo A. Breast cancer biomarkers: risk assessment, diagnosis, prognosis, prediction of treatment efficacy and toxicity, and recurrence. Curr Pharm Des 2014;20:4879–98. 8. Hardy K, Buckley S, Collins MJ, Estalrrich A, Brothwell D, Copeland L, et al. Neanderthal medics? Evidence for food, cooking, and medicinal plants entrapped in dental calculus. Naturwissenschaften 2012;99:617–26. 9. Fridlender M, Kapulnik Y, Koltai H. Plant derived substances with anti-cancer activity: from folklore to practice. Front Plant Sci 2015;6:799. 10. Mishra SR, Neupane D, Kallestrup P. Integrating complementary and alternative medicine into conventional health care system in developing countries. J Evid Based Complementary Altern Med 2015;20:76–9. 11. Routh S, Nandagopal K. Patent survey of resveratrol, taxol, podophyllotoxin, withanolides and their derivatives used in anticancer therapy. Recent Pat Biotechnol 2017;11:85–100. 12. Rai V, Tandon PK, Khatoon S. Effect of chromium on antioxidant potential of Catharanthus roseus varieties and production of their anticancer alkaloids: vincristine and vinblastine. Biomed Res Int 2014;2014:1–10.

Lycium barbarum (goji berry), human breast cancer, and antioxidant profile Chapter

35

405

13. Martino E, Della Volpe S, Terribile E, Benetti E, Sakaj M, Centamore A, et al. The long story of camptothecin: from traditional medicine to drugs. Bioorg Med Chem Lett 2017;27:701–7. 14. Cragg GM, Pezzuto JM. Natural products as a vital source for the discovery of cancer chemotherapeutic and chemopreventive agents. Med Princ Pract 2016;25(Suppl. 2):41–59. 15. Anurag Mishra RDL, Mishra A. Anticancer potential of plants and natural products: a review. J Diagnostic Tech Biomed Anal 2013;01: https://doi.org/ 10.4172/2327-4638.1000103. 16. Svejda B, Aguiriano-Moser V, Sturm S, H€oger H, Ingolic E, Siegl V, et al. Anticancer activity of novel plant extracts from Trailliaedoxa gracilis (W. W. Smith & Forrest) in human carcinoid KRJ-I Cells. Anticancer Res 2010;30:55–64. 17. van Weelden G, Bobinski M, Okła K, van Weelden WJ, Romano A, Pijnenborg JMA. Fucoidan structure and activity in relation to anti-cancer mechanisms. Mar Drugs 2019;17: pii: E32. 18. Wang Y, Qian J, Cao J, Wang D, Liu C, Yang R, et al. Antioxidant capacity, anticancer ability and flavonoids composition of 35 citrus (Citrus reticulata Blanco) varieties. Molecules 2017;22: pii: E1114. 19. Islam MT. Diterpenes and their derivatives as potential anticancer agents. Phyther Res 2017;31:691–712. 20. Xu XH, Li T, Fong CM, Chen X, Chen XJ, Wang YT, et al. Saponins from Chinese medicines as anticancer agents. Molecules 2016;21: pii: E1326. 21. Surh Y-J. Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 2003;3:768–80. 22. Dunn BK, Umar A, Richmond E. Introduction: cancer chemoprevention and its context. Semin Oncol 2016;43:19–21. 23. Penny LK, Wallace HM. The challenges for cancer chemoprevention. Chem Soc Rev 2015;44:8836–47. 24. Steward WP, Brown K. Cancer chemoprevention: a rapidly evolving field. Br J Cancer 2013;109:1–7. 25. Wawruszak A, Czerwonka A, Okła K, Rzeski W. Anticancer effect of ethanol Lycium barbarum (goji berry) extract on human breast cancer T47D cell line. Nat Prod Res 2016;30:1993–6. 26. Ma ZF, Zhang H, Teh SS, Wang CW, Zhang Y, Hayford F, et al. Goji berries as a potential natural antioxidant medicine: an insight into their molecular mechanisms of action. Oxid Med Cell Longev 2019;2019:2437397. 27. Sun Y, Rukeya J, Tao W, Sun P, Ye X. Bioactive compounds and antioxidant activity of wolfberry infusion. Sci Rep 2017;7:40605. 28. Potterat O. Goji (Lycium barbarum and L. chinense): phytochemistry, pharmacology and safety in the perspective of traditional uses and recent popularity. Planta Med 2010;76:7–19. 29. Luo Q, Cai Y, Yan J, Sun M, Corke H. Hypoglycemic and hypolipidemic effects and antioxidant activity of fruit extracts from Lycium barbarum. Life Sci 2004;76:137–49. 30. Cheng J, Zhou ZW, Sheng HP, He LJ, Fan XW, He ZX, et al. An evidence-based update on the pharmacological activities and possible molecular targets of Lycium barbarum polysaccharides. Drug Des Devel Ther 2014;9:33–78. 31. Gao Y, Wei Y, Wang Y, Gao F, Chen Z. Lycium barbarum: a traditional Chinese herb and a promising anti-aging agent. Aging Dis 2017;8:778–91. 32. Tang H-L, Chen C, Wang S-K, Sun G-J. Biochemical analysis and hypoglycemic activity of a polysaccharide isolated from the fruit of Lycium barbarum L. Int J Biol Macromol 2015;77:235–42. 33. Shen L, Du G. Lycium barbarum polysaccharide stimulates proliferation of MCF-7 cells by the ERK pathway. Life Sci 2012;91:353–7. 34. Chen S, Liang L, Wang Y, Diao J, Zhao C, Chen G, et al. Synergistic immunotherapeutic effects of Lycium barbarum polysaccharide and interferon-a 2b on the murine Renca renal cell carcinoma cell line in vitro and in vivo. Mol Med Rep 2015;12:6727–37. 35. Mao F, Xiao B, Jiang Z, Zhao J, Huang X, Guo J. Anticancer effect of Lycium barbarum polysaccharides on colon cancer cells involves G0/G1 phase arrest. Med Oncol 2011;28:121–6. 36. Georgiev KD, Slavov IJ, Iliev IA. Antioxidant activity and antiproliferative effects of Lycium barbarum’s (goji berry) fractions on breast cancer cell lines. Folia Med (Plovdiv) 2019;61:104–12. 37. Li G, Sepkovic DW, Bradlow HL, Telang NT, Wong GYC. Lycium barbarum inhibits growth of estrogen receptor positive human breast cancer cells by favorably altering estradiol metabolism. Nutr Cancer 2009;61:408–14. 38. Telang N, Li G, Sepkovic D, Bradlow HL, Wong GYC. Comparative efficacy of extracts from Lycium barbarum bark and fruit on estrogen receptor positive human mammary carcinoma MCF-7 cells. Nutr Cancer 2014;66:278–84. 39. Telang N, Li G, Katdare M, Sepkovic D, Bradlow L, Wong G. Inhibitory effects of Chinese nutritional herbs in isogenic breast carcinoma cells with modulated estrogen receptor function. Oncol Lett 2016;12:3949–57. 40. Moloney JN, Cotter TG. ROS signalling in the biology of cancer. Semin Cell Dev Biol 2018;80:50–64. 41. Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol Rev 2014;94:909–50. 42. Jiao R, Liu Y, Gao H, Xiao J, So KF. The anti-oxidant and antitumor properties of plant polysaccharides. Am J Chin Med 2016;44:463–88. 43. Wang S, Suh JH, Zheng X, Wang Y, Ho C-T. Identification and quantification of potential anti-inflammatory hydroxycinnamic acid amides from wolfberry. J Agric Food Chem 2017;65:364–72. 44. Zhang Q, Chen W, Zhao J, Xi W. Functional constituents and antioxidant activities of eight Chinese native goji genotypes. Food Chem 2016;200:230–6. 45. Islam T, Yu X, Badwal TS, Xu B. Comparative studies on phenolic profiles, antioxidant capacities and carotenoid contents of red goji berry (Lycium barbarum) and black goji berry (Lycium ruthenicum). Chem Cent J 2017;11:59. 46. Xin Y-F, Wan L-L, Peng J-L, Guo C. Alleviation of the acute doxorubicin-induced cardiotoxicity by Lycium barbarum polysaccharides through the suppression of oxidative stress. Food Chem Toxicol 2011;49:259–64. 47. Chao JC-J, Chiang S-W, Wang C-C, Tsai Y-H, Wu M-S. Hot water-extracted Lycium barbarum and Rehmannia glutinosa inhibit proliferation and induce apoptosis of hepatocellular carcinoma cells. World J Gastroenterol 2006;12:4478–84.

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48. Miao Y, Xiao B, Jiang Z, Guo Y, Mao F, Zhao J, et al. Growth inhibition and cell-cycle arrest of human gastric cancer cells by Lycium barbarum polysaccharide. Med Oncol 2010;27:785–90. 49. Chen H, Olatunji OJ, Zhou Y. Anti-oxidative, anti-secretory and anti-inflammatory activities of the extract from the root bark of Lycium chinense (Cortex Lycii) against gastric ulcer in mice. J Nat Med 2016;70:610–9. 50. Yi R, Liu X, Dong Q. A study of Lycium barbarum polysaccharides (LBP) extraction technology and its anti-aging effect. Afr J Tradit Complement Altern Med 2013;10:171–4. 51. Yousaf T, Rafique S, Wahid F, Rehman S, Nazir A, Rafique J, et al. Phytochemical profiling and antiviral activity of Ajuga bracteosa, Ajuga parviflora, Berberis lycium and Citrus lemon against hepatitis C virus. Microb Pathog 2018;118:154–8. 52. Ni W, Gao T, Wang H, Du Y, Li J, Li C, et al. Anti-fatigue activity of polysaccharides from the fruits of four Tibetan plateau indigenous medicinal plants. J Ethnopharmacol 2013;150:529–35. 53. Shi G, Zheng J, Wu J, Qiao HQ, Chang Q, Niu Y, et al. Beneficial effects of Lycium barbarum polysaccharide on spermatogenesis by improving antioxidant activity and inhibiting apoptosis in streptozotocin-induced diabetic male mice. Food Funct 2017;8:1215–26. 54. Hou Y-M, Wang J, Zhang X-Z. Lycium barbarum polysaccharide exhibits cardioprotection in an experimental model of ischemia-reperfusion damage. Mol Med Rep 2017;15:2653–8. 55. Liu Y, Cao L, Du J, Jia R, Wang J, Xu PYG. Protective effects of Lycium barbarum polysaccharides against carbon tetrachloride-induced hepatotoxicity in precision-cut liver slices in vitro and in vivo in common carp (Cyprinus carpio L.). Comp Biochem Physiol Part C Toxicol Pharmacol 2015;169:65–72. 56. Mi X, Zhong J, Chang RC-C, So K-F. Research advances on the usage of traditional Chinese medicine for neuroprotection in glaucoma. J Integr Med 2013;11:233–40. 57. Chen H, Li YJ, Sun YJ, Gong JH, Du K, Zhang YL, et al. Lignanamides with potent antihyperlipidemic activities from the root bark of Lycium chinense. Fitoterapia 2017;122:119–25. 58. Rebhun JF, Du Q, Hood M, Guo H, Glynn KM, Cen H, et al. Evaluation of selected traditional Chinese medical extracts for bone mineral density maintenance: a mechanistic study. J Tradit Complement Med 2019;9:227–35.

Chapter 36

Manuka honey, oxidative stress, 5-fluorouracil treatment, and colon cancer cells Sadia Afrina, Tamara Y. Forbes-Herna´ndezb, Francesca Giampierib,c,d, and Maurizio Battinob,c,e a

Department of Gynecology and Obstetrics, Johns Hopkins University, School of Medicine, Baltimore, MD, United States, b Department of Analytical and

Food Chemistry, Nutrition and Food Science Group, CITACA, CACTI, University of Vigo, Vigo Campus, Vigo, Spain, c Department of Clinical Sciences, Universita` Politecnica delle Marche, Ancona, Italy, d College of Food Science and Technology, Northwest University, Xi’an, Shaanxi, China, e

International Research Center for Food Nutrition and Safety, Jiangsu University, Zhenjiang, China

List of abbreviations 5-FU Bax Bcl-2 CDK CIMP CIN CRC Cyto c EGFR Erk1/2 FasL GPx GSH IL MAPK MDA MH MMP MSI mTOR NF-кB NO Nrf2 PARP PI3K Raf Ras ROS SOD TGF-b TNF-a

5-fluorouracil B-cell lymphoma 2 associated X protein B-cell lymphoma-2 cyclin-dependent kinase CpG island methylator phenotype chromosomal instability colorectal cancer cytochrome c epidermal growth factor receptor extracellular-signal-regulated kinase ½ first apoptosis signal ligand glutathione peroxidase reduced glutathione interleukin mitogen-activated protein kinase malondialdehyde Manuka honey metalloproteinases microsatellite instability mammalian target of rapamycin nuclear factor-kappa B nitric oxide nuclear related factor 2 poly (ADP-ribose) polymerase phosphatidylinositol 3-kinase raf proto-oncogene rat sarcoma virus oncogene reactive oxygen species superoxide dismutase transforming growth factor b tumor necrosis factor alpha

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00036-5 © 2021 Elsevier Inc. All rights reserved.

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Overview of etiology and risk factors of colorectal cancer (CRC) Worldwide, cancer is one of the deadliest diseases whereas CRC is the third most prevalent cancer in both men and women.1 More than 1 million CRC cases are diagnosed every year within 9.7% of all cancers and therefore over 693,933 deaths per annum confirming nearly 8.5% of the total number of cancer deaths.1 Several issues are related to the expansions and development of CRC, together with abundant alcohol consumption, high-fat intake, poor dietary fiber, red meat, overweight and lack of physical activity, older age, smoking, diabetes, inflammatory bowel disease (Crohn’s disease and ulcerative colitis), family history, hereditary nonpolyposis CRC, and familial adenomatous polyposis (Fig. 1).2 Diverse molecular mechanisms may elevate the incidence of CRC by altering various genetic and molecular pathways. Chromosomal instability (CIN), microsatellite instability (MSI), and CpG island methylator phenotype (CIMP) are the main habits in pathogenesis of CRC.3 Progression of CRC from polyp occurs by mutation in the adenomatous polyposis coli gene which subsequently activates the wingless-type pathway, as well as mutation of p53 (tumor suppressor gene), transforming growth factor b (TGF-b) and type II TGF-receptor gene remarkably responsible for CRC pathogenesis and development.4 More than 50% of CRC aberrantly activates the mitogen-activated protein kinase (MAPK) signaling pathway, which occurs by the mutation of the Kirsten rat sarcoma virus oncogene homolog (K-Ras) or B-raf protooncogene (B-Raf).4 Several signaling pathways such as phosphatidylinositol 3-kinase (PI3K), Protein kinase B (Akt), mammalian target of rapamycin (mTOR), and Ras-Raf-MEK-MAPKs are alternated in CRC4 and associated with overexpressed epidermal growth factor receptor (EGFR).5 Besides, an abnormal nuclear factor-kappa B (NF-кB) initiation has been identified in more than 50% of colitis-associated and CRCs.6

FIG. 1 Several risk factors associated with CRC development. Excessive alcohol consumption, high-fat intake, poor in dietary fiber, red meat, overweight and lack of physical activity, older age, smoking, diabetes, inflammatory bowel disease (Crohn’s disease and ulcerative colitis), family history, and hereditary nonpolyposis CRC are associated with CRC development.

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Oxidative stress and CRC In oxidative stress, there is an imbalance between production of reactive oxygen species (ROS) or other oxidants and their abolition by defensive mechanisms of antioxidant enzymes. In the carcinogenic pathway, oxidative stress has the potential impact to enhance the malignant transformation and proliferation of CRC cells7 (Fig. 2). Disorder in the redox equilibrium, such as increased production of ROS and nitrogen species, hampers the immune system and may lead to damaged lipids, proteins, and DNA of cells. Reproducing cells are highly sensitive against DNA damage by arresting the cell cycle or induction of transcription, replication error, initiation of signaling pathways, and genomic alteration, all of which are associated with CRC progression.8 ROS plays an important role in modulating multiple signal transduction pathways, such as NFkB, PI3K/Akt, heat shock proteins, and MAPK, which is associated with inflammation, proliferation, growth, differentiation, and apoptosis. In colon cancer cells, there is an unbalanced ROS level due to the defective cell metabolism and it acts as a pro-tumorigenic factor.9 The helpful or destructive role of ROS depends on their concentrations in the cells. The high ROS content in cancer cells renders them more susceptible to oxidative stress-induced cell death, and can be exploited for selective cancer therapy. ROS initiates lipid peroxidation by a chain of reactions that produces free radicals and other substances (malondialdehyde (MDA), hydroperoxides, lipoperoxides, and toxic aldehydes), which facilitate membrane permeability and increase inflammation.10 Additionally, lipid peroxidation may act as a signal transducer for cells proliferation and modulates gene expression in the DNA bases which may contribute to CRC.11 ROS restrains the proteolytic system by oxidizing structural proteins and the accretion of damaged proteins in cancer cells, acting as an inhibitor of the proteasome. This process induces accumulating misfolded and damaged proteins and affects the lysosomal system, which hampers protein turnover and gradually leads to further structural and functional alteration for promoting cancer initiation.12

FIG. 2 Oxidative stress induces CRC progression. Genetic alteration promoted by inflammation in the intestinal tract by generating pro-inflammatory cytokine, reactive oxygen species (ROS), and reactive nitrogen species (RNS). Increased production of ROS and nitrogen species hampers the immune system and may lead to damage of lipids, proteins, and DNA, generating a microenvironment favoring colonic epithelial proliferation, survival, and invasiveness.

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Management and treatment of 5-FU in CRC Several methods are available for diagnosis of early-stage CRC, adenomatous polyps, and precursor lesions for colon cancer, including fecal occult blood testing, colonoscopy (gold standard), flexible sigmoidoscopy, double-contrast barium enema X-ray, and computed tomography colonography. CRC metabolite or biomarkers, such as DNA, RNA, and proteins, are used for identifying the CIN, MSI, CIMP, B-Raf, and K-Ras mutation in cancer samples for classifying the tumor stage, prognosis of disease, and therapy management.13 At present, next-generation sequencing applications are available for CRC detection, by which healthy tissue can be distinguished, for example, from CRC by performing miRNA microarray and RT-qPCR. Hereditary CRC can be screened by DNA sequencing and identify gut microbiota composition by 16SrRNA sequencing.7 Depending on the patient’s features, cancer stage and type, numerous chemotherapies, drugs, and regimens are proposed for CRC management. Throughout the past four decades, 5-fluorouracil (5-FU) has been the earlier choice for CRC management and treatment because of its remarkable mechanism of action.14 5-FU acts as a thymidylate synthase inhibitor whereas its metabolites are assimilated into DNA and RNA. 5-FU decreases the CRC cell growth and progression by hampering the cancer cells proliferation and activating apoptosis.14 Successes reached by the therapeutic efficiency of 5-FU are scarce in patients with CRC, due to integration of progressive resistance to 5-FU and toxicity to adjacent healthy cells.15 However, with an early CRC stage efficiently responding to 5-FU more than 15%, the therapeutic effects rise up to 50% once it is combined with other chemopreventive drugs.16 The drug resistance percentage has amplified CRC treatment difficulty; therefore, an urgent devotion is needed to accompany the existing therapies. The use of several natural and synthetic drugs for CRC deterrence has reached notable care in the recent few years. Natural food products may represent an effective alternative because of their chemotherapeutic and preventive properties as well as less or absence of toxicity of side effects.2 Since ancient times, honey has been used as a food and medicinal product. More recently, there has been rising attention on the anticancer effectiveness of honey because of its diverse nutritional components with a good source of natural antioxidants.

Manuka honey (MH) MH is a monofloral dark honey, derived from the Manuka tree, Leptospermum scoparium, of the Myrtaceae family, which is cultivated as a shrub or a small tree through New Zealand and eastern Australia.17 In traditional medicine, MH has been used as a cleaning and healing agent in infection, wounds, abscesses, burns, and ulcers in different disease conditions, as well as antimicrobial and antioxidant agents due to its remarkable physicochemical properties and biological and therapeutic molecules.18

Nutritional composition of MH Nutritional composition can depend on the floral origin of MH and flavonoids and phenolic acid represents an effective botanical biomarker. The major identified flavonoids and phenolic acid of MH are illustrated in Table 1. According to different studies, MH is a rich source of flavonoids pinocembrin, pinobanksin, and chrysin, as well as other flavonoids also present in minor amounts, such as quercetin, kaempferol, 8-methoxykaempferol, luteolin, isorhamnetin, and galangin.19, 20 Several phenolic acid and volatile norisoprenoid compounds have been identified in MH (Table 1), which recognizes three chemotypes of L. scoparium in New Zealand. High levels of benzoic acid, 4-hydroxybenzoic acid, 2-methoxybenzoic acid, kojic acid, syringic acid, 4-methoxyphenyllactic acid dehydrovomifoliol, and methyl syringate have been characterized in MH by different research groups.20 Additionally, phenyllactic acid, phenylacetic acid, 4-methoxyphenyllactic acid, and leptosin were also determined in average amounts.25, 26 Methyl syringate and leptosin are good chemical markers for purifying and inducing myeloperoxidase inhibition activity of MH, while the biological activities and biosynthetic pathway or origin of these glycosides are quite unidentified.17 Additional interesting 1,2-dicarbonyl compounds, such as glyoxal, 3-deoxyglucosulose, and methylglyoxal, are also identified in MH which have nonperoxide antibacterial activity. These compounds are classically created through the caramelization or the Maillard reaction as degradation constituted from reducing carbohydrates.20

MH as a source of natural antioxidant There is a significant correlation between the antioxidant capacity and the phenolic compounds of MH24 since the antioxidant capacity of MH depends on good sources of phenolic compounds with an effective capacity to reduce free radicals and provide a relevant antioxidant capacity (Table 1). This honey has been used as a gold standard to test and calculate the antioxidant capacity of other honeys from diverse botanical and geographical roots.

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TABLE 1 Identified bioactive compounds of Manuka honey. Flavonoids

Phenolic acid

Other compounds

References

Pinocembrin, Pinobanksin Chrysin Quercetin Luteolin Apigenin Kaempferol Isorhammetin Leptosin Galangin

Gallic acid 4-Hydroxybenzoic acid Caffeic acid Syringic acid p-Coumaric acid trans-Ferulic acid trans-Cinnamic acid

Phenyllactic acid 4-Methoxyphenolactic acid 5-Hydroxymethylfurfural 2-Methoxybenzoic acid Kojic acid Phenylacetic acid Methyl syringate Dehydrovomifoliol Methylglyoxal Leptosin Glyoxal 3-Deoxyglucosulose

19–23

Total antioxidant capacity DPPH (0.06  0.01 mmol TE/100 g) FRAP (0.14  0.00 mmol TE/100 g) TEAC (0.22  0.00 mmol TE/100 g)

24

Abbreviations: DPPH, 2,2-diphenyl-1-picrylhydrazyl radical; FRAP, ferric reducing antioxidant power; TEAC, trolox equivalent antioxidant capacity.

The scavenger activity of MH against superoxide anion radicals has been examined through electronic paramagnetic resonance and methyl syringate induced the quenching properties of MH.27 MH induced protective effects against oxidative damage both in in vitro22, 28, 29 and in vivo models30, 31 (Fig. 3). In healthy dermal fibroblasts stressed with oxidant agents and in RAW 264.7 macrophages, MH exerted a protective effect against oxidative damage by (i) improving mitochondrial functionality, (ii) enhancing the damaged cell proliferation, (iii) decreasing apoptosis through reducing caspase-3, p38-MAPK, and extracellular signal-regulated kinase 1/2 (Erk1/2), (iv) reducing ROS and nitrite accumulation, (v) protecting cell biomolecules lipid, protein, and DNA, (vi) stimulating antioxidant enzyme, reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase activity and increasing the expression of antioxidant responsive element such as AMP-activated protein kinase (AMPK), Kelch-like ECH associated protein 1 (Keap1)-nuclear related factor 2 (Nrf2), sirtuin 1 (SIRT1), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a), (vii) suppressing the expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-a), interleukin (IL)-6, and IL-1b and toll-like receptor 4 (TLR4)/ NF-кB via inhibitor of kappa B (IкB) phosphorylation, and (viii) improving mitochondrial respiration and glycolysis of damaged cells.22, 28, 29 In an oxidative damaged rat model, MH remarkably reduced the ulcer index, protected the mucosa from lesions, and conserved the gastric mucosal glycoprotein levels.31 It increased both enzymatic and nonenzymatic antioxidant levels such as nitric oxide (NO), GSH, GPx, SOD, and catalase activity, while lipid peroxidation (MDA) and inflammatory markers (TNF-a, IL-6, and IL-1b) were decreased in liver30 and gastric mucosa31 after MH treatment. The above findings suggest a promising use of MH as an alternative natural supplement to recover the physiological oxidative status.

Chemopreventive effect of MH in colon cancer cells The anticancer activity of MH in colon cancer cells has been reported below by focusing on different mechanisms of action (Fig. 4).

Antiproliferative effect In CT-26, LoVo, and HCT-116 colon cancer cells, MH treatment suppressed cell proliferation in a dose- and timedependent manner.21, 32 The antiproliferative effects were associated by inhibiting the expression of EGFR, human epidermal growth factor receptor (HER2), and associated signaling p-Akt. The cell cycle was arrested after MH treatment at the S and G2/M phase via increasing p21 and p27 while decreased cyclin-dependent kinase (CDK)2, CDK4, cyclin

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FIG. 3 Protective effect of Manuka honey against oxidative damage. Manuka honey assists protective effects against oxidative damage through scavenging or quenching of oxygen free radicals, protecting biomolecules, activating the antioxidant enzyme, and increasing antioxidant responsive element expressions both in in vitro and in vivo models.

D1, and cyclin E were examined.21 The antiproliferative effects of 5-FU were significantly increased in the presence of MH, while the doses of 5-FU were lowered compared to a single dose of 5-FU.33

Apoptosis induction MH persuaded apoptotic death of colon cancer LoVo and HCT-116 cells through upregulation of p53 and caspase-3 and cleavage of poly (ADP-ribose) polymerase (c-PARP) expression.21 Both intrinsic and extrinsic apoptotic markers such as caspase-8, caspase-9, B-cell lymphoma 2 associated X protein (Bax), first apoptosis signal ligand (FasL), and cytochrome c (Cyto c) were increased after MH treatment. Moreover, increased p-p38MAPK and p-Erk1/2 expressions were found after MH treatment for inducing apoptotic death. MH also induced synergistic apoptotic effects by reducing the chemotherapeutic drug concentration.33

Alteration of oxidative stress Increased ROS accumulation was shown after MH treatment of colon cancer cells, while in noncancer cells there was not any oxidative stress.34 MH started oxidative stress associated with colon cancer cells death by suppressing both antioxidant enzymes activities and expression of Nrf2, SOD, catalase, and heme oxygenase 1, increasing the damage of cellular

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FIG. 4 Anticancer effect of Manuka honey in colon cancer cells. Chemopreventive action of Manuka honey actions in colon cancer cells by targeting on cellular functions and signal transduction pathways associated with antiproliferative, apoptotic, oxidative stress, metabolism alteration, and antimetastasis mechanism.

biomolecules (lipid, protein, and DNA), and decreasing metabolic phenotype through suppressing mitochondrial respiration and glycolysis. Additionally, additive effects were also evaluated when MH was combined with 5-FU.33

Antimetastatic effects MH inducing antimetastatic effects were demonstrated by suppressing the migration and invasion ability as well as epithelial mesenchymal transition of colon cancer cells.34 The above effects occurred by suppressing matrix metalloproteinases (MMP)-2, MMP-9, N-cadherin, and b-catenin expressions and increasing E-cadherin in HCT-116 and LoVo cells. Remarkably, MH augmented the antimigration and invasion activity of 5-FU compared to single drugs.33

Effect of MH on other cancer cells In breast cancer cells (MDA-MB-231 and MCF-7), MH elevated the enzymatic activity of caspase cascade -3/7, -6, -8, and -9 and increased the expression of Bax while B-cell lymphoma-2 (Bcl-2) expression was decreased.35 Additionally, MH promoted the translocation of Cyto c from mitochondria to cytosol and of Bax from cytosol to mitochondria.35 In melanoma cancer cells (B16.F1), MH-activated mitochondria-dependent apoptosis through increasing caspase-3/7, caspase-9 enzyme activities, PARP cleavage and DNA fragmentation, and decreasing Bcl-2 expression.32

Conclusions Based on the results obtained in the evaluation of the anticancer potential of MH on in vitro colon cancer models, the following conclusions can be drawn: (i) MH possesses a high content of phenolic and antioxidant capacity; the bioactive compounds of MH depend on its floral and geographical origins which have a significant impact on the antiproliferative potential.

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(ii) Evidence indicated that MH induced cytotoxic effects in colon cancer cells by arresting the cell cycle at the S and G2/M phase, activating apoptosis through increasing the expression of p53, caspase-3, and c-PARP and inducing oxidative stress by generating ROS, reducing antioxidant enzyme activity and inducing DNA, protein, and lipid damage. In addition, the expression of the p-p38MAPK and p-Erk1/2 pathways was increased while that of the EGFR, HER2, and p-Akt pathways was decreased after the treatment of MH. MH acted also as a regulator of energy metabolism in colon cancer cells both in anaerobic and aerobic pathways and indicated an alternative strategy for treatment of colon cancer. These findings provide fundamental insights into the molecular mechanism of MH induced colon cell death. (iii) The antimetastatic effects of MH against CRC cells were evaluated. MH inhibited the migration ability, as well as suppressed invasion abilities, observed by decreasing MMP-2 and MMP-9 expression and regulating the expression of EMT-related genes, including E-cadherin, N-cadherin, and b-catenin. Based on these results, MH may serve as an effective therapeutic agent for colorectal metastasis treatment. (iv) The development of new approaches to increase the effectiveness of 5-FU combining with polyphenol-containing foods is vitally required for increasing the effectiveness of conventional chemotherapy, overcoming the cancer cell resistance, and reducing the severity of adverse toxicity. The antiproliferation, apoptosis induction, increased ROS production, and antimetastatic activity were enhanced after co-treatment of 5-FU in the presence of MH compared to single doses of 5-FU. These interesting and promising findings encourage our knowledge about the chemopreventive effects of MH and could be useful for further studies to highlight the phenolic compounds of MH and the possible molecular mechanisms as well as in vivo studies against colon cancer.

Summary points l l l l

l l

l

l

This chapter provides an overview of the chemopreventive effects of MH in colon cancer cells. MH is a good source of natural antioxidants which have significant human health benefits. MH inducing antiproliferative effects were associated by suppressing cells growth and arresting the cell cycle. MH increased oxidative stress and decreased oxidative defense for inducing colon cancer cells death, while in healthy cells it increased the antioxidant defense system against oxidative stress. MH-activated apoptosis through the intrinsic and extrinsic apoptotic pathways. MH decreased colon cancer cells metabolism as well as suppressed cells migration, invasion, and epithelial mesenchymal transition. Remarkable synergistic effects were evaluated after MH treatment of colon cancer cells when administrated together with 5-FU. The anticancer effect of MH was also evaluated on other cancers both in in vitro and in vivo models.

References 1. Siegel RL, Miller KD, Fedewa SA, Ahnen DJ, Meester RGS, Barzi A, et al. Colorectal cancer statistics, 2017. CA Cancer J Clin 2017;67:177–93. 2. Afrin S, Giampieri F, Gasparrini M, Forbes-Herna´ndez TY, Cianciosi D, Reboredo-Rodriguez P, et al. Dietary phytochemicals in colorectal cancer prevention and treatment: a focus on the molecular mechanisms involved. Biotechnol Adv 2018; https://doi.org/10.1016/j.Biotechadv.2018.11.011. 3. Lao VV, Grady WM. Epigenetics and colorectal cancer. Nat Rev Gastroenterol Hepatol 2011;8:686. 4. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, et al. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319:525–32. 5. Cohen RB. Epidermal growth factor receptor as a therapeutic target in colorectal cancer. Clin Colorectal Cancer 2003;2:246–51. 6. Kojima M, Morisaki T, Sasaki N, Nakano K, Mibu R, Tanaka M, et al. Increased nuclear factor-kb activation in human colorectal carcinoma and its correlation with tumor progression. Anticancer Res 2004;24:675–82. 7. Li SKH, Martin A. Mismatch repair and colon cancer: mechanisms and therapies explored. Trends Mol Med 2016;22:274–89. 8. Valko M, Rhodes C, Moncol J, Izakovic MM, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact 2006;160:1–40. 9. Nogueira V, Hay N. Molecular pathways: reactive oxygen species homeostasis in cancer cells and implications for cancer therapy. Clin Cancer Res 2013;19:4309–14. 10. Mena S, Ortega A, Estrela JM. Oxidative stress in environmental-induced carcinogenesis. Mutat Res Genet Toxicol Environ Mutagen 2009;674:36–44. 11. Marnett LJ. Lipid peroxidation—DNA damage by malondialdehyde. Mutat Res 1999;424:83–95.

Manuka honey and colon cancer Chapter

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415

12. Brunk UT, Terman A. The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis. Eur J Biochem 2002;269:1996–2002. 13. Ma´rmol I, Sa´nchez-de-Diego C, Pradilla Dieste A, Cerrada E, Rodriguez Yoldi M. Colorectal carcinoma: a general overview and future perspectives in colorectal cancer. Int J Mol Sci 2017;18:197. 14. Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 2003;3:330. 15. Ohtsu A. Chemotherapy for metastatic gastric cancer: past, present, and future. J Gastroenterol 2008;43:256–64. 16. Stoehlmacher J, Park DJ, Zhang W, Yang D, Groshen S, Zahedy S, et al. A multivariate analysis of genomic polymorphisms: prediction of clinical outcome to 5-FU/oxaliplatin combination chemotherapy in refractory colorectal cancer. Br J Cancer 2004;91:344. 17. Kato Y, Umeda N, Maeda A, Matsumoto D, Kitamoto N, Kikuzaki H. Identification of a novel glycoside, leptosin, as a chemical marker of manuka honey. J Agric Food Chem 2012;60:3418–23. 18. Alvarez-Suarez JM, Gasparrini M, Forbes-Herna´ndez TY, Mazzoni L, Giampieri F. The composition and biological activity of honey: a focus on manuka honey. Foods 2014;3:420–32. 19. Chan CW, Deadman BJ, Manley-Harris M, Wilkins AL, Alber DG, Harry E. Analysis of the flavonoid component of bioactive New Zealand manuka (Leptospermum scoparium) honey and the isolation, characterisation and synthesis of an unusual pyrrole. Food Chem 2013;141:1772–81. 20. Oelschlaegel S, Gruner M, Wang P-N, Boettcher A, Koelling-Speer I, Speer K. Classification and characterization of manuka honeys based on phenolic compounds and methylglyoxal. J Agric Food Chem 2012;60:7229–37. 21. Afrin S, Giampieri F, Gasparrini M, Hernandez TF, Cianciosi D, Rodrı´guez PR, et al. The inhibitory effect of manuka honey on human colon cancer hct-116 and lovo cell growth. Part 1: the suppression of cell proliferation, promotion of apoptosis and arrest of the cell cycle. Food Funct 2018;9:2145–57. 22. Alvarez-Suarez JM, Giampieri F, Cordero M, Gasparrini M, Forbes-Herna´ndez TY, Mazzoni L, et al. Activation of AMPK/Nrf2 signalling by manuka honey protects human dermal fibroblasts against oxidative damage by improving antioxidant response and mitochondrial function promoting wound healing. J Funct Foods 2016;25:38–49. 23. Mavric E, Wittmann S, Barth G, Henle T. Identification and quantification of methylglyoxal as the dominant antibacterial constituent of manuka (Leptospermum scoparium) honeys from New Zealand. Mol Nutr Food Res 2008;52:483–9. 24. Afrin S, Forbes-Hernandez TY, Gasparrini M, Bompadre S, Quiles JL, Sanna G, et al. Strawberry-tree honey induces growth inhibition of human colon cancer cells and increases ros generation: a comparison with manuka honey. Int J Mol Sci 2017;18:613. 25. Tuberoso CIG, Bifulco E, Jerkovic I, Caboni P, Cabras P, Floris I. Methyl syringate: a chemical marker of asphodel (Asphodelus microcarpus salzm. Et viv.) monofloral honey. J Agric Food Chem 2009;57:3895–900. 26. Stephens JM, Schlothauer RC, Morris BD, Yang D, Fearnley L, Greenwood DR, et al. Phenolic compounds and methylglyoxal in some New Zealand Manuka and Kanuka honeys. Food Chem 2010;120:78–86. 27. Inoue K, Murayama S, Seshimo F, Takeba K, Yoshimura Y, Nakazawa H. Identification of phenolic compound in Manuka honey as specific superoxide anion radical scavenger using electron spin resonance (esr) and liquid chromatography with coulometric array detection. J Sci Food Agric 2005;85:872–8. 28. Afrin S, Gasparrini M, Forbes-Herna´ndez TY, Cianciosi D, Reboredo-Rodriguez P, Manna PP, et al. Protective effects of Manuka honey on LPStreated RAW 264.7 macrophages. Part 1: enhancement of cellular viability, regulation of cellular apoptosis and improvement of mitochondrial functionality. Food Chem Toxicol 2018;121:203–13. 29. Gasparrini M, Afrin S, Forbes-Herna´ndez TY, Cianciosi D, Reboredo-Rodriguez P, Amici A, et al. Protective effects of manuka honey on LPS-treated RAW 264.7 macrophages. Part 2: control of oxidative stress induced damage, increase of antioxidant enzyme activities and attenuation of inflammation. Food Chem Toxicol 2018;120:578–87. 30. Jubri Z, Rahim NBA, Aan GJ. Manuka honey protects middle-aged rats from oxidative damage. Clinics 2013;68:1446–54. 31. Almasaudi SB, El-Shitany NA, Abbas AT, Abdel-dayem UA, Ali SS, Al Jaouni SK, et al. Antioxidant, anti-inflammatory, and antiulcer potential of Manuka honey against gastric ulcer in rats. Oxid Med Cell Longev 2016;2016:3643824. 32. Fernandez-Cabezudo MJ, El-Kharrag R, Torab F, Bashir G, George JA, El-Taji H, et al. Intravenous administration of manuka honey inhibits tumor growth and improves host survival when used in combination with chemotherapy in a melanoma mouse model. PLoS One 2013;8:e55993. 33. Afrin S, Giampieri F, Forbes-Hernandez TY, Gasparrini M, Amici A, Cianciosi D, et al. Manuka honey synergistically enhances the chemopreventive effect of 5-fluorouracil on human colon cancer cells by inducing oxidative stress and apoptosis, altering metabolic phenotypes and suppressing metastasis ability. Free Radic Biol Med 2018;126:41–54. 34. Afrin S, Giampieri F, Gasparrini M, Hernandez TF, Cianciosi D, Rodrı´guez PR, et al. The inhibitory effect of manuka honey on human colon cancer HCT-116 and LoVo cell growth. Part 2: induction of oxidative stress, alteration of mitochondrial respiration and glycolysis, and suppression of metastatic ability. Food Funct 2018;9:2158–70. 35. Aryappalli P, Al-Qubaisi SS, Attoub S, George JA, Arafat K, Ramadi KB, et al. The IL-6/STAT3 signaling pathway is an early target of manuka honey-induced suppression of human breast cancer cells. Front Oncol 2017;7:167.

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Chapter 37

Piplartine (piperlongumine), oxidative stress, and use in cancer Daniel Pereira Bezerra Gonc¸alo Moniz Institute, Oswaldo Cruz Foundation (IGM-FIOCRUZ/BA), Salvador, Bahia, Brazil

Introduction Piplartine, also known as piperlongumine, is an amide alkaloid that has a natural occurrence in several Piper species (Piperaceae), including Piper longum L. (long pepper), a medicinal plant that belongs to the Indian Ayurvedic system. The chemical structure of piplartine is shown in Fig. 1. This molecule has attracted considerable attention in recent times due to its potential antineoplastic properties, and has now been considered an important prototype for new anticancer drugs.1, 2 Moreover, antithrombotic, antinociceptive, anxiolytic, antidepressant, neuroprotective, antiatherosclerotic, antihyperlipidemic, antidiabetic, antiinflammatory, gastroprotective, antibacterial, antifungal, leishmanicidal, trypanocidal, and schistosomicidal activities have also been reported for this molecule and/or its analogs.1, 2 Although piplartine had been first isolated in 1961 by the Indian chemists C.K. Atal and S.S. Banga, its antineoplastic potential stood out only after the publication of an article in an issue of Nature by Raj and collaborators,3 describing the potent and selective ability of this molecule to induce oxidative stress-mediated cell death in cancer cells.1, 2 In the last 10 years, more than 200 articles related to this plant-derived molecule have been published (Fig. 2). Surprisingly, in 2018, Nature published a retraction note on the article by Raj and collaborators3 due to problems with two figures and the unavailability of their original data.4 Two previous corrections related to this article have also been published in 2012 and 2015 by Nature.5, 6 Nevertheless, this manuscript seems to have been well designed and well performed, and piplartine continues to be highlighted in the research area of new antineoplastic drugs. Currently, the effect of this molecule in cancer cells has been reproduced in diverse models by different researcher groups. Fig. 3 shows the timeline of the discovery and preclinical development of piplartine as an antineoplastic drug candidate. In this chapter, we summarize the main features of piplartine in the induction of oxidative stress and its usage perspective in cancer therapy.

Oxidative stress induction Reactive oxygen species (ROS) are molecules, radicals, or ions that have a single unpaired electron in their outermost shell of electrons and consequently are highly reactive. These include superoxide, hydrogen peroxide, and hydroxyl radicals.7 In physiological conditions, the intracellular levels of ROS are kept at low levels to prevent cellular damage. Cellular antioxidant systems, including antioxidant enzymes that specifically scavenge different kinds of ROS and nonenzymatic molecules, for example, glutathione and vitamins A, C, and E, execute cellular ROS detoxification. Importantly, cancer cells have higher ROS levels than noncancer cells, which can be attributed to some factors, including highest metabolic activity, mitochondrial dysfunction, peroxisome activity, oncogene activity, increased activity of oxidases, cyclooxygenases, and lipoxygenases.7 Therefore, oxidative stress is considered as a selective cellular target in cancer therapy. Piplartine has been reported as an agent able to cause selective oxidative stress in cancer cells. Using the proteomic approach, glutathione S-transferase pi 1 (GSTp1), carbonyl reductase 1 (CBR1), glutathione S-transferase zeta 1 (GSTZ1), glutathione S-transferase M3 (GSTM3), glyoxalase I (GLO1), glutathione S-transferase omega 1 (GSTO1), and peroxiredoxin 1 (PRDX1) were found changed in piplartine-treated cancer cells. Piplartine also binds directly with GSTp1, inhibits glutathione S-transferase activity, decreases the reduced glutathione levels, and increases the oxidized glutathione levels in cancer cells. Augmentation of superoxide anion, hydrogen peroxide, and nitric oxide levels was also observed in piplartinetreated cancer cells, and coincubation with the antioxidant N-acetyl-L-cysteine reduced ROS production and cell death caused by piplartine.1, 3, 8–10 Interestingly, piplartine does not increase ROS levels in normal cells. In addition to the Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00037-7 © 2021 Elsevier Inc. All rights reserved.

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O 9

15

H3CO

10

14

O 6

7 8

5

N 1

11

13

H3CO

12

2

4 3

OCH 3

FIG. 1 Chemical structure of piplartine.

40 Number of published articles

FIG. 2 Number of published articles with piplartine versus year of publication. These data were obtained in Pubmed using “piplartine” and/or “piperlongumine” as keywords on July 30th, 2019. The articles with piperlonguminine that were wrongly linked with piperlongumine search were removed from the analysis.

30

20

10

1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019

0

Year of publication

FIG. 3 Timeline of the discovery and preclinical development of piplartine as an antineoplastic drug candidate.

glutathione S-transferase activity inhibition caused by piplartine, it interacts with thioredoxin reductase 1 (TrxR1) to induce ROS-mediated apoptosis in gastric and colon cancer cells.11, 12 In a structure/activity relationships study performed with some piplartine analogs, electrophilicity of the C2–C3 olefin was found to be critical for ROS elevation, glutathione depletion, and cellular toxicity of piplartine. On the other hand, analogs without reactive C7–C8 olefin can increase ROS levels, but presented less cytotoxicity than piplartine, indicating a ROS-independent mechanism caused by piplartine.13 Lee and collaborators14 found that piplartine could interact directly with Kelch-like ECH-associated protein-1 (Keap1) causing heme oxygenase-1 (HO-1) upregulation mediated by nuclear factor-erythroid-2-related factor-2 (Nrf2) via a ROS-independent pathway in breast cancer cells. For these authors, HO-1 is responsible for the action of piplartine in breast cancer cells, sparing its normal counterparts.14 In pancreatic cancer cells, piplartine treatment caused JNK-mediated cell death and activated Nrf-2 transcription of HO-1 as a compensatory survival pathway.15 Using transcriptome analysis, Dhillon and collaborators16 also observed that oxidative stress and endoplasmic reticulum stress are essential for piplartine effect in pancreatic cancer cells.

Cancer cell death induction Piplartine is able to induce cell death (with IC50 values range of 7 mM) in cancer cells from different histological types, and many cell signaling pathways have been associated with piplartine-induced cell death, including the p53, NF-kB,

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FIG. 4 Main cell death pathways altered by piplartine in cancer cells.

MAPK, STAT3, and PI3K/Akt/mTOR pathways. Interestingly, most of them are linked with the oxidative stress caused by piplartine. Fig. 4 summarizes the main cell death pathways altered by piplartine in cancer cells. The p53 tumor suppressor protein is a transcription factor that can induce apoptotic cell death during oxidative stress conditions. Piplartine induces both p53-dependent and p53-independent cell death in cancer cells, since wild-type p53 expression was improved after being treated with piplartine in U2OS, MFC-7, and HCT116 cells. Moreover, piplartine caused apoptotic cell death in p53-null Saos-2 cells.3, 17 Phospho-p53 (Ser 15) was also found increased in piplartinetreated AMC-HN3 and AMC-HN9 cells alone or combined with cisplatin. Moreover, piplartine triggers apoptosis in both p53-null UMSCC-1 cells and in UMSCC-1 cells transfected with wild-type p53, as well as in both AMC-HN9 cells expressing endogenous wild-type p53 and AMC-HN9 cells transfected with p53-siRNA.18 In addition, piplartine causes cell death in UMSCC1 (TP53 deficient), UMSCC17A (wild-type TP53), FaDu (TP53 mutation), and UMSCC10A (TP53 mutation) cancer cells.19 Factor nuclear kappa B (NF-kB) signaling has been reported as a strategic target of piplartine in hematological cancer cells. In fact, NF-kB activation has been found in different types of cancers, which can act in cancer cell survival, proliferation, invasion, angiogenesis, and metastasis. Moreover, since ROS has many inhibitory or stimulatory roles in NF-kB signaling, piplartine can inhibit this pathway by increasing intracellular oxidative stress. Han and collaborators20, 21 were pioneers in demonstrating that piplartine inhibited the growth of Burkitt lymphoma cells, but not normal B cells, by reduction of NF-kB, MYC, and LMP-1 activities. Similarly, piplartine caused apoptosis in B-cell acute lymphoblastic leukemia cells, but not normal B cells, along with elevation of ROS. Significant downregulation of multiple transcription factors, including AP-1, MYC, NF-kB, SP1, STAT1, STAT3, STAT6, and YY1, were observed in these cells treated with piplartine.22 In addition, piplartine was able to inhibit selectively activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL) cells by inhibition of NF-kB p65 activity.23 Han and collaborators24 reported that piplartine inhibits the NF-kB pathway by targeting IKK as well as NF-kBassociated biomarkers in a human myeloid cell line KBM-5. Doxorubicin resistance in K562/A02 human leukemia cells was also reversed by piplartine treatment through reduction of P-glycoprotein, MDR1, MRP1, survivin, and p-Akt expressions, arrest of the cell cycle at G2/M, and suppression of the transcriptional activities of NF-kB and twist.25 Using primary human acute myelogenous leukemia specimens, Pei and collaborators26 also found that piplartine caused complete glutathione depletion and led to cell death in leukemia stem cells from acute myelogenous leukemia cells, in contrast to significantly less toxicity in normal hematopoietic stem cells. Additionally, the NF-kB pathway has also been reported as a target for piplartine in prostate,27 breast,28 CNS,29 lung,30 tongue,24 kidney,24 and pancreas31 cancers. Many authors have identified that mitogen-activated protein kinases (MAPK) signaling has also an important role in the cell death induced by piplartine. MAPK signaling has different functions in cell proliferation, differentiation, and apoptosis. Currently, the MAPK pathway is known to comprise four subpathways: extracellular signal-regulated kinase (ERK) (ERK1 and ERK2), Jun kinase (JNK/SAPK) (JNK1, JNK2, and JNK3), p38 MAPK (a, b, d, and g), and Big MAP kinase 1 (BMK1 or ERK5). Additionally, activation of MAPK signaling has been associated with intracellular ROS increase. Randhawa and collaborators32 have demonstrated for the first time that piplartine causes cell death by ERK activation in cancer cells. They observed that piplartine at lower concentrations leads to ERK1/2 activation resulting in apoptosis, and at higher

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concentrations induces necrosis without activating ERK1/2 in colon carcinoma cells HT-29. Piplartine also kills glioblastoma multiforme cells (LN229, U87, and 8MG), but not astrocytes in cultures, via ROS accumulation by activation of JNK/SAPK and p38 MAPK.33 Li and collaborators34 also observed that piplartine induces cell death by JNK/SAPKmediated pathways in colon carcinoma cells HCT116. Furthermore, piplartine is also able to induce cell death by autophagy. Wang and collaborators35 demonstrated that the cell death induced by piplartine can be inhibited by the autophagy inhibitor 3-methyladenine. Piplartine also enhances autophagy activity without blocking autophagy flux, and the antioxidant N-acetyl-cysteine reduces piplartine-induced autophagy and cell death, suggesting an essential role for intracellular ROS in PL-induced autophagy. Moreover, piplartine active p38 MAPK by ROS-induced stress response and the block of p38 MAPK signaling reduce piplartine-mediated autophagy.35 Piplartine also eliminates cells of bone marrow mononuclear cells from patients with myeloid leukemias, but not from patients with myelodysplastic syndrome. Cell death was accompanied by a mechanism dependent on ROS and MAPK signaling, and augmentation of the expression of the apoptotic (Bax, Bcl-2, and caspase-3) and autophagic proteins (Beclin-1 and LC3B) was observed in these cells.36 Piplartine can also cause autophagic and apoptotic cancer cell death via inhibition of the PI3K/Akt/mTOR, PKCz, and p38 pathways.28, 37–40 Recently, Kumar and Agnihotri41 demonstrated that piplartine also inhibits tumor cell growth in a dimethylhydrazine/dextran sulfate sodium-induced colon carcinogenesis animal model by inhibition of the Ras/PI3K/Akt/mTOR pathways. Piplartine also eliminates head and neck and melanoma cancer cells via ROS- and JNK-dependent pathways.18, 42 Chen and collaborators43 observed that piplartine selectively increased cell death and inhibited migration/invasion in hepatocellular carcinoma cells by increase of ROS, along with activated/upregulated downstream PERK/Ire 1a/Grp78, p38/JNK/ Erk, and CHOP. Interestingly, ER stress responses or MAPK signaling inhibitors reduce the piplartine effect in cell migration/invasion, but not in cell death.43 Similarly, piplartine caused cell cycle arrest at the G2/M phase and triggered ROS/JNK/ERK-mediated apoptosis in cholangiocarcinoma cells.44 More recently, Oliveira and collaborators45 synthetized a novel platinum complex with a piplartine analog that displayed oxidative stress and apoptotic cell death by the ERK/p38 pathway in human acute promyelocytic leukemia HL-60 cells. Moreover, two ruthenium complexes with piplartine induced oxidative stress and triggered MAPK-mediated apoptosis by a p53-dependent pathway.8, 46 Signal transducer and activator of transcription 3 (STAT3) is also a drug target in cancer therapy, and its functions in cancer cells include cell growth, apoptosis resistance, DNA damage response, and metastasis. Inhibition of STAT3 nuclear translocation, ligand-induced and constitutive STAT3 phosphorylation, and modulation expression of multiple STAT3regulated genes were associated with piplartine-induced cell death in breast cancer cells.47 Moreover, piplartine treatment also reduced JAK2-STAT3 activation in nonsmall cell lung and breast cancer cells,48, 49 and eliminated growth, invasion, and migration of gastric cancer cells by inhibition of JAK1,2/STAT3 signaling.50 Piplartine also binds directly to the STAT3 Cys712 residue and inactive STAT3 signaling in multiple myeloma cells.51 In renal carcinoma cells, piplartine reduced c-Met signaling as observed by inhibition of its downstream components Erk/MAPK, STAT3, NF-kB, and Akt/mTOR.52 Alpay and collaborators53 also observed a significant inhibition of COX-2 and the induction of caspase-3 cleavage in acute promyelocytic leukemia Nb4 cells, and a synergistic effect with combination of alpha lipoic acid was found. A similar effect was observed with a combination of piplartine with a curcumin analog.54 In addition, Liao and collaborators55 synthetized a hybrid compound of piplartine with a histone deacetylase inhibitor, and observed that it causes cell death in acute myelogenous leukemia cell lines mediated by oxidative stress, reduction of DNA repair and pro-survival protein expressions, and upregulation of proapoptotic proteins.55 Meegan and collaborators56 demonstrated that piplartine is also a tubulin-destabilizing agent in breast cancer cells. Moreover, protein regulator of cytokinesis 1 (PRC1) has been reported as a target of piplartine in gastric cancer,57 and HER family receptors HER1, HER2, and HER3 have been downregulated by piplartine treatment in breast cancer cells through generation of ROS.58 Induction of cell death by ferroptosis seems to be another mechanism to kill cancer cells caused by piplartine. Yamaguchi and collaborators59 observed that piplartine-induced cell death in human pancreatic cancer cells is inhibited by ferroptosis inhibitors (ferrostatin-1 and liproxstatin-1) and the iron chelator, deferoxamine, and association of piplartine with sulfasalazine, a ferroptosis inducer, improved piplartine-induced cell death. Other mechanisms of piplartine-induced cell death include induction of C/EBP homologous protein (CHOP) by a ROS-dependent mechanism and enhance TRAILinduced cell death in breast cancer cells,60 activation of AMPK via phosphorylation and inactivation of acetyl-CoA carboxylases in HepG2 cells,61 induction of nuclear translocation of the FOXO3A transcription factor in different cancer cells,62 and inhibition of the transporter proteins Chromosome Region Maintenance1 (CRM1) in Hela cancer cells.63

Antitumor, antiangiogenic, and antimetastatic effects In vivo antitumor effects of piplartine have been widely assessed in both syngeneic and xenograft mouse models (Table 1). In the syngeneic mouse model, the antitumor effect of new drugs is studied in the presence of a functional immune system.

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TABLE 1 In vivo antitumor activity of piplartine in mice bearing cancer cells. Cancer cells

Histological type

Origin

Dose (mg/kg/day)

Days of treatment

Route

Inhibition rate (%)

References

Syngeneic models Sarcoma 180

Sarcoma

Mouse

50/100

7

i.p.

28/52 (increased 5-FU effect)

64, 76

B16-F10

Melanoma

Mouse

1.5

21

i.p.

50

3

MMTV-PyVT

Breast adenocarcinoma

Mouse

2.4

13

i.p.

75 (reduced breast metastasis)

3

H22

Hepatocellular carcinoma

Mouse

1.5/2.5/3.5

14

i.p.

50

43

MTT

Pheochromocytoma

Mouse

24

28

i.p.

95 (for tumor grow) and 50 (for lung metastases)

66

Xenograft models EJ

Bladder carcinoma

Human

1.5

21

i.p.

80

3

T24

Bladder carcinoma

Human

1.5/3.5

14

i.p.

28/30

67

MDAMB436

Breast carcinoma

Human

1.5

21

i.p.

50

3

MCF-7

Breast carcinoma

Human

8

14

i.p.

50

62

A549

Lung carcinoma

Human

1.5

21

i.p.

50

3

A549

Lung carcinoma

Human

2

14

i.p.

38.31

68

A549

Lung carcinoma

Human

30

5 days/week for 3 weeks

i.p.

75

48

A549/DTX

Lung carcinoma (docetaxel-resistant)

Human

20/60

28

i.p.

34/65

39

A549

Lung carcinoma

Human

2.5/5

Twice per week for 3 weeks

i.p.

33/70

30

A549

Lung carcinoma

Human

5

21

i.p.

41

69

HCC827

Lung carcinoma

Human

10

Every 2 days

i.p.

60

70

H1975

Lung carcinoma

Human

10

Every 2 days

i.p.

60

70

SF-295

Glioblastoma

Human

50/100

4

i.p.

62.2/61.5

65

HCT-8

Colon carcinoma

Human

50/100

4

i.p.

33.7/50.8

65

HCT116

Colon carcinoma

Human

20

21

i.p.

32.03

46

HCT-116

Colon carcinoma

Human

5

21

i.p.

60.6

71

PC-3

Prostate carcinoma

Human

20

16

i.p.

30

37

AMC-HN9

Parotid gland carcinoma

Human

2.5

21

i.p.

50 (increased cisplatin effect)

18

PANC-1

Pancreas carcinoma

Human

2.4

30

i.p.

50

72

BxPC-3

Pancreas carcinoma

Human

10

24

i.p.

30 (increased gemcitabine effect)

31

MIA PaCa-2

Pancreas carcinoma

Human

5

28

i.p.

37 (increased gemcitabine effect in orthotopic Model)

73

Continued

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TABLE 1 In vivo antitumor activity of piplartine in mice bearing cancer cells—cont’d Cancer cells

Histological type

Origin

Dose (mg/kg/day)

Days of treatment

Route

Inhibition rate (%)

References

HGC27

Stomach carcinoma

Human

3.6

16

i.p.

66

74

SGC-7901

Stomach carcinoma

Human

4/12

15

i.p.

50/75

11

NCI-H929

Plasmacytoma

Human

50

5 consecutive days a week for 3 weeks

i.p.

60

51

RPMI-8226

Plasmacytoma

Human

50

5 consecutive days a week for 2 weeks

i.p.

85

51

SAS

Tongue squamous cell carcinoma

Human

2.4

13

i.p.

55

75

On the other hand, human cancer cell heterogeneity is kept and well evaluated in xenograft mouse models. These complementary mouse models allow a fast analysis of cancer response to a new drug in in vivo protocols. The first in vivo antitumor effect of piplartine was performed by Bezerra and collaborators64 using mice-bearing sarcoma 180 cells. Piplartine inhibited tumor growth in a range of 28%–52% at doses of 50 and 100 mg/kg. In addition, piplartine was also able to increase the antitumor effect of 5-fluorouracil, cisplatin, and gemcitabine in both syngeneic and xenograft mouse models.18, 31, 73, 76 Using xenograft mouse models, the antitumor effect of piplartine was evaluated in human cancer cells from different histological types, including the bladder, breast, lung, nervous central system, colon, prostate, parotid gland, pancreas, stomach, plasmacytoma, and tongue.3, 18, 37, 51, 62, 65, 68, 72, 74, 75 In all models, piplartine administration was well tolerated by the animals and no important side effects have been reported. Importantly, in vivo antiangiogenic activity was also observed after piplartine treatment by the reduction of the expression of VEGF.3 In addition, using a transgenic mouse model of spontaneous breast cancer, MMTV-PyVT, piplartine administration inhibited not only the formation of blood vessels, as observed by the reduction of the expression of CD31, but also the development of spontaneous metastasis to the lungs, indicating antimetastatic activity.3 Corroborating with these data, piplartine treatment was reduced by metastasis development in the mouse pheochromocytoma model using MTT cells.66 Methoxy poly(ethylene glycol)-grafted chitosan nanoparticles containing piplartine also inhibited metastasis in the mouse colon model using the CT26 cell.77 Piplartine also reduced bladder cancer cell T24 migration/invasion via disturbing F-actin reorganization and by the ROS, Erk, and PKC pathways. In addition, it inhibited epithelial mesenchymal transition by decreased levels of Slug, b-catenin, ZEB1, and N-Cadherin expression in T24-bearing mice.67 Moreover, piplartine reduced TGF-b-induced epithelial-to-mesenchymal transition by downregulating Snail1 and Twist1 and upregulating E-cadherin in breast cancer MCF-7 cells and lung cancer A549 cells.78 In hepatocellular carcinoma cells, piplartine suppressed invasion by increasing the expression of a long noncoding RNA LINC01391, leading the inactivation of the Wnt/b-catenin pathway by physical interaction with a T-cell factor (ICAT).79

Conclusion Piplartine is an important prototype in the development of new antineoplastic drugs, acting by induction of oxidative stress and triggering cell death by different pathways. In addition, piplartine suppresses tumor development in in vivo models through induction of cancer cell death and inhibition of the formation of blood vessels, and eliminates metastasis along with low side effects. Although, to date, there are no reports of clinical trials with this molecule or its derivatives, the results found so far suggest a good clinical outcome for patients with a different type of cancer treated with piplartine.

References 1. Bezerra DP, Pessoa C, de Moraes MO, Saker-Neto N, Silveira ER, Costa-Lotufo LV. Overview of the therapeutic potential of piplartine (piperlongumine). Eur J Pharm Sci 2013;48:453–63.

Piplartine (piperlongumine), oxidative stress, and use in cancer Chapter



37

423

2. Piska K, Gunia-Krzyzak A, Koczurkiewicz P, Wo´jcik-Pszczoła K, Pękala E. Piperlongumine (piplartine) as a lead compound for anticancer agents— synthesis and properties of analogues: a mini-review. Eur J Med Chem 2018;156:13–20. 3. Raj L, Ide T, Gurkar AU, Foley M, Schenone M, Li X, et al. Selective killing of cancer cells by a small molecule targeting the stress response to ROS. Nature 2011;475:231–4. 4. Raj L, Ide T, Gurkar AU, Foley M, Schenone M, Li X, et al. Retraction note: selective killing of cancer cells by a small molecule targeting the stress response to ROS. Nature 2018;561:420. 5. Raj L, Ide T, Gurkar AU, Foley M, Schenone M, Li X, et al. Corrigendum: selective killing of cancer cells by a small molecule targeting the stress response to ROS. Nature 2012;481:534. 6. Raj L, Ide T, Gurkar AU, Foley M, Schenone M, Li X, et al. Corrigendum: selective killing of cancer cells by a small molecule targeting the stress response to ROS. Nature 2015;526:596. 7. Liou GY, Storz P. Reactive oxygen species in cancer. Free Radic Res 2010;44:479–96. 8. Costa COS, Araujo Neto JH, Baliza IRS, Dias RB, Valverde LF, Vidal MTA, et al. Novel piplartine-containing ruthenium complexes: synthesis, cell growth inhibition, apoptosis induction and ROS production on HCT116 cells. Oncotarget 2017;8:104367–92. 9. Harshbarger W, Gondi S, Ficarro SB, Hunter J, Udayakumar D, Gurbani D, et al. Structural and biochemical analyses reveal the mechanism of glutathione S-Transferase pi 1 inhibition by the anti-cancer compound piperlongumine. J Biol Chem 2017;292:112–20. 10. Prejano` M, Marino T, Russo N. On the inhibition mechanism of glutathione transferase P1 by piperlongumine. Insight from theory. Front Chem 2018;6:606. 11. Zou P, Xia Y, Ji J, Chen W, Zhang J, Chen X, et al. Piperlongumine as a direct TrxR1 inhibitor with suppressive activity against gastric cancer. Cancer Lett 2016;375:114–26. 12. Wang H, Jiang H, Corbet C, de Mey S, Law K, Gevaert T, et al. Piperlongumine increases sensitivity of colorectal cancer cells to radiation: involvement of ROS production via dual inhibition of glutathione and thioredoxin systems. Cancer Lett 2019;450:42–52. 13. Adams DJ, Dai M, Pellegrino G, Wagner BK, Stern AM, Shamji AF, et al. Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs. Proc Natl Acad Sci U S A 2012;109:15115–20. 14. Lee HN, Jin HO, Park JA, Kim JH, Kim JY, Kim B, et al. Heme oxygenase-1 determines the differential response of breast cancer and normal cells to piperlongumine. Mol Cells 2015;38:327–35. 15. Mohammad J, Singh RR, Riggle C, Haugrud B, Abdalla MY, Reindl KM. JNK inhibition blocks piperlongumine-induced cell death and transcriptional activation of heme oxygenase-1 in pancreatic cancer cells. Apoptosis 2019;24:730–44. 16. Dhillon H, Mamidi S, McClean P, Reindl KM. Transcriptome analysis of piperlongumine-treated human pancreatic cancer cells reveals involvement of oxidative stress and endoplasmic reticulum stress pathways. J Med Food 2016;19:578–85. 17. DA Silva MF, Munari FM, Scariot FJ, Echeverrigaray S, Aguzzoli C, Pich CT, et al. Piperlongumine induces apoptosis in colorectal cancer HCT 116 cells independent of Bax, p21 and p53 status. Anticancer Res 2018;38:6231–6. 18. Roh JL, Kim EH, Park JY, Kim JW, Kwon M, Lee BH. Piperlongumine selectively kills cancer cells and increases cisplatin antitumor activity in head and neck cancer. Oncotarget 2014;5:9227–38. 19. Hang W, Yin ZX, Liu G, Zeng Q, Shen XF, Sun QH, et al. Piperlongumine and p53-reactivator APR-246 selectively induce cell death in HNSCC by targeting GSTP1. Oncogene 2018;37:3384–98. 20. Han SS, Son DJ, Yun H, Kamberos NL, Janz S. Piperlongumine inhibits proliferation and survival of Burkitt lymphoma in vitro. Leuk Res 2013;37:146–54. 21. Han SS, Tompkins VS, Son DJ, Kamberos NL, Stunz LL, Halwani A, et al. Piperlongumine inhibits LMP1/MYC-dependent mouse B-lymphoma cells. Biochem Biophys Res Commun 2013;436:660–5. 22. Han SS, Han S, Kamberos NL. Piperlongumine inhibits the proliferation and survival of B-cell acute lymphoblastic leukemia cell lines irrespective of glucocorticoid resistance. Biochem Biophys Res Commun 2014;452:669–75. 23. Niu M, Shen Y, Xu X, Yao Y, Fu C, Yan Z, et al. Piperlongumine selectively suppresses ABC-DLBCL through inhibition of NF-kB p65 subunit nuclear import. Biochem Biophys Res Commun 2015;462:326–31. 24. Han JG, Gupta SC, Prasad S, Aggarwal BB. Piperlongumine chemosensitizes tumor cells through interaction with cysteine 179 of IkBa kinase, leading to suppression of NF-kB-regulated gene products. Mol Cancer Ther 2014;13:2422–35. 25. Kang Q, Yan S. Piperlongumine reverses doxorubicin resistance through the PI3K/Akt signaling pathway in K562/A02 human leukemia cells. Exp Ther Med 2015;9:1345–50. 26. Pei S, Minhajuddin M, Callahan KP, Balys M, Ashton JM, Neering SJ, et al. Targeting aberrant glutathione metabolism to eradicate human acute myelogenous leukemia cells. J Biol Chem 2013;288:33542–58. 27. Ginzburg S, Golovine KV, Makhov PB, Uzzo RG, Kutikov A, Kolenko VM. Piperlongumine inhibits NF-kB activity and attenuates aggressive growth characteristics of prostate cancer cells. Prostate 2014;74:177–86. 28. Shrivastava S, Kulkarni P, Thummuri D, Jeengar MK, Naidu VG, Alvala M, et al. Piperlongumine, an alkaloid causes inhibition of PI3 K/Akt/mTOR signaling axis to induce caspase-dependent apoptosis in human triple-negative breast cancer cells. Apoptosis 2014;19:1148–64. 29. Liu QR, Liu JM, Chen Y, Xie XQ, Xiong XX, Qiu XY, et al. Piperlongumine inhibits migration of glioblastoma cells via activation of ROS-dependent p38 and JNK signaling pathways. Oxid Med Cell Longev 2014;2014:653732. 30. Zheng J, Son DJ, Gu SM, Woo JR, Ham YW, Lee HP, et al. Piperlongumine inhibits lung tumor growth via inhibition of nuclear factor kappa B signaling pathway. Sci Rep 2016;6:26357. 31. Wang Y, Wu X, Zhou Y, Jiang H, Pan S, Sun B. Piperlongumine suppresses growth and sensitizes pancreatic tumors to gemcitabine in a xenograft mouse model by modulating the NF-kappa B pathway. Cancer Prev Res (Phila) 2016;9:234–44.

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32. Randhawa H, Kibble K, Zeng H, Moyer MP, Reindl KM. Activation of ERK signaling and induction of colon cancer cell death by piperlongumine. Toxicol In Vitro 2013;27:1626–33. 33. Liu JM, Pan F, Li L, Liu QR, Chen Y, Xiong XX, et al. Piperlongumine selectively kills glioblastoma multiforme cells via reactive oxygen species accumulation dependent JNK and p38 activation. Biochem Biophys Res Commun 2013;437:87–93. 34. Li W, Wen C, Bai H, Wang X, Zhang X, Huang L, et al. JNK signaling pathway is involved in piperlongumine-mediated apoptosis in human colorectal cancer HCT116 cells. Oncol Lett 2015;10:709–15. 35. Wang Y, Wang JW, Xiao X, Shan Y, Xue B, Jiang G, et al. Piperlongumine induces autophagy by targeting p38 signaling. Cell Death Dis 2013;4:e824. 36. Xiong XX, Liu JM, Qiu XY, Pan F, Yu SB, Chen XQ. Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways. Acta Pharmacol Sin 2015;36:362–74. 37. Makhov P, Golovine K, Teper E, Kutikov A, Mehrazin R, Corcoran A, et al. Piperlongumine promotes autophagy via inhibition of Akt/mTOR signalling and mediates cancer cell death. Br J Cancer 2014;110:899–907. 38. Wang XQ, Wang YC, Guo YT, Tang X. Effect of piperlongumine on drug resistance reversal in human retinoblastoma HXO-RB44/VCR and SORb50/CBP cell lines. Int J Clin Exp Pathol 2015;8:2525–34. 39. Wang F, Mao Y, You Q, Hua D, Cai D. Piperlongumine induces apoptosis and autophagy in human lung cancer cells through inhibition of PI3K/Akt/ mTOR pathway. Int J Immunopathol Pharmacol 2015;28:362–73. 40. Wang H, Wang Y, Gao H, Wang B, Dou L, Li Y. Piperlongumine induces apoptosis and autophagy in leukemic cells through targeting the PI3K/Akt/ mTOR and p38 signaling pathways. Oncol Lett 2018;15:1423–8. 41. Kumar S, Agnihotri N. Piperlongumine, a piper alkaloid targets Ras/PI3K/Akt/mTOR signaling axis to inhibit tumor cell growth and proliferation in DMH/DSS induced experimental colon cancer. Biomed Pharmacother 2019;109:1462–77. 42. Song X, Gao T, Lei Q, Zhang L, Yao Y, Xiong J. Piperlongumine induces apoptosis in human melanoma cells via reactive oxygen species mediated mitochondria disruption. Nutr Cancer 2018;70:502–11. 43. Chen Y, Liu JM, Xiong XX, Qiu XY, Pan F, Liu D, et al. Piperlongumine selectively kills hepatocellular carcinoma cells and preferentially inhibits their invasion via ROS-ER-MAPKs-CHOP. Oncotarget 2015;6:6406–21. 44. Thongsom S, Suginta W, Lee KJ, Choe H, Talabnin C. Piperlongumine induces G2/M phase arrest and apoptosis in cholangiocarcinoma cells through the ROS-JNK-ERK signaling pathway. Apoptosis 2017;22:1473–84. 45. Oliveira MS, Barbosa MIF, de Souza TB, Moreira DRM, Martins FT, Villarreal W, et al. A novel platinum complex containing a piplartine derivative exhibits enhanced cytotoxicity, causes oxidative stress and triggers apoptotic cell death by ERK/p38 pathway in human acute promyelocytic leukemia HL-60 cells. Redox Biol 2019;20:182–94. 46. Baliza IRS, Silva SLR, Santos LDS, Neto JHA, Dias RB, Sales CBS, et al. Ruthenium complexes with piplartine cause apoptosis through MAPK signaling by a p53-dependent pathway in human colon carcinoma cells and inhibit tumor development in a xenograft model. Front Oncol 2019;9:582. 47. Bharadwaj U, Eckols TK, Kolosov M, Kasembeli MM, Adam A, Torres D, et al. Drug-repositioning screening identified piperlongumine as a direct STAT3 inhibitor with potent activity against breast cancer. Oncogene 2015;34:1341–53. 48. Lewis KM, Bharadwaj U, Eckols TK, Kolosov M, Kasembeli MM, Fridley C, et al. Small-molecule targeting of signal transducer and activator of transcription (STAT) 3 to treat non-small cell lung cancer. Lung Cancer 2015;90:182–90. 49. Chen D, Ma Y, Li P, Liu M, Fang Y, Zhang J, et al. Piperlongumine induces apoptosis and synergizes with doxorubicin by inhibiting the JAK2-STAT3 pathway in triple-negative breast cancer. Molecules 2019;24. pii: E2338. 50. Song B, Zhan H, Bian Q, Gu J. Piperlongumine inhibits gastric cancer cells via suppression of the JAK1,2/STAT3 signaling pathway. Mol Med Rep 2016;13:4475–80. 51. Yao Y, Sun Y, Shi M, Xia D, Zhao K, Zeng L, et al. Piperlongumine induces apoptosis and reduces bortezomib resistance by inhibiting STAT3 in multiple myeloma cells. Oncotarget 2016;7:73497–508. 52. Golovine K, Makhov P, Naito S, Raiyani H, Tomaszewski J, Mehrazin R, et al. Piperlongumine and its analogs down-regulate expression of c-Met in renal cell carcinoma. Cancer Biol Ther 2015;16:743–9. 53. Alpay M, Yurdakok-Dikmen B, Kismali G, Sel T. Antileukemic effects of piperlongumine and alpha lipoic acid combination on Jurkat, MEC1 and NB4 cells in vitro. J Cancer Res Ther 2016;12:556–60. 54. Pignanelli C, Ma D, Noel M, Ropat J, Mansour F, Curran C, et al. Selective targeting of cancer cells by oxidative vulnerabilities with novel curcumin analogs. Sci Rep 2017;7:1105. 55. Liao Y, Niu X, Chen B, Edwards H, Xu L, Xie C, et al. Synthesis and antileukemic activities of piperlongumine and HDAC inhibitor hybrids against acute myeloid leukemia cells. J Med Chem 2016;59:7974–90. 56. Meegan MJ, Nathwani S, Twamley B, Zisterer DM, O’Boyle NM. Piperlongumine (piplartine) and analogues: antiproliferative microtubuledestabilising agents. Eur J Med Chem 2017;125:453–63. 57. Zhang B, Shi X, Xu G, Kang W, Zhang W, Zhang S, et al. Elevated PRC1 in gastric carcinoma exerts oncogenic function and is targeted by piperlongumine in a p53-dependent manner. J Cell Mol Med 2017;21:1329–41. 58. Jin HO, Park JA, Kim HA, Chang YH, Hong YJ, Park IC, et al. Piperlongumine downregulates the expression of HER family in breast cancer cells. Biochem Biophys Res Commun 2017;486:1083–9. 59. Yamaguchi Y, Kasukabe T, Kumakura S. Piperlongumine rapidly induces the death of human pancreatic cancer cells mainly through the induction of ferroptosis. Int J Oncol 2018;52:1011–22. 60. Jin HO, Lee YH, Park JA, Lee HN, Kim JH, Kim JY, et al. Piperlongumine induces cell death through ROS-mediated CHOP activation and potentiates TRAIL-induced cell death in breast cancer cells. J Cancer Res Clin Oncol 2014;140:2039–46.

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61. Ryu J, Kim MJ, Kim TO, Huh TL, Lee SE. Piperlongumine as a potential activator of AMP-activated protein kinase in HepG2 cells. Nat Prod Res 2014;28:2040–3. 62. Liu Z, Shi Z, Lin J, Zhao S, Hao M, Xu J, et al. Piperlongumine-induced nuclear translocation of the FOXO3A transcription factor triggers BIMmediated apoptosis in cancer cells. Biochem Pharmacol 2019;163:101–10. 63. Niu M, Xu X, Shen Y, Yao Y, Qiao J, Zhu F, et al. Piperlongumine is a novel nuclear export inhibitor with potent anticancer activity. Chem Biol Interact 2015;237:66–72. 64. Bezerra DP, Castro FO, Alves AP, Pessoa C, Moraes MO, Silveira ER, et al. In vivo growth-inhibition of sarcoma 180 by piplartine and piperine, two alkaloid amides from Piper. Braz J Med Biol Res 2006;39:801–7. 65. Bezerra DP, Ferreira PM, Machado CM, de Aquino NC, Silveira ER, Chammas R, et al. Antitumour efficacy of Piper tuberculatum and piplartine based on the hollow fiber assay. Planta Med 2015;81:15–9. 66. Bullova P, Cougnoux A, Abunimer L, Kopacek J, Pastorekova S, Pacak K. Hypoxia potentiates the cytotoxic effect of piperlongumine in pheochromocytoma models. Oncotarget 2016;7:40531–45. 67. Liu D, Qiu XY, Wu X, Hu DX, Li CY, Yu SB, et al. Piperlongumine suppresses bladder cancer invasion via inhibiting epithelial mesenchymal transition and F-actin reorganization. Biochem Biophys Res Commun 2017;494:165–72. 68. Wu Y, Min X, Zhuang C, Li J, Yu Z, Dong G, et al. Design, synthesis and biological activity of piperlongumine derivatives as selective anticancer agents. Eur J Med Chem 2014;82:545–51. 69. Xu X, Fang X, Wang J, Zhu H. Identification of novel ROS inducer by merging the fragments of piperlongumine and dicoumarol. Bioorg Med Chem Lett 2017;27:1325–8. 70. Zhou L, Li M, Yu X, Gao F, Li W. Repression of hexokinases II-mediated glycolysis contributes to piperlongumine-induced tumor suppression in nonsmall cell lung cancer cells. Int J Biol Sci 2019;15:826–37. 71. Zou Y, Zhao D, Yan C, Ji Y, Liu J, Xu J, et al. Novel Ligustrazine-based analogs of piperlongumine potently suppress proliferation and metastasis of colorectal cancer cells in vitro and in vivo. J Med Chem 2018;61:1821–32. 72. Dhillon H, Chikara S, Reindl KM. Piperlongumine induces pancreatic cancer cell death by enhancing reactive oxygen species and DNA damage. Toxicol Rep 2014;1:309–18. 73. Mohammad J, Dhillon H, Chikara S, Mamidi S, Sreedasyam A, Chittem K, et al. Piperlongumine potentiates the effects of gemcitabine in in vitro and in vivo human pancreatic cancer models. Oncotarget 2017;9:10457–69. 74. Duan C, Zhang B, Deng C, Cao Y, Zhou F, Wu L, et al. Piperlongumine induces gastric cancer cell apoptosis and G2/M cell cycle arrest both in vitro and in vivo. Tumour Biol 2016;37:10793–804. 75. Chen YJ, Kuo CC, Ting LL, Lu LS, Lu YC, Cheng AJ, et al. Piperlongumine inhibits cancer stem cell properties and regulates multiple malignant phenotypes in oral cancer. Oncol Lett 2018;15:1789–98. 76. Bezerra DP, de Castro FO, Alves AP, Pessoa C, de Moraes MO, Silveira ER, et al. In vitro and in vivo antitumor effect of 5-FU combined with piplartine and piperine. J Appl Toxicol 2008;28:156–63. 77. Lee HL, Hwang SC, Nah JW, Kim J, Cha B, Kang DH, et al. Redox- and pH-responsive nanoparticles release piperlongumine in a stimuli-sensitive manner to inhibit pulmonary metastasis of colorectal carcinoma cells. J Pharm Sci 2018;107:2702–12. 78. Park MJ, Lee DE, Shim MK, Jang EH, Lee JK, Jeong SY, et al. Piperlongumine inhibits TGF-b-induced epithelial-to-mesenchymal transition by modulating the expression of E-cadherin, Snail1, and Twist1. Eur J Pharmacol 2017;812:243–9. 79. Fan X, Song J, Zhao Z, Chen M, Tu J, Lu C, et al. Piplartine suppresses proliferation and invasion of hepatocellular carcinoma by LINC01391modulated Wnt/b-catenin pathway inactivation through ICAT. Cancer Lett 2019;460:119–27.

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Chapter 38

Antioxidant of Pleurotus ostreatus (Jacq.) P. Kumn and lymphoid cancer cells Md. Moyen Uddin Pka,b,c,d,e, Jane O’Sullivanf, Rumana Pervine, and Matiar Rahmane a

Institute of Biological Science, University of Rajshahi, Rajshahi, Bangladesh, b Biochemistry, Primeasia University, Dhaka, Bangladesh, c Independent

University of Bangladesh, Dhaka, Bangladesh, d Clinical Biochemistry (Diagnostic), Anwer Khan Modern Medical College & Hospital, Dhaka, Bangladesh, e Biochemistry & Molecular Biology, University of Rajshahi, Rajshahi, Bangladesh, f Department of Anaesthesiology and Critical Care, Tallaght University Hospital, Dublin, Ireland

List of abbreviations 4-HNE AP-1 ATP CAT DNA GSH GSSG H2O2 LPO MDA MOMP mtDNA MTT NF-KB Nrf2 P53 PKC PUFA ROS SNPs SOD VDAC Vit-C Vit-E

4-hydroxy-2-nonenal activator protein 1 adenosine triphosphate catalase deoxy ribonucleic acid reduced glutathione glutathione disulfide hydrogen peroxide lipid peroxidation malonaldehyde mitochondrial outer membrane permeabilization mitochondrial DNA 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide nuclear factor kappa-light-chain-enhancer of activated B cells nuclear factor 2 tumor protein, P53 protein kinase C polyunsaturated fatty acids reactive oxygen species single-nucleotide polymorphisms superoxide dismutase voltage-dependent anion channels vitamin C vitamin E

Introduction Lymphoid cancer, cancer of the immune system’s cells, which starts in the lymphocytes. In Canada, risk of developing lymphoid cancer is 1 in 30 and has been doubling every 20 years over 70 years. Lymphoid cancer is one of the leading cancers in children and young adults.1, 2 While most cancer investigations have focused on antineoplastic drug developments that have led to a substantial improvement in the predictions of breast cancer in recent decades, preventive methods are highly needed. The pursuit of medicinal products and nutritional interventions to prevent cancer thus can develop acceptable methods for control of low toxicity incidence of breast cancer.3 Hence, an enormous study has been carried out in plant products over the last century, and some active elements in the pharmaceutical industry have been identified by scientists.4 An elevated consumption in fruits, vegetables, and mushrooms is connected with a reduced incidence of Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00038-9 © 2021 Elsevier Inc. All rights reserved.

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chronic diseases such as cancer. In the field of cancer prevention and treatment due to their low toxicity, natural nutritional agents, particularly fruits, vegetables, and mushrooms have attracted considerable attention from scientists and the public.4 The Pleurotus ostreatus is a traditional Chinese medicine, a delicious mushroom and lots of proteins, carbohydrates, minerals, and vitamins as well as low fats. It contains polysaccharides, lectin, polypeptides, amino acids, and phenol oxidase in its principal constituents.5 P. ostreatus has beneficial impacts including antitumor, antioxidant, decreasing blood fat, antivirus, antiinflammatory characteristics, antibiotic characteristics, and immune-regulation. In Ehrlich’s tumor and sarcoma 180, the Polysaccharide portion of P. ostreatus has a powerful antitumor activity, and the antitumor was postulated to be b-glucan.6 Recently, by changing the oxidant/antioxidant state in experimental rats, we already proved the anticancer impact of P. ostreatus in our laboratory.7 This review seeks to evaluate current information carefully and provide a thorough overview of our current understanding of the anticancer effect of P. ostreatus. Despite expanding knowledge about the role of edible mushrooms in cancer cell biology, our understanding of P. ostreatus’ effect on immune responses to antitumors continues an evolving field.

Applications to other cancers or conditions P. ostreatus protein extracts demonstrated therapeutic effectiveness against human colorectal adenocarcinoma cells and cells of human monocytic leukemia.5 Cao et al.6 investigated that the colony-forming potential of the BGC-823 cells was significantly reduced, after P. ostreatus poly therapy. Pornariya et al.7 studied the antioxidant properties of fungi, P. ostreatus obtained from a local farm in Thailand. Studies of histopathology confirmed the hepatoprotective effect of P. ostreatus extract. Such findings suggest that a P. ostreatus extract can significantly reduce hepatotoxicity.8 Uddin Pk et al.9 isolated exopolysaccharides and internal polysaccharides from P. ostreatus and investigated in vitro antioxidant activity revealed strong antioxidant ability, which was demonstrated by the EC50 value for DPPH, ABTS scavenging activity and iron chelating activity. Antimicrobial activity of P. ostreatus extracts was determined by disc diffusion method Gram-negative bacteria and Gram-positive bacteria. The acetone extracts had antimicrobial activity against only Bacillus subtilis and Escherichia coli, while the other extracts inhibited the growth of most oral bacteria, indicating a significant growth inhibition of Streptococcus sanguinis.10

Cancer Cancer formation is a multiphase approach. This technique involves the basic modification of genome DNA, followed by selective mutated cell growth. This can be either due to an increase in division and/or a decrease in apoptosis of mutated cells. The mutated cell further splits so that the newly shaped lesion produces further epigenetic and genetic alterations. The earlier study recognized modifications that occur during tumorigenesis leading to the naming of demonstrable cellular and pathological phases of initiation, promotion, and progression Fig. 1.8 The initiation of a genotoxic event involves the formation of a mutated preneoplastic cell. Initiated cell formation is an irreversible but dose-dependent process. The promotion involves the spread of the selective clonal cell by increasing cell growth through either increased cell proliferation and/or reduced apoptosis in the target cell population.9 These stage events Oxidative stress

Normal cells

Initiated cells

Initiation FIG. 1 Role of oxidative stress in the process of carcinogenesis.

Focal lesion

Promotion

Neoplasia

Progression

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are dose-dependent and reversible when the tumor promotion stimulus is removed. The third phase of progression involves cell and molecular changes from the preneoplastic to the neoplastic state. This is an irreversible stage, involving genetic instability, nuclear-ploidy changes, and chromosome integrity disruption. Subsequent research has shown that carcinogenesis is much more complicated. The use of the three-stage process is, however, useful to underline the role of carcinogenesis modifiers. Based on our knowledge of carcinogenesis multistage, cancer-producing chemicals can work at any stage of the process or selective stages or promotional phases (Fig. 1).10

Oxidative stress (OxS) OxS, in turn, can result in genetic mutations and/or cell growth changes. A link has been found between increased radicals of reactive oxygen and the formation of cancer. Many studies have shown that reactive oxygen species (ROS) play an important role in the development of tumors.11 Due to exogenous and endogenous factors of ROS, the human body is constantly under OxS, which is closely linked to all aspects of cancer. Furthermore, chemically active carcinogenic agents have been found to overcome both cellular antioxidant systems and/or DNA repair systems, causes OxS. ROC-generating potential endogenous factors are inflammation, mitochondrial oxidative phosphorylation, peroxisomes, cytokines, and cellular metabolism (Fig. 2). Radiation, metals, pathogens, chemical therapeutics, and other xenobiotic chemical products form part of the exogenous sources of reagent oxygen. ROS sources, both endogenous and exogenous, may interact and alter all stages of the processes. The role of ROS in cancer has been challenging over the past 20 years where high levels of ROS can promote mutations of DNA and genetic instability. In particular, cancer cell lines have produced increased ROS that can increase tumorigenesis by activating signaling pathways. Increased ROS formation has been observed to be a hallmark of many tumors

Oxidative Stress

Exogenous

Endogenous 1. Mitochondria 2. Cytokines 3. Peroxisomes 4. Inflammation

1. Radiation 2. Xenobiotics 3. Pathogens 4. Chemotherapeutics

SNPs

Antioxidant

1. DNA repair 2. Oxidative enzymes

1. Enzymatic (CAT, SOD) 2. Nonenzymatic (Vit-E, Vit-C, GSH)

ROS

Altered genes expression

DNA Damage

Cancer FIG. 2 Sources of ROS and role in cancer development.

Cell proliferation

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Molecular oxygen

O2 e–

O2

.. Superoxide radical Superoxide dismutase

OH

.

Fe2+

Hydroxyl radical

Catalase, PRX(s), GPX(s) H2O2 Hydrogen peroxide

H2O Water

Thiol oxidation Cellular damage

Cellular signaling FIG. 3 Formation and interconversion of reactive oxygen species (ROS).

and cancer cell lines12 through harmful proteins, lipids, and DNA, thus establishing that ROS can be cancerous through genomic instability.13 Most intracellular ROSs come from the single electron oxygen (O2) reductions to the form of radical superoxide ðO2 ∙
Þ, with two molecules converted by superoxide dismutases into one molecule of hydrogen peroxide (H2O2) and one water (H2O) molecule. The Fenton reaction also allows hydrogen peroxide to accept another free Fe2+ electron to become a hydroxyl radical (HO ∙). These three primary forms of ROS have various reactivities that may have differential effects on cell physiology. The mitochondrial oxidative metabolism converts approximately 5% of molecular oxygen to ROS (primarily superoxide). Superoxide is dismuted into hydrogen peroxide (H2O2) by superoxide dismutase, which is then converted into a hydroxyl radical (Fig. 3).

Biomarkers of OxS The most common way of measuring free radicals and OxS is to determine the biomarkers for free radicals. OxS biomarkers are extremely clinically important and an assessment of body tissue and fluid is used to diagnose pathologies, diseases, and types of cancer. The polyunsaturated fatty acids (PUFA) are the primary target of ROS which are oxidated by lipid peroxidation (LPO) in cell membranes. LPO products, including malondialdehyde (MDA), 4-hydroxynoneal (4-HNE), and acrolein, cause changes to enzymes, receptors and therefore cell lesions through binding to proteins and functional alterations. Studies have shown that MDA increases in various cancers, including breast cancer, as an indicator of LPO and biomarker of OxS.14–16 Nucleic acids are the target of ROS too. DNA reacts with hydroxyl radicals, which lead to changes in deoxyribose and cross-linkings. Thymine glycol and 8-hydroxideoxide guanosine (8-OHdG) are metabolites resulting from oxidative DNA damage. A common product of DNA oxidant damage and a major indicator of oxidant stress is 8-hydroxyguanosine (8-OHdG). It can be used as a tumor marker and therefore in clinical diagnostics.10, 17 Glutathione, which exists in two forms: GSH and oxidized (GSSG) is an antioxidant compound and is a reducing compound of the body. In healthy people, their reduced form (GSH) is the highest GSH level. An increase in GSSG can, therefore, be a sign of OxS in cells and tissue. Studies have shown that GSH decreases in the blood of breast cancer patients and therefore acts as the OxS biomarker.18

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Cancer state

ROS ROS Nrf2

Nrf2 Nrf2

Nrf2 Nrf2

Nrf2

Nrf2

Nrf2 Nrf2

Nrf2

(A)

Nrf2

Nrf2

Nrf2

Nrf2

Maf

Maf

(B)

FIG. 4 Duel role of Nrf2 in cancer progression and prevention. (A) Cellular homeostasis and tumor suppression and (B) protect against high ROS level, enhance tumorigenesis.

OxS and cell proliferation Cell proliferation is induced by modulation of multiple signaling pathways at levels of ROS and ROS-induced alteration of genetic expression.19 Kinases including protein kinase C (PKC), which regulate the modification of cell cycles, can be activated by ROS. This way regulates cell proliferation and survival linked to ROS-induced carcinogenesis activation.20 Transcription factors have also been activated on signal pathways following ROS exposure (Fig. 4). More specifically, ROS targets transcription factors such as Nrf2, NF-kB, AP-1, and HIF-1a. In a cell, transcription factors are activated by ROS level and thus cell death or cellular proliferation depends on the ROS level.21 Nrf2 has an important role in the prevention of cancer and the suppression of tumors in a normal cell but also has the responsibility of tumorigenesis. Previous studies have found that high Nrf2 levels in lung, pancreatic and endometrium cancer cells have been detected.22, 23 In the tumor tissue of lung, breast, gallbladder, liver, ovarian, endometrial, head and neck, airways, and skin cancer, KEAP1 gene mutations were identified. Nrf2 signals interruption affects the in vitro progression of the cell cycle and the proliferation of cancer cell lines.24, 25

OxS and apoptosis The process of apoptosis is highly controlled and essential for multicellular organisms to develop and survive. These organisms often need to reject cells that have accumulated mutations, which are superfluous or potentially harmful or become infected by pathogens.26 Apoptosis presents a distinctive set of morphological and biochemical characteristics by which cells undergo self-destruction. Thus, adequate apoptosis regulation is crucial for keeping cell homeostasis normal. Nevertheless, apoptosis deregulation has been connected to a range of disease conditions, including cancer.27, 28 Any kind of extrinsic and intrinsic signals can trigger apoptosis. The various stresses include ROS, DNA damage, and viral infection. Exposure to xenobiotics such as pesticides, pollutants, and chemotherapy may also lead to apoptosis, often mediated by ROS.29 ROS is crucial in cell signaling and in regulating the principal pathways of mitochondrial mediation of apoptosis, death receptors, and the endoplasmic reticulum. ROS such as H2O2 has been linked with cell survival induction through p53 activation at lower dosages, while higher doses trigger death processes like apoptosis.30 When the cell is exposed to stress or damaging DNA, p53 is released and stabilized through posttranslational modifications before associated with DNA, thus preventing proteasomal degradation. When stress is low, p53 leads to cell cycle arrest, repair of DNA, and senescence. However, when damages are too severe, p53 can regulate apoptosis transcriptionally through downregulating proapoptotic proteins such as Bcl-2, Bcl-XL, etc. P53 enables a proapoptotic gene transcription of the pathways that are essential for apoptosis induction such as Bax, Bid, Puma, Noxa, and Apaf-1; it also activates external proapoptotic factors like Fas, FasL, DR-4, and DR-5. Cytosolic p53 can also translocate into mitochondria in which it can directly interact with antiapoptotic

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proteins like Bcl-2, Mcl-1, and the proapoptotic proteins Bax and Bak, allowing MOMP, the release of proapoptotic factors and apoptosis. Cytosolic p53 can also directly activate Bax by causing its reorganization.31 Thus, p53 enhances the permeability of the mitochondrial membrane and further releases of mitochondria proapoptotic factor. H2O2-induced Apoptosis has been associated with several cell typologies including rat neural AF5, glioma, colon cancer, and the human cervical HeLa cells with increased protein expression of p53 and Puma and Noxa and Bax, as well as p53 phosphorylation at Ser15 and SER46.32 Post translations of their catalytic site cysteine residues appear to be included in the redox regulation of caspase activity.32, 33 In most caspases, catalytic site cysteines are susceptible to oxidation whereas S-glutathionylation is susceptible to procaspase-9, procaspase-3, and caspase-3.33 Although oxidants such as H2O234 can activate caspases, their enzyme activity of caspases-3, -8, and -9, can also be irreversibly inactivated by oxidants.32 Also, caspase-3 activity, mediated by S-glutathiolation, can be regulated by cGSH which decreases accessibility for proteolytic cleavage, which results in apoptosis resistance. However, there is still no clarification on the overall control of the process and its consequences for apoptosis induction.33

ROS and mtDNA damage In response to a range of cellular stresses, including mitochondrial DNA damage (mtDNA), growth factor deprivation, heat shock, ER stress, and developmental evidence, the mitochondrial pathway of apoptosis is activated.33 The mitochondrial pathway was closely linked to ROS activation.35 The most intracellular ROS is produced by mitochondria as a result of the leakage of the chain of transportation of respiratory electron.36 Here, ROS damage mtDNA would affect mtRNA transcription of protein from the electron transport chain resulting in interruption of the functioning of the respiratory chain, further increases in ROS generation and the loss of the mitochondrial membrane potential as well as impairment of ATP synthesis.35 These events rise to the highest point in apoptosis by the mitochondrial way. ROS like superoxide and hydrogen peroxide induce apoptosis through releasing cytochrome c from mitochondria.37 ROS targets protein components of mTP in the inner membrane of mitochondria and undergoes oxidative change of target proteins such as VDAC, ANT, cyclophilin D, which will stimulate mitochondrial permeability transition pore (mTP). Due to hyperpolarization of mitochondrial membrane by H2O2 causes membrane potential destruction, increased translocation of Bax and Bad and increased cytochrome c release.34 Resulting from a significant loss of cytochrome c from mitochondria causes a further increase of free radical generation through the disruption of the electron transport chain. Caspase-dependent and caspase-independent apoptosis are induced by ROS, H2O2. In caspase-dependent apoptosis, ROS triggers the loss of mitochondrial membrane permeability, cytochrome c release into cytosol, and activation of caspase-3 with caspase-9. On the other hand, AIF released from mitochondria to nucleus in caspase-independent apoptosis.38 Also, ROS oxidizes lipid component of the inner membrane of mitochondria (IMM) such as phospholipid cardiolipin which binds cytochrome c. IMM is the active site for cellular metabolism, which is related to various chronic diseases like cancer. Normally, cytochrome c carries an electron in the electron transport chain while oxidized phospholipid cardiolipin decreases the activity of cytochrome c resulting it initiates the apoptotic cascade.39 Uncontrolled ROS production increase Ca2+-induced cell destruction. During OxS, the pore opening in IMM is induced by mitochondrial Ca2+ accumulation. This increases osmotic swelling and IMM rupture results in the release of cytochrome c protein and subsequent cell damage.40

Antioxidant defense in cancer development A body-produced defense mechanism to neutralize the effects of ROS is antioxidants. It could be enzymatic and enzymefree. Vitamin C, vitamin E, selenium, zinc, beta carotene, carotene, taurine, and glutathione are nonenzymatic sources of antioxidants. SOD, catalase, glutaredoxin, and glutathione reductase are included in the enzyme antioxidant.41 As the body ages, the amount of antioxidants decreases causing disturbance to the balance of antioxidants and prooxidant molecules. OxS, on the other hand, may cause oxidizing damage to DNA, proteins, and lipids. Many clinical conditions have increased oxidizing stress indices, suggesting that an overwhelming antioxidant defense system initiates and spreads pathogens involving many diseases. In the human body, the effect of an oxidant is counterbalanced by a variety of antioxidants.42, 43 Enzymatic antioxidant defenses are given below, Table 1. As superoxide is the main ROS from a range of sources, its dismutation by SOD for individual cells is of prime importance. The human lungs widely express the three forms of SOD, CuZn-SOD, Mn-SOD, and EC-SOD. The mitochondrial matrix of Mn-SOD is located. EC-SOD is mainly localized within the extracellular matrix, particularly in areas with high amounts of collagen type 1 fibers and pulmonary and systemic vessels.44 Overall, CuZn-SOD, and Mn-SOD are generally considered to be bulk radicals scavengers of superoxide. The relatively high EC-SOD level in the lung can constitute a fundamental element for protecting the lung matrix with its special binding on the extracellular matrix components.45

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TABLE 1 Enzymatic antioxidants and their role in biological functions. Antioxidant defense

Role

Catalase, CAT

Catalase is a catalyst for the conversion of hydrogen peroxide into oxygen and water. It cancels the effect of intracellular hydrogen peroxide

Glutathione, GSH

Reduction of glutathione contributes to a neutralization of hydrogen peroxide produced on the inside of the cell. These enzymes are a key element in the prevention of increased oxidative stress

Superoxide dismutase (SOD)

Superoxide dismutase is a general body enzyme that catalyzes superoxide disputation

Glutathione peroxidase (GPx)

Glutathione peroxidases (GPX) are selenium-driven hydroperoxidase enzymes that help reduce H2O2 and hydroperoxides of fatty acids

Thioredoxin (TRX)

Thioredoxin can protect cells from anti-Fas antibody, tumor necrosis factor (TNF) hydraulic peroxide, active neutrophils, and ischemic damage

The increasing GSH level is associated with a proliferating response in many normal and malignant cells and is essential for cell cycle progression.46 Much remains speculative about the molecular mechanism by which GSH modulates cell proliferation. One key GSH-function mechanism for DNA synthesis involves maintaining the rate-limiting enzyme in DNA synthesis, reduced glutaredoxin or thioredoxin, required for ribonucleotide reductase activity.47 Besides, GSH status has been correlated with growth in liver and metastatic melanoma cells and a direct correlation between the cell and metastatic activity associated levels of GSH was shown. Indeed, intrasplenic inoculation in C57BL/6J syngenic mice of B16 melanoma (b16 M) cells induces the formation of metastatic focus by colonizing various organs. However, the metastases were significantly larger in terms of number and size as the inoculation in vivo of B16 M cells with high GSH content.48 In the face of an OxS, a high percentage of tumor cells with a high GSH content was able to remain the main task force in the metastatic invasion.49 Thus, it is plausible that maintaining high levels of GSH intracellularly could be critical for metastatic cell extravascular growth. Also, the maintenance of mitochondrial GSH homeostasis may be a limiting factor in the immediate aftermath of intra-sinusoidal cell survival and interaction with endothelial vascular cells. Mitochondrial dysfunctions are a common event in the mechanism of cell death and recently have been identified as a key step to killing conventional treatment-resistant nonsmall cell lungs (NSCLC) carcinomas.50

P. ostreatus, oyster mushroom For centuries, mushrooms have been known as precious food and the origin of a broad spectrum of bioactive compounds of many medicines in different cultures.51–53 P. ostreatus is an important source of protein, carbohydrates, minerals, vitamins, fats, and volatile compounds based on its high nutritionally based value. The major antioxidants reported in P. ostreatus include phenolic compounds and polysaccharides.54 Several antioxidant compounds reported in P. ostreatus and mycelium have been shown in Fig. 5. Jayakumar et al. (2011) explored the antioxidant characteristics of aqueous and 95% ethanol extracts of P. ostreatus and the aqueous extracts showed the largest quantity of total polyphenols and stronger antioxidant activity than the ethanol extracts.54 Reis et al. researched the antioxidant activity and the total quantity of polyphenol, mycelium methanol extracts and P. ostreatus fruiting body. In particular, fruiting bodies showed the greatest antioxidant activity and reducing power, while mycelium showed the greatest chelating activity.55, 56 Polysaccharides from P. ostreatus have been of particular interest including anticancer activities.57 The basic structure of mushrooms and mushrooms-derived polysaccharides has a beta-1,3 backbone with the beta-1,6 branching.7, 55

Extraction and purification of polysaccharides Mushroom polysaccharide consists of two main polysaccharide kinds as the structural elements of the fungal cell wall. The celluloses and matrix-like glycoprotein are a rigid cellulose fibrilla, a-glucan, or b-glucan. The selection of mushroom polysaccharides is usually based on the cell wall structure. A reliable procedure has been developed for successful polysaccharide mining of either cultivar mycelia or fruit body.1 The process of extraction usually involves 80% ethanol to remove low molecular substances from the pest material and 3–5 repeated water extractions (100°C, 2–4 h). Alternatively, 5% sodium hydroxide (80°C, 6 h) or 2% ammonium oxalate (100°C, 6 h) are used. Using a mixture of methods such as

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HO

O

O

OH

HO

O HO

HO

O HO

O HO

HO

OH

O O HO

HO

O n

OH

a

O

O

OH H

HO

b c

f

HO

OH

e

d

CH3 HO

H

H3C

O CH3

CH3

CH3

H

CH3

CH3 CH3

FIG. 5 Antioxidant compounds of the edible mushroom Pleurotus ostreatus. (a) Polysaccharides, (b) phenolic compounds, (c) ergosterol, (d) beta carotene, (e) tocopherol, and (f) ascorbic acid.

ethanol precipitation, fractional precipitation, acidic precipitation with acetic acid, ion-exchange chromatography, gel filtration, and chromatography of affinity, extracted polysaccharides can be further purified. The precipitation of ethanol excludes polysaccharides from the impurities. Acidic and neutral polysaccharides can be separated on a DEAE-cellulose column by anion-exchange chromatography. First, a suitable running buffer elucidates the neutral polysaccharide in the blend; then the acid polysaccharide is eluted at a greater salt concentration.2 Using gel filtration and affinity chromatography, neutral polysaccharides can be further divided into a-glucans (adsorbed fraction) and b-glucans (nonadsorbed fraction). Affinity chromatography is a bioselective adsorption method and the subsequent regeneration from an immobilized ligand of a compound. This method now enables some carbohydrates to be extremely specific and efficiently purified.3 Previous studies have indicated that the mushroom sample fractionation for polysaccharides usually began with the extraction of warm water. Pk et al. described the isolation and characterization and anticancer effect of antioxidant polysaccharide from P. ostreatus.7

Treatment of cancer The effectiveness of antioxidants is currently being investigated to prevent carcinogenesis. Issues to be dealt with in the future include the development of easy, accurate methods of measuring and evaluating the extent of OxS within the body and the clinical application of knowledge gained experimentally to cancer prevention and therapy. The application of mushrooms with potential therapeutic properties raises the scientific and clinical community’s international interest on two main grounds. First, mushrooms are as effective as cancer or degenerative disease against numerous diseases and metabolism disruptions. These therapeutic effects seem to have several pharmacologically complex effects on a variety of cell and molecular objectives.58 The most significant medicinal effects of mushrooms and their metabolites are their antitumor characteristics.59, 60 The pharmaceutical utilization of food mushroom extracts appears to be less costly, natural, and usually with minimum undesirable side effects. Moreover, a potential new source of antimicrobial agents might be purified bioactive products derived from food-serving mushrooms. One of the most commonly cultivated edible mushroom is the oyster mushroom.60 Various properties, including antitumor, hypocholesterolemic, antiatherogenic, and antioxidant activity have been associated with this mushroom. Also, the cytotoxicity of crude extract on PC-3 cells has been shown, and the antiproliferative and proapoptotic effects of aqueous polysaccharides extract on HT-29 cell and lymphoid cancer cells.56 At the cellular and molecular levels, the possible modes of action of mushroom polysaccharides against cancer cells were assessed by EL-Deeb et al. (2019).57 They showed that P. ostreatus polysaccharides caused cytotoxic impacts of NK-cells against lung and breast cancer proteins with the greatest impact on breast cancer cells. Activation of NK cells for cytokine secretion was associated with upregulation of KIR2DL genes, while the cytotoxic activation impact of NK cells against cancer cells was correlated with the upregulation of NKG2D and induction of manufacturing of IFNg and NO. In the presence of IL2, these cytotoxic effects were enhanced. Analysis of the most active partly purified fraction shows that it consists predominantly of glucans. These findings show that bioactive 6-linked glucans in P. ostreatus extracts

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activate the cytotoxicity of NK-cells by regulating the activation and induction of IFNg and NO. These trials create a beneficial position in the activation and induction of an innate immune response against breast and lung cancer cells for bioactive P. ostreatus polysaccharides.

Summary points A large amount of mushrooms have been increasingly used as a source of medicinal compounds and therapeutic adjuvants over the past few decades. l

l

l

l l

Recently, much attention has been paid to the biological activities of polysaccharides extracted from mushrooms, P. ostreatus. Mushroom polysaccharides’ most promising activities are immunomodulation and anticancer effects. However, there is still obviously no recognition of the mode of action for the antitumor nature of mushroom-isolated polysaccharides. P. ostreatus mushroom polysaccharides can induce cytotoxicity mediated by NK-cell against lung and breast cancer cells with increased activity toward breast cancer. The cytotoxic effects of NK cells against cancer cells are mediated by upregulation and induction of NKG2D and IFNg and are enhanced with IL2. Mushroom polysaccharide antitumor is frequently acknowledged as enhancing various immune responses in vivo and in vitro and acting as a modifier of biological reactions. Therefore, extensive studies are required to produce mushroom food supplements for antitumor impacts. In addition to benefiting from the antitumor effect of mushroom extracts, these precious compounds will also enable customers to obtain medicinal advantages.

References 1. Dalla-Favera R. Lymphoid malignancies: many tumor types, many altered genes, many therapeutic challenges. J Clin Invest 2012;122(10):3396–7. 2. BC Cancer Agency. The centre for lymphoid cancer is a multi-disciplinary research program focused on the development of new treatments and diagnostics for lymphoid cancers [Internet]. BC Cancer Agency; 2018. Available from: http://www.bccrc.ca/dept/cflr. 3. Amin ARMR, Kucuk O, Khuri FR, Shin DM. Perspectives for cancer prevention with natural compounds. J Clin Oncol 2009;27(16):2712–25. 4. Cooper AJ, Sharp SJ, Lentjes MAH, Luben RN, Khaw K-T, Wareham NJ, et al. A prospective study of the association between quantity and variety of fruit and vegetable intake and incident type 2 diabetes. Diabetes Care 2012;35(6):1293–300. 5. Sun Y, Liu J. Purification, structure and immunobiological activity of a water-soluble polysaccharide from the fruiting body of Pleurotus ostreatus. Bioresour Technol 2009;100(2):983–6. 6. Cao XY, Liu JL, Yang W, Hou X, Li QJ. Antitumor activity of polysaccharide extracted from Pleurotus ostreatus mycelia against gastric cancer in vitro and in vivo. Mol Med Rep 2015;12(2):2383–9. https://doi.org/10.3892/mmr.2015.3648. 7. Pornariya C, Kanok-Orn I. Amino acids and antioxidant properties of the oyster mushrooms, Pleurotus ostreatus, and Pleurotus sajor-caju. Sci Asia 2009;35:326–31. https://doi.org/10.2306/scienceasia1513-1874.2009.35.326. 8. DiGiovanni J. Multistage carcinogenesis in mouse skin. Pharmacol Ther 1992;54(1):63–128. 9. Uddin Pk MM, Islam MS, Pervin R, Dutta S, Talukder RI, Rahman M. Optimization of extraction of antioxidant polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm and its cytotoxic activity against murine lymphoid cancer cell line. PLoS ONE 2019;14(1):e0209371. https://doi.org/ 10.1371/journal.pone.0209371. 10. Klaunig JE, Kamendulis LM, Hocevar BA. Oxidative stress and oxidative damage in carcinogenesis. Toxicol Pathol 2010;38(1):96–109. 11. Ishikawa K, Takenaga K, Akimoto M, Koshikawa N, Yamaguchi A, Imanishi H, et al. ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis. Science 2008;320:661–4. https://doi.org/10.1126/science.1156906. 12. Szatrowski TP, Nathan CF. Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res 1991;51:794–8 (0008–5472 (Print)). 13. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci U S A 1993;90(17):7915–22. 14. Gonenc A, Tokgoz D, Aslan S, Torun M. Oxidative stress in relation to lipid profiles in different stages of breast cancer. Indian J Biochem Biophys 2005;42:190–4 (0301–1208 (Print)). 15. Rao CSS, Kumari DS. Changes in plasma lipid peroxidation and the antioxidant system in women with breast Cancer. Int J Basic Appl Sci 2012; 1(4):429–38. 16. Abdel-Salam OME, Youness ER, Hafez HF. The antioxidant status of the plasma in patients with breast cancer undergoing chemotherapy. Open J Mol Integr Physiol 2011;1(3):29–35. 17. Yoshikawa T, Naito Y. What is oxidative stress? J Jpn Med Assoc 2002;45(7):271–6. 18. Blein S, Berndt S, Joshi AD, Campa D, Ziegler RG, Riboli E, et al. Factors associated with oxidative stress and cancer risk in the breast and prostate cancer cohort consortium (BPC3). Free Radic Res 2014;48(3):380–6. 19. Fiorani M, Cantoni O, Tasinato A, Boscoboinik D, Azzi A. Hydrogen peroxide-and fetal bovine serum-induced DNA synthesis in vascular smooth muscle cells: positive and negative regulation by protein kinase C isoforms. Biochim Biophys Acta 1995;1269(1):98–104. 20. Wu W-S, Tsai RK, Chang CH, Wang S, Wu J-R, Chang Y-X. Reactive oxygen species mediated sustained activation of protein kinase C alpha and extracellular signal-regulated kinase for migration of human hepatoma cell Hepg2. Mol Cancer Res 2006;4(10):747–58.

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21. Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol 2007;47:89–116. 22. Nioi P, Nguyen T. A mutation of Keap1 found in breast cancer impairs its ability to repress Nrf2 activity. Biochem Biophys Res Commun 2007; 362(4):816–21. 23. Padmanabhan B, Tong KI, Ohta T, Nakamura Y, Scharlock M, Ohtsuji M, et al. Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer. Mol Cell 2006;21(5):689–700. 24. Li QK, Singh A, Biswal S, Askin F, Gabrielson E. KEAP1 gene mutations and NRF2 activation are common in pulmonary papillary adenocarcinoma. J Hum Genet 2011;56(3):230–4. 25. Wong TF, Yoshinaga K, Monma Y, Ito K, Niikura H, Nagase S, et al. Association of Keap1 and Nrf2 genetic mutations and polymorphisms with endometrioid endometrial adenocarcinoma survival. Int J Gynecol Cancer 2011;21(8):1428–35. 26. Gibson SB. Investigating the role of reactive oxygen species in regulating autophagy. Methods Enzymol 2013;528:217–35. 27. Circu ML, Aw TY. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med 2010;48(6):749–62. 28. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26(4):239–57. 29. Fulda S, Gorman AM, Hori O, Samali A. Cellular stress responses: cell survival and cell death. Int J Cell Biol 2010;2010:214074. 30. Kaminskyy VO, Zhivotovsky B. Free radicals in cross talk between autophagy and apoptosis. Antioxid Redox Signal 2014;21(1):86–102. 31. Luna-Vargas MPA, Chipuk JE. The deadly landscape of pro-apoptotic BCL-2 proteins in the outer mitochondrial membrane. FEBS J 2016; 283(14):2676–89. 32. Pallepati P, Averill-Bates D. Mild thermotolerance induced at 40 degrees C increases antioxidants and protects HeLa cells against mitochondrial apoptosis induced by hydrogen peroxide: Role of p53. Arch Biochem Biophys 2010;495(2):97–111. 33. Circu ML, Aw TY. Glutathione and modulation of cell apoptosis. Biochim Biophys Acta 2012;1823(10):1767–77. 34. Bernardi P, Di Lisa F. The mitochondrial permeability transition pore: molecular nature and role as a target in cardioprotection. J Mol Cell Cardiol 2015;78:100–6. 35. Orrenius S, Gogvadze V, Zhivotovsky B. Calcium and mitochondria in the regulation of cell death. Biochem Biophys Res Commun 2015; 460(1):72–81. 36. Dickinson BC, Chang CJ. Chemistry and biology of reactive oxygen species in signaling or stress responses. Nat Chem Biol 2011;7(8):504–11. 37. Bolisetty S, Jaimes EA. Mitochondria and reactive oxygen species: physiology and pathophysiology. Int J Mol Sci 2013;14(3):6306–44. 38. Sevrioukova IF. Apoptosis-inducing factor: structure, function, and redox regulation. Antioxid Redox Signal 2011;14(12):2545–79. 39. Paradies G, Paradies V, Ruggiero FM, Petrosillo G. Oxidative stress, cardiolipin and mitochondrial dysfunction in nonalcoholic fatty liver disease. World J Gastroenterol 2014;20(39):14205–18. 40. Gogvadze V, Orrenius S, Zhivotovsky B. Multiple pathways of cytochrome c release from mitochondria in apoptosis. Biochim Biophys Acta 2006;1757(5):639–47. 41. Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. vol. 2015. Oxford University Press; 2015. 961 p. 42. Siems WG, Grune T, Esterbauer H. 4-Hydroxynonenal formation during ischemia and reperfusion of rat small intestine. Life Sci 1995;57(8):785–9. 43. Wang M, Dhingra K, Hittelman WN, Liehr JG, de Andrade M, Li D. Lipid peroxidation-induced putative malondialdehyde-DNA adducts in human breast tissues. Cancer Epidemiol Biomarkers Prev 1996;5(9):705–10. 44. Lu SC, Ge JL. Loss of suppression of GSH synthesis at low cell density in primary cultures of rat hepatocytes. Am J Physiol 1992;263:C1181–9 (0002–9513 (Print)). 45. Holmgren A. Regulation of ribonucleotide reductase. In: Horecker BL, Stadtman ER, editors. Current topics in cellular regulation. vol. 19. Academic Press; 1981. p. 47–76. 46. Carretero J, Obrador E, Anasagasti MJ, Martin JJ, Vidal-Vanaclocha F, Estrela JM. Growth-associated changes in glutathione content correlate with liver metastatic activity of B16 melanoma cells. Clin Exp Metastasis 1999;17(7):567–74. 47. Marengo B, De Ciusis C, Ricciarelli R, Romano P, Passalacqua M, Marinari UM, et al. DNA oxidative damage of neoplastic rat liver lesions. Oncol Rep 2010;23:1241–6 (1791–2431 (Electronic)). 48. Carretero J, Obrador E, Esteve JM, Ortega A, Pellicer JA, Sempere FV, et al. Tumoricidal activity of endothelial cells. Inhibition of endothelial nitric oxide production abrogates tumor cytotoxicity induced by hepatic sinusoidal endothelium in response to B16 melanoma adhesion in vitro. J Biol Chem 2001;276(28):25775–82 (0021–9258 (Print)). 49. Costantini P, Jacotot E, Decaudin D, Kroemer G. Mitochondrion as a novel target of anticancer chemotherapy. J Natl Cancer Inst 2000; 92(13):1042–53. 50. Joseph B, Marchetti P, Formstecher P, Kroemer G, Lewensohn R, Zhivotovsky B. Mitochondrial dysfunction is an essential step for killing of nonsmall cell lung carcinomas resistant to conventional treatment. Oncogene 2002;21(1):65–77. 51. El Enshasy HA, Hatti-Kaul R. Mushroom immunomodulators: unique molecules with unlimited applications. Trends Biotechnol 2013;31(12):668–77. 52. Islam MR, Pk MMU. In vitro doses and incubations dependent thrombolytic potential study of edible mushrooms Pleurotus ostreatus, Ganoderma lucidum and Lentinula edodes available in Bangladesh. J Pharm Res Int 2018;7(1):44–51. 53. Islam S, MMU P. Antihyperglycemic activity of edible mushroom, Lentinus edodes in alloxan induced diabetic swiss albino mice. Int J Pharm Clin Res 2014;6:121–6. 54. Jayakumar T, Thomas PA, Sheu JR, Geraldine P. In-vitro and in-vivo antioxidant effects of the oyster mushroom Pleurotus ostreatus. Food Res Int 2011;44(4):851–61.

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55. Reis FS, Martins A, Barros L, Ferreira ICFR. Antioxidant properties and phenolic profile of the most widely appreciated cultivated mushrooms: a comparative study between in vivo and in vitro samples. Food Chem Toxicol 2012;50(5):1201–7. 56. Lavi I, Friesem D, Geresh S, Hadar Y, Schwartz B. An aqueous polysaccharide extract from the edible mushroom Pleurotus ostreatus induces antiproliferative and pro-apoptotic effects on HT-29 colon cancer cells. Cancer Lett 2006;244(1):61–70. 57. El-Deeb NM, El-Adawi HI, El-Wahab AEA, Haddad AM, El Enshasy HA, He YW, et al. Modulation of NKG2D, KIR2DL and cytokine production by Pleurotus ostreatus glucan enhances natural killer cell cytotoxicity toward cancer cells. Front Cell Dev Biol 2019;7:165. 58. Rajewska J. Bałasinska B [Biologically active compounds of edible mushrooms and their beneficial impact on health]. Postepy Hig Med Dosw (Online) 2004;58:352–7. 59. Sliva D. Ganoderma lucidum in cancer research. Leuk Res 2006;30(7):767–8. 60. Sarangi I, Ghosh D, Bhutia SK, Mallick SK, Maiti TK. Anti-tumor and immunomodulating effects of Pleurotus ostreatus mycelia-derived proteoglycans. Int Immunopharmacol 2006;6(8):1287–97.

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Chapter 39

“Skin cancer, polyphenols, and oxidative stress” or Counteraction of oxidative stress, inflammation, signal transduction pathways, and extracellular matrix remodeling that mediate skin carcinogenesis by polyphenols Neena Philipsa, Richard Richardsona, Halyna Siomyka, David Bynuma, and Salvador Gonzalezb a

School of Natural Sciences, University College, Fairleigh Dickinson University, Teaneck, NJ, United States, b Dermatology Service, Memorial

Sloan-Kettering Cancer Center, New York, NY, United States

List of abbreviations ROS MMP ECM UV MAPK NF-kB STAT ERK1/2 JNK AP-1 MAE COX EGCG IL TGF-b VEGF

reactive oxygen species matrixmetalloprotienases extracellular matrix ultraviolet mitogen activated protein kinase nuclear factor-kappa beta signal transduction and activation of transcription extracellular signal-regulated kinase 1/2 c-Jun-N-terminal-kinase activator protein-1 michelia alba extract cyclooxygenase epigallocatechin-3-gallate interleukin transforming growth factor-b vascular endothelial growth factor

Introduction The characteristics of malignancy are loss of cellular regulation, and metastasis.1 One of the major causes of skin cancer (melanoma and nonmelanoma) is the cellular accumulation reactive oxygen species (ROS), which outbalances the cellular antioxidant system, and the subsequent oxidative damage, inflammation, activation of oxidative/inflammatory signal transduction pathways, and the degradation/remodeling of the extracellular matrix (ECM) for angiogenesis and metastasis. The predominant structural ECM proteins, collagen, and elastin fibers, are remodeled by matrixmetalloprotienases (MMP) and elastases. This chapter reviews the fundamental biology, the alterations, and counteraction by polyphenols in the mechanism to carcinogenesis or its prevention as (a) oxidative stress, inflammation, and associated signal transduction pathways and (b) ECM remodeling and associated growth factors.

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00039-0 © 2021 Elsevier Inc. All rights reserved.

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Oxidative stress, inflammation, and associated signal transduction pathways: Fundamental biology, the alteration, and counteraction by polyphenols Oxidative stress and polyphenols The reactive oxygen species, which cause oxidative stress, include hydroxyl radicals, superoxide, and hydrogen peroxide; and the counteracting cellular antioxidants include catalase, glutathione peroxidase, glutathione, ascorbate, a-tocopherol, and carotene. Phytonutrients, most of which are rich in polyphenols, have been gaining popularity for improving the cellular antioxidant score and thereby preventing oxidative stress and cancer. The structure of the phenolic compounds possess at least one aromatic ring with one or more hydroxyl groups and have been categorized as phenolic acids, monophenols, polyphenols, and associated substructures; and polyphenols have been classified as flavonoids (xanthohumol), anthocyanin (grapes), catechins (tea leaves), flavones (luteolin), flavonols (quercetin), isoflavones (genistein), lignans (flax seeds), proanthocyanidins (red wine), procyanidins (apple), stilbenes (resveratrol), tannins (nuts, tea).2 The structure, as well as the number or location of the hydroxyl groups, may correlate with the radical scavenging potential of polyphenols.3–5 The cellular oxidative stress occurs with intrinsic aging though more so from the exposure of skin to environmental pollutants and ultraviolet (UV) radiation. With intrinsic aging, there is cellular and mitochondrial DNA damage and diminished expression of protective hormones and growth factors that strengthen the ECM.6 The environmental pollutants include benzene, dioxins, heavy metals, aryl, or chlorinated hydrocarbons. Benzene is metabolized by skin cells and its metabolites, and copper increases oxidative stress and the expression of ECM proteolytic enzymes by dermal fibroblasts.7, 8 The UVA radiation increases cellular ROS, and oxidative chain reaction.2, 9 The damage by UVB irradiation is mediated initially by reactive oxygen species, and then because of the increased reactive nitrogen species, and lipid peroxidant species and reduced counteracting catalase and glutathione levels.10 The ROS attack the DNA, proteins, and lipids directly, and stimulate inflammation.2, 11–13, 57–59 The oxidative damage includes 8-oxo-7, 8-dihydro-20 -deoxyguanosine (8-oxodG) that generates a GC to TA transversion mutation during replication, pyrimidine dimers, carbonyl amino acid derivatives, lipid inflammatory mediators/peroxides, advanced glycation end products, calcification, and mechanical fatigue.2,11–13 The repair of oxidative DNA damage (8-oxoG) is reduced because of the glutathione depletion in fibroblasts and melanoma cells.14 The application of oligomeric proanthocyanidins (OPCs) as creams reduces oxidative stress in skin, while a combination of 5 phytochemicals (protandim, rich in polyphenols) potently induces cellular antioxidant enzymes for cancer prevention15, 16 (Fig. 1). Green tea polyphenols improve DNA repair and induce the expression of tumor suppressor gene through the inhibition of histone methylation2, 17, 18 (Fig. 1). The Humulus lupulus (Cannabinaceae) (HOP) extract and its phenolic components, alpha-acid (humulone), beta-acid (lupulone), xanthoflavonoids, xanthohumunol, and isoxanthohumunol, possess direct antioxidant activity in the removal of hydrogen peroxide60; (Fig. 1). The alpha-acid demonstrate the highest antioxidant activity, the beta-acid, xanthoflavonoids, and xanthohumunol show similar activity, and isoxanthohumunol the least activity.60 In addition, the HOP extract and its components significantly and similarly inhibit the viability of melanoma cells.60 The inhibition of cell viability could be through the activation of extrinsic or intrinsic apoptotic pathways.1 The extrinsic pathway is through the activation of Fas-associated death domain (FADD) and TNF-associated death domain (TRADD) that activate the initiator caspases and inactivate the antiapoptotic Bcl-2, which allows for the oligomerization of the Bax proteins, the release of cytochrome C from the mitochondria to the cytoplasm and the subsequent activation of effector caspases and thereby apoptosis.1 The intrinsic pathway is activated is by Bcl-2 homology (BH) 3 only proteins that inactivate the antiapoptotic Bcl-2 and thereby activate Bax proteins to mediate apoptosis.1 These BH3 proteins include BAD that is released on the inhibition of the cell growth stimulatory protein kinase B (PKB), Bim that is released from altered cell integrin signaling, and Puma that is released following DNA damage.1 Nicotinamide and its hydroxyl derivatives, 2,6-dihydroxynicotinamide, 2, 4, 5, 6-tetrahydroxynicotinamide, and 3-hydroxypicolinamide possess anticarcinogenic properties.61–63 Nicotinamide, 2,6-dihydroxynicotinamide, and 2,4,5,6-tetrahydroxynicotinamide have direct antioxidant activities, with the 2,6-dihydroxynicotinamide exhibiting the highest antioxidant activity61, 63 (Fig. 1). Similarly, 1a,25 dihydroxyvitamin D3 (vitamin D) has direct antioxidant activity through the prevention of the oxidative effect of hydrogen peroxide; as well as cellular antioxidative effects through the inhibition of DNA/RNA oxidative damage in nonirradiated, UVA-radiated, and UVB-radiated dermal fibroblasts64 (Fig. 1). In addition, vitamin D inhibits lipid peroxidation in nonirradiated and UVA-radiated fibroblasts; and membrane damage in UVA-radiated, and UVB-radiated fibroblasts64 (Fig. 1).

Inflammation and polyphenols ROS are inducers of pro-inflammatory genes. The inflammatory mediators are released from keratinocytes, fibroblasts, tumor cells, leukocytes, and the endothelial lining of blood vessels. The mediators include the plasma mediators

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FIG. 1 Oxidative stress and phenolic molecules.

Oligomeric proanthocyanidins reduce oxidative stress 15

Protandim induces cellular antioxidant enzymes 16

Oxidative stress and polyphenols

Green tea polyphenols improve DNA repair and inhibit histone methylation 18

Humulus lupulus exhibits direct antioxidant activity 60

Nicotinamide/ its hydroxy derivatives, and vitamin D exhibit direct antioxidant activity 61, 64 . Vitamin D inhibits cellular oxidative stress effects

(bradykinin, plasmin, and fibrin), lipid mediators (prostaglandins, leukotrienes, and platelet-activating factor), and the inflammatory cytokines [interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a)].19 The initial inflammatory response to environmental agents, including carcinogens, is through innate or nonspecific immunity.1, 19, 57, 58 The specific immune response is mediated by the T-lymphocytes, which includes the cytotoxic T cells and helper T (Th) cells, and the B-lymphocytes (B cells) or the antibody-producing cells.1, 19, 57, 58 Interleukin2 (IL-2) and interferon-g (IFN-g), which are stimulated by interleukin-12 (IL-12), activate Th1 cells that mediate tissue damage.1, 19, 57, 58 The interleukin-4 (IL-4) activates the Th2 cells that activate eosinophils and stimulate the B cells to produce IgE antibodies, which bind to mast cells and basophils to cause the release of inflammatory mediators such as histamines, leukotrienes, and cytokines.1, 19, 57–59 Dupilumab (Dupixent, Sanofi and Regeneron Pharmaceuticals, Inc.), an antibody to the IL-4 a receptor chain, inhibits IL-4 signaling, and thereby the inflammatory cascade.57, 58 UV radiation directly induces inflammation, and the release of prostaglandins, histamine, and active phospholipase.20 The UV radiation also activates the tissue leukocytes to generate prostaglandins and inflammatory cytokines.2 A major mechanism to the anticarcinogenic effect of polyphenols is the inhibition of inflammation. In our laboratory, xanthohumol [a flavonoid from Hop plant Humulus lupulus L. (Cannabinaceae)], and P. leucotomos (rich in mono- and polyphenols) have shown anti-cancer activities.21–24 The anti-cancer effects of these polyphenols are similar to those of ascorbate or lutein.25, 26 The effects of lutein are similar to that of superoxide dismutase, indicating the inhibition of ROS as one of its mechanism.25 The anticancer mechanism of xanthohumol includes the inhibition of cyclooxygenases/prostaglandins and reactive oxygen/reactive nitrogen species28 (Fig. 2). Vitamin D significantly inhibits IL-1 and IL-8 expression in UVAradiated dermal fibroblasts, indicating its cellular anti-inflammatory activity64 (Fig. 2).

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FIG. 2 Inflammation and phenolic molecules.

Xanthohumol inhibits cyclooxygenases/prost aglandins, and NF-kB 28, 35

Inflammation and polyphenols

Green tea polyphenol inhibits NF-kB, and IL-1 36

Olive oil and red wine polyphenols inhibits NF-kB 37

Vitamin D inhibits IL-1 and IL-8 expression in UVA-radiated fibroblasts 64

Signal transduction pathways and polyphenols The predominant pathways activated by reactive oxygen species, and inflammatory cytokines for carcinogenesis include the mitogen-activated protein kinase (MAPK), the nuclear factor-kappa beta (NF-kB)/p65, and the JAK/STAT (Signal Transduction and Activation of Transcription).1, 2, 11, 19 The MAPK pathway is comprised of the extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun-N-terminal-kinase (JNK), and p38 proteins. The activation of MAP kinase pathway, through the receptor tyrosine kinase, results in the activation of transcription factor activator protein-1 (AP-1) that activates expression of MMPs.11 The JNK and p38 pathways play a major role in the UVA radiation-mediated increase in AP-1 and COX-2 expression and are targets for chemoprevention of skin cancer.29 The inhibition of MAPK is a mechanism to the anticancer activity of polyphenols. Michelia alba extract (MAE) inhibits UVB-induced ERK and JNK kinase in the prevention of ECM degradation.30 Resveratrol and black tea polyphenol decrease the expression of phosphorylated ERK1/2, JNK, and p38, and increase phosphorylated p53 and apoptosis in skin tumors and thereby suppress skin carcinogenesis.31 Grape skin polyphenols inhibit MAPK pathway to inhibit cancer cell migration and metastasis.32 The NF-kB pathway is activated by oxidative stress and inflammation through the activation of cytoplasmic I-kB kinase. Active I-kB kinase phosphorylates and degrades I-kB, the inhibitor of NF-kB (p65/p50 heterodimeric protein) transcription factor.1 The NF-kB activation is associated with UVA and UVB radiation mediated oxidative modification of cellular membrane components.33 The release of NF-kB, from its inhibitor (I-kB), results in the translocation of active NF-kB to the nucleus to activate the inflammatory cytokines and prostaglandins.1 The active NF-kB also activates nitric oxide synthase, cyclooxygenase, and histone acetylase.1,33 The inhibition of NF-kB activation is a mechanism to the anticancer property of dietary polyphenols.34 Xanthohumol inhibits NF-kB transcription factor35 (Fig. 2). Epigallocatechin-3-gallate (EGCG), the major polyphenolic in green tea, decreases NF-kB, inflammation, interleukin-1 b (IL-1b) secretion, and cell growth of melanoma cells36 (Fig. 2). The antioxidant polyphenols from olive oil (oleuropein and hydroxytyrosol) and red wine (resveratrol and quercetin) inhibit NF-kB and thereby COX-2, MMP-9, and tumor angiogenesis37 (Fig. 2). The STAT pathway is activated by the inflammatory cytokines.19 Many of the cytokines bind to their cytokine receptors on cell surfaces to activate JAK kinase that activates STAT transcription factors through phosphorylation.1 The phosphorylation of STAT results in its dimerization and translocation into the nucleus to modulate gene expression.1 The different cytokines activate different STAT proteins; one of its effects being the activation of Bcl-xL to prevent apoptosis and allow

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for cell proliferation.1 The IL-4 signaling is through the activation of JAK1/JAK3 that activates STAT6, which activates the GATA transcription factor that stimulates Th2 cells.19, 57–59 The polyphenols apigenin and luteolin inhibit the cytokine IL-6/STAT3 pathway and thereby endothelial cell proliferation, migration, and differentiation for angiogenesis.38

ECM remodeling and associated growth factors: Fundamental biology, the alteration, and counteraction by polyphenols Collagen/elastin and polyphenols Collagen and elastin are the structural proteins of the ECM. The deterioration/remodeling of the collagen and elastin fibers facilitates angiogenesis and metastasis and the damaged collagen and elastin proteins serve as additional sensitizers of photooxidative stress.39 Collagen is the predominant ECM protein, central to the interstitial ECM as well as to the basement membrane. Collagens are homo or heterotrimeric triple helical proteins, composed of repeating Gly-X-Y motifs where X or Y being proline or hydroxyproline.1 The specific properties of the different collagens are based on the lengths of the triple helical segments, interruptions to the triple helix, and amino acid modifications. There are about 28 types of collagen.1 Collagens are classified as fibrillar collagens (types I, II, III, V), fibril-associated collagens (types VI, IX), sheet forming anchoring collagens (types IV, VII, XV), transmembrane collagens (types XIII, XVII), and host defense collagens.1 The dermal collagen fibers are formed of the types I (90%), III, V, and VII collagens.11 The elastin fibers that provide stretch-recoil properties to the skin are composed predominantly of an elastin core (90%) surrounded by fibrillin microfibrils. Elastin is expressed as soluble hydrophobic tropoelastin, rich in proline, valine, lysine, alanine, glycine, leucine, and isoleucine, transferred to the microfibril scaffold, and cross-linked by lysyl oxidase and transglutaminase.13, 40 The fibrillins are cysteine-rich highly disulfide bonded glycoproteins, with calcium-binding epidermal growth factor like domains.40, 41 They are secreted in pro-forms, assemble into microfibrils, and associate with other microfibril-associated glycoproteins.40, 41 The loss of proper elastin fibers occurs with the exposure of skin to UV radiation.13 UV radiation also depletes the microfibrillar network in the epidermal-dermal layer and the dermis, which contributes to the aberrant elastic fibers.41 Xanthohumol dramatically stimulates the expression of types I, III, and V collagens, elastin, fibrillin-1, and fibrillin-2 in dermal fibroblasts21 (Fig. 3). P. leucotomos stimulates the expression of type I, III, and V collagens, and elastin in nonirradiated, UVA radiated, and UVB radiated dermal fibroblasts21, 22, 24 (Fig. 3). FIG. 3 Collagen/elastin and phenolic molecules.

Collagen/elastin and polyphenols

Xanthohumol stimulates the expression of types I, III and V collagens, and elastin, fibrillin-1, fibrillin-2 21

P. leucotomos stimulates the expression of type I, III, and V collagens 21,22,24

Nicotinamide/its hydroxy derivatives and vitamin D stimulates collagen and/or elastin 61, 62, 64

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Nicotinamide, 2,6-dihydroxynicotinamide, 2, 4, 5, 6-tetrahydroxynicotinamide, and 3-hydroxypicolinamide simultaneously stimulate the expression of type I collagen, type III collagen, and type V collagen in dermal fibroblasts, and thereby strengthens the structural integrity of the ECM61 (Fig. 3). The stimulation of the type I collagen expression was at protein and promoter levels, suggesting a transcriptional regulatory mechanism.61 UVA radiation significantly inhibits the expression of type I and III collagens, though not type V collagen, in dermal fibroblasts.61 Nicotinamide, 2,6-dihydroxynicotinamide, 2, 4, 5, 6-tetrahydroxynicotinamide, and 3-hydroxypicolinamide maintained its predominant stimulatory effect on the expression of types I, III, and V collagens in the UVA-radiated fibroblasts.61 The 2,6-dihydroxynicotinamide showed higher stimulatory effect than the other nicotinamide derivatives, on the expression of fibrillar collagen in nonirradiated and UVA radiated fibroblasts, suggesting the importance of the hydroxyl groups in these locations for higher activity.61 Vitamin D significantly stimulates the transcription, and thereby the protein levels, of type I collagen in nonirradiated and UVA-radiated fibroblasts to strengthen the integrity of the ECM64 (Fig. 3). The heat shock protein (HSP)-47 is essential to the folding of collagen fibers.1 The 2,4,5,6-tetra hydroxynicotinamide and 3-hydroxypicolinamide derivatives of nicotinamide stimulate the expression of HSP-47 in nonirradiated fibroblasts, suggesting aiding the formation of collagen fibers.61 UVA-radiation inhibits the expression of HSP-47 in dermal fibroblasts.61 Nicotinamide, 2,6-dihydroxynicotinamide, 2,4,5,6-tetrahydroxynicotinamide, and 3-hydroxypicolinamide significantly and dramatically stimulate the expression of HSP-47 in UVA-radiated fibroblasts, suggesting its supportive role in the strengthening of the ECM in response to UVA-radiation.61 Vitamin D significantly stimulates the expression of HSP-47 in UVA-radiated fibroblasts, suggesting its role in the upregulation of the expression of type I collagen in these cells.64 Nicotinamide, 2,6-dihydroxynicotinamide, 2, 4, 5, 6-tetrahydroxynicotinamide, and 3-hydroxypicolin simultaneously stimulate the expression of elastin, fibrillin-1 and fibrillin-2 in nonirradiated as well as UVA-radiated fibroblasts, suggesting the formation of proper elastic fibers62 (Fig. 3). The stimulation of the elastin expression was at protein and promoter levels, suggesting a transcriptional regulatory mechanism.62 Nicotinamide and 2,6-dihydroxynicotinamide show higher stimulatory effect than the other two nicotinamide derivatives, on the expression of elastin.62

Matrixmetalloproteinases/elastases The ECM proteolytic enzymes (MMPs/elastases) are produced by epidermal keratinocytes, dermal fibroblasts, and melanoma in the mediation of ECM remodeling and skin cancer.7, 8, 21–26, 42–44 The basal levels of MMPs increase with aging, and are further increased by environmental pollutants and UV radiation; resulting in the fragmentation of collagen and elastin fiber proteins for carcinogenesis. The MMPs are zinc metalloproteinases that are secreted extracellularly in an inactive form, with the propeptide linked to the zinc in the catalytic domain. One of the mechanisms of activation of MMPs is through proteolysis of its propeptide by plasmin, which is formed from plasminogen following the secretion of plasminogen activator by tumor cells.1, 45 The MMPs are classified on the basis of their substrate specificity, or their regulatory promoter elements. The categories based on substrate specificity are the interstitial collagenases (predominantly MMP-1) that cleave the fibrillar collagens, the gelatinases (MMP-2, 9) that cleave the basement membrane collagens and elastin, the stromelysines (MMP-3, 10) that degrade the basement membrane proteins, the membrane-type MMPs with a transmembrane domain that act on pro-MMPs and fibrillar collagens, and the other MMPs including the matrilysin (MMP-7) and metalloelastase (MMP-12) that degrade the basement membrane proteins and elastin.45 MMPs are categorized on the basis of the presence of AP-1 or TATA sequences in the promoters into group I MMPs (MMP-1, 3, 7, 9, 10, 12, 13, 19, and 26) that contain TATA box and activator protein-1 (AP-1 site), group II MMPs (MMP-8, 11, 21) without the AP-1 site, and group III (MMP-2, 14, 28) without the TATA box and AP-1 site.46 The transcription factor AP-1, stimulated largely by the MAPK pathway, stimulates the transcription of several MMPs that collectively degrade the ECM, such as MMP-1, MMP-2/9, and MMP-3.33 Further, AP-1 inhibits the transcription of type I collagen gene.11 Hence, the damage to the ECM and tissue integrity is from the enhanced degradation of ECM by MMPs as well as the reduced expression of the structural ECM proteins. The pro- and active forms of MMPs are inhibited by the tissue inhibitors of MMPs or TIMPs.45, 46 The genes for four of the TIMPs (1–4) have been mapped to chromosomes X, 17, 22, and 3.47 TIMP-1 and TIMP-3 are inducible, TIMP-2 is constitutive, and TIMP-4 is restricted in its expression to certain tissues.47 The TIMPs (1–4) bind to all of the MMPs, though TIMP-1 has preference for MMP-1 and TIMP-2 for MMP2.45, 47 The N-terminal conserved region of TIMPs is essential to binding to the active site of MMPs.47 The remodeling of collagen and elastin, for angiogenesis, metastasis, and tissue destruction, is largely from the increased expression or activation of MMPs and reduced expression of TIMPs.48, 49 The inhibition of expression and activity of MMPs and the stimulation of expression of TIMPs have been active targets in managing carcinogenesis. Specific MMP-1 gene silencing with siRNAs inhibits the expression of MMP-1 in melanoma cells.23

Skin cancer, polyphenols, and oxidative stress Chapter

Xanthohumol directly inhibits MMP-1, MMP-2, MMP-3, and MMP-9 activities 21

39

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FIG. 4 Matrixmetalloproteinases (MMP)/ elastases and phenolic molecules.

P. leucotomos inhibits activities of MMPs, and expression of MMP-1 and MMP-2, and stimulates TIMP-1 22

MMP/elastases and polyphenols

Apigenin inhibits the expression of MMP-2 30

Luteolin inhibits the expression of MMP-2 30

Michelia alba extract inhibits expression of MMP-1, MMP-3, and MMP-9 38

Nicotinamide/its hydroxy derivatives, and vitamin D inhibit MMPs and /or Elastase activities 62, 64

Synthetic flavonoid inhibits cancer cell invasiveness and stimulates TIMP-2 expression 49

An MMP-9 inhibitor prevents epithelial cancer cell migration and tubular network formation.48 A human serum albumin/ TIMP-2 fusion protein inhibits MMP-2 expression and thereby inhibits in vivo vascularization/angiogenesis.50 Polyphenols provide a means to accomplish the inhibition of MMPs and stimulation of TIMPs. The activities of MMP-1, MMP-2, MMP-3, and MMP-9 are directly inhibited by xanthohumol and P. leucotomos21,22 (Fig. 4). P. leucotomos inhibits the expression of MMP-1 in nonirradiated, UVA or UVB radiated keratinocytes, and fibroblasts.24 Further, P. leucotomos inhibits the expression of MMP-2 while stimulating TIMP-1 and/or TIMP-2 expression in dermal fibroblasts, and melanoma cells22 (Fig. 4). P. leucotomos counteracts the stimulation of MMP-1 by the growth inhibitory concentration of ascorbate in melanoma cells, suggesting the beneficial effect of the combination in the management of cancer.23 The polyphenols apigenin and luteolin inhibit the expression of MMP-2 whereas Michelia alba extract (MAE) inhibits the UVB radiation induced expression of MMP-1, MMP-3, and MMP-930, 38 (Fig. 4). A synthetic flavanoid (SR13179) simultaneously inhibits cancer cell invasiveness and stimulates TIMP-2 expression49 (Fig. 4). Nicotinamide, 2,6-dihydroxynicotinamide, 2,4,5,6-tetrahydroxynicotinamide, and 3-hydroxypicolinamide directly inhibit elastase activity.62 In addition, nicotinamide and 2,6-dihydroxynicotinamide also inhibit MMP-1 and MMP-3 activities62 (Fig. 4). Thus nicotinamide and its hydroxyl derivatives have dual protective roles in the strengthening of the ECM, through the stimulation of the formation of collagen and elastin fibers as well as the inhibition of elastase and MMPs that degrade the collagen and elastin fibers. Vitamin D directly inhibits elastase activity; as well as inhibits cellular elastase activity in non-radiated, and UVA-radiated fibroblasts; suggesting its role in preventing the proteolysis of elastic fibers64 (Fig. 4).

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Transforming growth factor-b (TGF-b) and polyphenols TGF-b is a key regulator of the cell cycle, and the expression of collagen, elastin, and MMPs. Carcinogenesis is associated with altered expression of TGF-b gene, TGF-b receptors, or transcription factors (Smads). It is expressed in high levels in tumors, and mediates epithelial-mesenchymal transition, angiogenesis, and tumor invasion.51 There are three isoforms of TGF-b.1, 7, 21 TGF-b is secreted in an inactive form and forms a disulfide bonded dimer with a latency associated protein (LAP) as its prodomain and associates with latent TGF-b binding protein (LTBP).1 TGF-b is activated by the dissociation of the mature TGF-b dimer from LAP and LTBP.1 The active TGF-b binds to transmembrane serine/threonine kinase receptors on target cells.1 There are three types of receptors that specifically bind TGF-b; however, only type I (TbRI) and type II (TbRII) receptors have intrinsic serine/threonine kinase activity whereas type III (TbRIII) receptor facilitates TGF-b binding to TbRII. Once TGF-b binding has occurred, TbRI and TbRII form a heteromeric complex and phosphorylate intracellular Smads.1 The phosphorylated Smad 2/3 bind Smad 4 translocates to the nucleus and bind TGF-b regulatory elements.1 The inhibitory Smads, Smad 6 and  7, interact with TbRI to prevent phosphorylation and subsequent activation of Smad 2/3.1 TGF-b has differential effects in different cell types.42–44, 52 It stimulates the expression of MMPs in keratinocytes and epithelial cells, whereas it inhibits the expression of MMPs in normal dermal fibroblasts.42–44 TGF-b counteracts the effects of UV radiation on the expression of ECM remodeling enzymes and inhibits the expression of MMP-1 at the protein, mRNA, and promoter levels in normal dermal fibroblasts.43, 52 TGF-b is coordinately stimulated with MMP-1 in cancer cells.53, 54 Cancer metastasis is associated with enhanced MMPs and TGF-b expression.52–54 P. leucotomos inhibits the expression of TGF-b in melanoma cells22,23 (Fig. 5). Further, P. leucotomos counteracts the stimulation of TGF-b by the growth inhibitory concentration of ascorbate in melanoma cells, suggesting the beneficial effect of the combination in the management of cancer23 (Fig. 5).

Vascular endothelial growth factor (VEGF) and polyphenols VEGF is central to angiogenesis for cancer growth and metastasis, and its overexpression is associated with poor prognosis.55 VEGF is regulated by various factors, TGF-b, hypoxia-inducible factor-1, and MMPs, and binds to its receptor tyrosine kinase.1, 48 The VEGF receptor tyrosine kinase activates the mitogen-activated protein kinase (MAPK) pathway, which results in the activation of several transcription factors including the serum response factor that activates c-fos gene expression for malignancy.1 Epigallocatechin gallate (EGCG) in green tea inhibits cancer cell growth by preventing the activation of VEGF receptor56 (Fig. 6). This effect is similar to the VEGF antibodies, such as Avastin, that inhibit angiogenesis, tumor growth, and metastasis.48 Xantho-flavonoid, a component of Humulus lupulus, exhibits antiangiogenic potential through the inhibition of the expression of VEGF in melanoma cells60 (Fig. 6). P. leucotomos inhibits the expression of VEGF in melanoma cells, as does ascorbate, and the combination of P. leucotomos and ascorbate inhibits it further, suggesting the beneficial effect of the combination in the inhibition of angiogenesis and thereby metastasis of cancer cells65 (Fig. 6). FIG. 5 Transforming growth factor (TGF-b) and phenolic molecules.

P. leucotomos inhibits the expression of TGFβ 22

TGF-α and polyphenols P. leucotomos counteracts stimulation of TGF- β by cytotoxic ascorbate concentration 23

Skin cancer, polyphenols, and oxidative stress Chapter

Xantho-flavonoid inhibits VEGF expression in melanoma cells 60

Vascular endothelial growth factor (VEGF) and polyphenols

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FIG. 6 Vascular endothelial growth factor (VEGF) and phenolic molecules.

Green tea polyphenol prevents activation of VEGF receptor 56

P. leucotomos inhibits VEGF expression in melanoma cells 65

Nanobiomaterials and polyphenols A limitation of polyphenols is its bioavailability and specific targeting, topically and systemically, and thereby its effectiveness.66 The combination of polyphenols with nanobiomaterials, which are biodegradable, would improve bioavailability as well as allow for the specific targeting of polyphenols.66, 67 The characteristics of nanobiomaterials would allow for the targeted sustained release of actives and its biodegradability without toxicity.66, 67 The investigated nanobiomaterials include chitosan, polymeric nanoparticles, nanoconjugates, liposomes, and delivery with a phage system.66, 67 Chitosan, a sugar, has been widely used in medicine and is biodegradable. The polymeric nanobiomaterials, poly lactide-co-glycolide acid (PLGA) and poly ehylene glicol (PEG), increase bioavailability by increasing half-life.66, 67 The nanoconjugates, created by adding functional groups to polymers, allows for the specific targeting of the active to tissues/cells.66, 67 The liposomes, with its phospholipid bilayer, for the entry of the active through the cell membrane.1, 66, 67 The carbon-based nanomaterial, such as graphene, can be engineered to improve functionality.66, 67 The phage system may become the preferred mechanism for the targeted delivery of actives. It has been widely used in recombinant DNA technology, and for the synthesis of pharmaceutical products.1, 66, 67 Polyphenols conjugated with chitosan, liposomes or graphene have improved effectiveness due to greater antioxidant activity, sustained release, and/or specific targeting of cancer cells for destruction.68–70

Conclusion Oxidative stress from the increased cellular ratio of ROS to antioxidants is the major factor in the etiology of cancer. It occurs with intrinsic aging but more so from exposure of skin to environmental pollutants and UV radiation. The ROS directly damage the DNA, proteins and lipids, induce inflammation, and activate the MAPK, NF-kB, and JAK/STAT pathways to activate the expression of MMPs, TGF-b, and VEGF that collectively remodel the ECM, composed primarily of collagen and elastin, for tumor growth, angiogenesis, and metastasis. Polyphenols are increasingly playing a role in cancer prevention through the restoration of the cellular antioxidant balance, anti-inflammation, inhibition of the MAPK, NF-kB, and JAK/STAT pathways, inhibition of MMPs, TGF-b, and VEGF, and the inhibition of cancer growth, angiogenesis and metastasis. The bioavailability of the polyphenols is increased by conjugating them with nanobiomaterials such as chitosan, polymeric nanoparticles, nanoconjugates, liposomes, and delivery with a phage system.

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Summary points l

l l l

l

l l

ROS, which facilitates carcinogenesis, are induced with intrinsic aging and more so with exposure of skin to environmental pollutants and UV radiation; and are counteracted by polyphenols. ROS damage DNA, proteins and lipids; counteracted and damage repaired by polyphenols. ROS induce inflammation and inflammatory mediators, counteracted by polyphenols. ROS induce oxidative and inflammatory pathways such as MAPK, NF-kB, and STAT pathways that activate transcription factors that increase cell proliferation and metastasis; counteracted by polyphenols. ROS induce MMPs, TGF-b, and VEGF that remodel the ECM composed of structural collagen and elastin for tumor growth, angiogenesis and metastasis; counteracted by polyphenols. Polyphenols, and their combinations, are effective in natural cancer prevention or as supplements in cancer treatment. Polyphenols have increased effectiveness in combination with nanobiomaterials.

Acknowledgment Hui Jia and David Sherer, for participating in the literature search.

References 1. Lodish H, Berk A, Kaiser CA, Krieger M, Scott MP, Bretscher A, et al. Molecular cell biology. 6th ed. W. H Freeman and Company; 2008. 2. Nichols JA, Katiyar SK. Skin photoprotection by natural polyphenols: anti-inflammatory, antioxidant and DNA repair mechanisms. Arch Dermatol Res 2010;302:71–83. 3. Cheng LX, Tang JJ, Luo H, Jin XL, Dai F, Yang J, et al. Antioxidant and antiproliferative activities of hydroxyl-substituted Schiff bases. Bioorg Med Chem Lett 2010;20:2417–20. 4. Chen ZY, Chan PT, Ho KY, Fung KP, Wang J. Antioxidant activity of natural flavonoids is governed by number and location of their aromatic hydroxyl groups. Chem Phys Lipids 1996;79:157–63. 5. Gaza´k R, Sedmera P, Vrbacky´ M, Vosta´lova´ J, Drahota Z, Marhol P, et al. Molecular mechanisms of silybin and 2, 3-dehydrosilybin antiradical activity—role of individual hydroxyl groups. Free Radic Biol Med 2009;46:745–58. 6. Philips N, Devaney J. Beneficial regulation of type I collagen and matrixmetalloproteinase 1 expression by estrogen, progesterone, and its combination in skin fibroblasts. J Am Aging Assoc 2003;26:59–62. 7. Philips N, Hwang H, Chauhan S, Leonardi D, Gonzalez S. Stimulation of cell proliferation, and expression of matrixmetalloproteinase-1 and interluekin-8 genes in dermal fibroblasts by copper. Connect Tissue Res 2010;51:224–9. 8. Philips N, Burchill D, O’Donoghue D, Keller T, Gonzalez S. Identification of benzene metabolites in dermal fibroblasts: regulation of cell viability, apoptosis, lipid peroxidation, and expression of MMP-1 and elastin by benzene metabolites. Skin Pharmacol Physiol 2004;17:147–52. 9. Krutmann J. The role of UVA rays in skin aging. Eur J Dermatol 2001;11:170–1. 10. Terra VA, Souza-Neto FP, Pereira RC, Silva TNX, Costa ACC, Luiz RCC, et al. Time-dependent reactive species formation and oxidative stress damage in the skin after UVB irradiation. J Photochem Photobiol B Biol 2012;109:34–41. 11. Callaghan TM, Wilhelm KP. A review of ageing and an examination of clinical methods in the assessment of ageing skin. Part I: cellular and molecular perspectives of skin ageing. Int J Cosmet Sci 2008;30:313–22. 12. Briganti S, Picardo M. Antioxidant activity, lipid peroxidation and skin diseases. What’s new? J Eur Acad Dermatol Venereol 2003;17:663–9. 13. Yaar M, Gilchrest BA. Photoageing: mechanism, prevention and therapy. Br J Dermatol 2007;157:874–87. 14. Eiberger W, Volkmer B, Amouroux R, Dherin C, Radicella JP, Epe B. Oxidative stress impairs the repair of oxidative DNA base modifications in human skin fibroblasts and melanoma cells. DNA Repair 2008;7:912–21. 15. Van Wijk EPA, Van Wijk R, Bosman S. Using ultra-weak photon emission to determine the effect of oligomeric proanthocyanidins on oxidative stress of human skin. J Photochem Photobiol B Biol 2010;98:199–206. 16. Liu J, Gu X, Robbins D, Li G, Shi R. Protandim: a fundamentally new antioxidant approach in chemoprevention using mouse two-stage skin carcinogenesis as a model. PLoS One 2009;4(4): e5284. 17. Choudhury SR, Balasubramanian S, Chew YC, Han B, Marquez VE, Eckert RL. ()-Epigallocatechin-3-gallate and DZNep reduce polycomb protein level via a proteasome-dependent mechanism in skin cancer cells. Carcinogenesis 2011;32:1525–32. 18. Thakur VS, Gupta K, Gupta S. The chemopreventive and chemotherapeutic potentials of tea polyphenols. Curr Pharm Biotechnol 2012;13:191–9. 19. Kindt TJ, Goldsby RA, Osborne BA. Kuby immunology. 6th ed. W. H. Freeman and Company; 2007. 20. Hruza LL, Pentland AP. Mechanisms of UV-induced inflammation. Invest Dermatol 1993;100:35S–41S. 21. Philips N, Samuel M, Arena R, Chen Y, Conte J, Natrajan P, et al. Direct inhibition of elastase and matrixmetalloproteinases., and stimulation of biosynthesis of fibrillar collagens, elastin and fibrillins by xanthohumol. J Cosmet Sci 2010;61:125–32. 22. Philips N, Conte J, Chen Y, Natrajan P, Taw M, Keller T, et al. Beneficial regulation of matrixmetalloproteinases and its inhibitors., fibrillar collagens and transforming growth factor-b by P. Polypodium leucotomos, directly or in dermal fibroblasts, ultraviolet radiated fibroblasts, and melanoma cells. Arch Dermatol Res 2009;301(7):487–95.

Skin cancer, polyphenols, and oxidative stress Chapter

39

449

23. Philips N, Dulaj L, Upadhya T. Growth inhibitory mechanism of ascorbate and counteraction of its matrix metalloproteinases-1 and transforming growth factor-beta stimulation by gene silencing or P. leucotomos. Anticancer Res 2009;29:3233–8. 24. Philips N, Smith J, Keller T, Gonzalez S. Predominant effects of Polypodium leucotomos on membrane integrity, lipid peroxidation, and expression of elastin and matrixmetalloproteinase-1 in ultraviolet radiation exposed fibroblasts, and keratinocytes. J Dermatol Sci 2003;32:1–9. 25. Philips N, Keller T, Hendrix C, Hamilton S, Arena R, Tuason M, et al. Regulation of the extracellular matrix remodeling by lutein in dermal fibroblasts, melanoma cells, and ultraviolet radiation exposed fibroblasts. Arch Dermatol Res 2007;299:373–9. 26. Philips N, Keller T, Holmes C. Reciprocal effects of ascorbate on cancer cell growth and the expression of matrix metalloproteinases and transforming growth factor-beta. Cancer Lett 2007;256:49–55. 27. Lemay, M., Murray, M. A., Davies, A., Roh-Schmidt, H., Randolph, R. (2004). In vitro and ex vivo cyclooxygenase inhibition by a hops extract. Asia Pac J Clin Nutr 13, S110. 28. Gerhauser C, Alt A, Heiss E, Gamal-Eldeen A, Klimo K, Knauft J, et al. Cancer chemopreventive activity of Xanthohumol, a natural product derived from hop. Mol Cancer Ther 2002;1:959–69. 29. Bachelor MA, Bowden GT. UVA mediated activation of signaling pathways involved in skin tumor promotion and progression. Semin Cancer Biol 2004;14:131–8. 30. Chiang HM, Chen HC, Lin TJ, Shih IC, Wen KC, Ellis LZ, et al. Michelia alba extract attenuates UVB-induced expression of matrix metalloproteinases via MAP kinase pathway in human dermal fibroblasts. Food Chem Toxicol 2012;50:4260–9. 31. George J, Singh M, Srivastava AK, Bhui K, Roy P, Chaturvedi PK. Resveratrol and black tea polyphenol combination synergistically suppress mouse skin tumors growth by inhibition of activated MAPKs and p53. PLoS One 2011;6:e23395. 32. Sun T, Chen QY, Wu LJ, Yao XM, Sun XJ. Antitumor and antimetastatic activities of grape skin polyphenols in a murine model of breast cancer. Food Chem Toxicol 2012;50:3462–7. 33. Fisher GJ, Datta SC, Talwar HS, Wang ZQ, Varani J, Kang S, et al. Molecular basis of sun-induced premature skin ageing and retinoid antagonism. Nature 1996;379:335–9. 34. Guo W, Kong E, Meydani M. Dietary polyphenols, inflammation, and cancer. Nutr Cancer 2009;61(6):807–10. 35. Albini A, Dell’Eva R, Vene R, Ferrari N, Buhler DR, Noonan DM, et al. Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF-kappaB and Akt as targets. FASEB J 2006;20:527–9. 36. Ellis LZ, Liu W, Luo Y, Okamoto M, Qu D, Dunn JH, et al. Green tea polyphenol epigallocatechin-3-gallate suppresses melanoma growth by inhibiting inflammasome and IL-1b secretion. Biochem Biophys Res Commun 2011;414:551–6. 37. Scoditti E, Calabriso N, Massaro M, Pellegrino M, Storelli C, Martines G, et al. Mediterranean diet polyphenols reduce inflammatory angiogenesis through MMP-9 and COX-2 inhibition in human vascular endothelial cells: a potentially protective mechanism in atherosclerotic vascular disease and cancer. Arch Biochem Biophys 2012;527:81–9. 38. Lamy S, Akla N, Ouanouki A, Lord-Dufour S, Beliveau R. Diet-derived polyphenols inhibit angiogenesis by modulating the interleukin-6/STAT3 pathway. Exp Cell Res 2012;318:1586–96. 39. Wondrak GT, Roberts MJ, Cervantes-Laurean D, Jacobson MK, Jacobson EL. Proteins of the extracellular matrix are sensitizers of photo-oxidative stress in human skin cells. J Invest Dermatol 2003;121:578–86. 40. Kielty CM, Sherratt MJ, Shuttleworth CA. Elastic fibres. J Cell Sci 2002;115:2817–28. 41. Watson RE, Griffiths CE, Craven NM, Shuttleworth CA, Kielty CM. Fibrillin-rich microfibrils are reduced in photoaged skin. Distribution at the dermal-epidermal junction. J Invest Dermatol 1999;112:782–7. 42. Philips N, Tuason M, Chang T, Lin Y, Tahir M, Rodriguez SG. Differential effects of ceramide on cell viability and extracellular matrix remodeling in keratinocytes and fibroblasts. Skin Pharmacol Physiol 2009;22:151–7. 43. Philips N, Keller T, Gonzalez S. TGF b like regulation of matrix metalloproteinases by anti transforming growth factor-b and anti transforming growth factor-b1 antibodies in dermal fibroblasts: implications to wound healing. Wound Repair Regen 2004;12:53–9. 44. Philips N. An anti TGF-b increased the expression of transforming growth factor-b, matrix metallproteinase-1, and elastin, and its effects were antagonized by ultraviolet radiation in epidermal keratinocytes. J Dermatol Sci 2003;33:177–9. 45. Herouy Y. Matrixmetalloproteinases in skin pathology. Int J Mol Med 2001;7:3–12. 46. Yan C, Boyd DD. Regulation of matrix metalloproteinase gene expression. J Cell Physiol 2007;211:19–26. 47. Verstappen J, Von den Hoff JW. Tissue inhibitors of metalloproteinases (TIMPs): their biological functions and involvement in oral diseases. J Dent Res 2006;85:1074–84. 48. Karroum A, Mirshahi P, Faussat A, Therwath A, Mirshahi M, Hatmi M. Tubular network formation by adriamycin-resistant MCF-7 breast cancer cells is closely linked to MMP-9 and VEGFR-2/VEGFR-3 over-expressions. Eur J Pharmacol 2012;685:1–7. 49. Waleh NS, Murphy BJ, Zaveri NT. Increase in tissue inhibitor of metalloproteinase-2 (TIMP-2). Levels and inhibition of MMP-2 activity in a metastatic breast cancer cell line by an anti-invasive small molecule SR13179. Cancer Lett 2010;289:111–8. 50. Lee MS, Jung JI, Kwon SH, Lee SM, Morita K. TIMP-2 fusion protein with human serum albumin potentiates anti-angiogenesis-mediated inhibition of tumor growth by suppressing MMP-2 expression. PLoS One 2012;7:e35710. 51. Fuxe J, Karlsson MCI. TGF-b-induced epithelial-mesenchymal transition: a link between cancer and inflammation. Semin Cancer Biol 2012;22:455–61. 52. Philips N, Arena R, Yarlagadda S. Inhibition of ultraviolet radiation mediated extracellular matrix remodeling in fibroblasts by transforming growth factor b. Bios 2009;80:1–5. 53. Philips N, Tahir M, Stellatella J, Stephan K, Givant J, Zhou L, et al. Differential regulation of growth factors and matrix metalloproteinase-1 by estrogen, progesterone, and tamoxifen in normal and cancerous endometrial cells. J Cancer Mol 2009;4:169–73.

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54. Philips N, McFadden K. Inhibition of transforming growth factor-beta and matrix metalloproteinases by estrogen and prolactin in breast cancer cells. Cancer Lett 2004;206:63–8. 55. Ali EM, Sheta M, El Mohsen MA. Elevated serum and tissue VEGF associated with poor outcome in breast cancer patients. Alex J Med 2011;47:217–24. 56. Shimizu M, Shirakami Y, Sakai H, Yasuda Y, Kubota M, Adachi S, et al. ()-Epigallocatechin gallate inhibits growth and activation of the VEGF/ VEGFR axis in human colorectal cancer cells. Chem Biol Interact 2010;185:247–52. 57. Philips N, Samuel P, Samuel M, Perez G, Khundoker R, Alahmade G. Interleukin-4 signaling pathway and effects in allergic diseases. In: Current signal transduction therapy. 13:Bentham Science Publishers; 2018. p. 1–5. 58. Philips N, Samuel M. Inhibition of interleukin-4 signalling in the treatment of atopic dermatitis and allergic asthma. Glob J Allergy 2017;3(1):019–21. 59. Geha RS, Jabara HH, Brodeur SR. The regulation of immunoglobulin E class-switch recombination. Nat Rev Immunol 2003;3:721–32. 60. Philips N, Samuel P, Lozano T, Gvaladze A, Guzman B, Siomyk H, et al. Effects of Humulus lupulus extract or its components on viability, lipid peroxidation, and expression of vascular endothelial growth factor in melanoma cells and fibroblasts. Madridge J Clin Res 2017;1:15–9. 61. Philips N, Chalensouk-Khaosaat J, Gonza´lez S. Stimulation of the fibrillar collagen and heat shock proteins by nicotinamide or its derivatives in non-irradiated or UVA radiated fibroblasts, and direct anti-oxidant activity of nicotinamide derivatives. Cosmetics 2015;2:146–61. 62. Philips N, Chalensouk-Khaosaat J, Gonza´lez S. Stimulation of the elastin and fibrillin in non-irradiated or UVA radiated fibroblasts, and direct inhibition of elastase or matrix metalloptoteinases activity by nicotinamide or its derivatives. J Cosmet Sci 2017;68:1–10. 63. Philips N, Samuel M, Parakandi H, Siomyk H, Gopal S, Jia H, et al. Vitamins in the therapy of inflammatory and oxidative disease. In: Frontiers in clinical drug research-anti allery agents. vol. 1. Bentham Science Publishers; 2013. 64. Philips N, Ding X, Kandalai P, Marte I, Krawczyk H, Richardson R. Beneficial regulation of extracellular matrix and heat shock proteins, and the inhibition of oxidative stress effects and inflammatory cytokines by 1a,25 dihydroxyvitamin D3 in non-irradiated and ultraviolet radiated dermal fibroblasts. Cosmetics 2019;6:46. 65. Philips N, Siomyk H, Jia H, Parakandi H. Inhibition of angiogenesis in cancer management by antioxidants: ascorbate and P. leucotomos. In: Anti-angiogenesis drug discovery and development. vol. 2. Bentham science; 2014. p. 132–46. 66. Philips N, Chalensouk-Khaosaat J, Devmurari A, Patel H. Polyphenolic nanobiomaterials as emerging therapies for combating physiology and clinical aspects of photoaging and photocarcinogenesis. In: Skin aging and photoaging: physiology, clinical aspects and emerging therapies. Nova Publishers Inc; 2015. ISBN 978-1-63482-907-6 Ch. 6. 67. Su-Eon J, Hyo-Eon J, Soon-Sun H. Targeted delivery system of nanobiomaterials in anticancer therapy: from cells to clinics. Biomed Res Int 2014;2014:814208 PMC. 68. Qin Y, Wang H, Karuppanapandian T, Wook K. Chitosan green tea polyphenol complex as a released control compound for wound healing. Chin J Traumatol 2010;13:91. 69. Zou LQ, Liu W, Liu WL, Liang RH, Li T, Liu CM, et al. Characterization and bioavailability of tea polyphenol nanoliposome prepared by combining an ethanol injectionmethod with dynamic high-pressure microfluidization. J Agric Food Chem 2014;62:934–41. 70. Abdolahad M, Janmaleki M, Mohajerzadeh S, Akhavan O, Abbasi S. Polyphenols attached graphene nanosheets for high efficiency NIR mediated photodestruction of cancer cells. Mater Sci Eng 2013;33:1498.

Chapter 40

Pterostilbene and cancer chemoprevention Rong-Jane Chena and Ying-Jan Wangb a

Department of Food Safety/Hygiene and Risk Management, College of Medicine, National Cheng Kung University, Tainan, Taiwan, b Department of

Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan

List of abbreviations ABAP ABTS AGE Akt AMACR AMPK AOM ASC AP-1 Atg BCG BLT2 CHOP COX-2 CYP1A1 DAMPs DMBA DPPH EGF EGFR EMT ER ERK1/2 HIF-1a HRG-b1 hTERT HUVEC IKK IL iNOS JAK lncRNAs LPS MAPK miRNAs/miR NMI MMPs mTOR NFkB NLR

2,20 -azo-bis(2-amidinopropane) 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) advanced glycation and products active human protein kinases alpha-methylacyl-CoA recemase AMP-activated protein kinase azoxymethane apoptosis-associated speck-like activator protein 1 autophagy related gene Bacille Calmette-Guerin leukotriene B4 C/EBP-homologous protein cyclooxygenase-2 cytochrome P450, family 1, subfamily A, polypeptide 1 damage-associated molecular patterns 7, 12 dimethylbenz[a] antracene 2,2-diphenyl-1-picrylhydrazyl epidermal growth factor epidermal growth factor receptor epithelial-mesenchymal transition endoplasmic reticulum extracellular signal-regulated kinase hypoxia inducible factor 1, alpha subunit heregulin-b1 human telomerase reverse transcriptase human vascular endothelial cells IkB kinase interleukin inducible nitric oxide synthase Janus-activated kinase long noncoding RNAs lipopolysaccharide mitogen-activated protein kinases microRNAs non-muscle-invasive matrix metalloproteinases the mammalian TOR nuclear factor-kB Nod-like receptor

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00040-7 © 2021 Elsevier Inc. All rights reserved.

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NO NOX Nrf2 OIS oxLDL PAH PAMPs PGs PI3K PPARa RNS ROS SA-b-gal SOD Stat TAMs TIS TNFa TPA u-PA VEGF

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nitric oxide NADPH oxidase NF-E2 related factor 2 oncogene-induced senescence oxidized low-density lipoprotein polycyclic aromatic hydrocarbons pathogen-associated molecular patterns prostaglandins phosphatidylinositol-3-kinase peroxisome proliferator-activated receptor reactive nitrogen species reactive oxygen species senescence-associated b-galactosidase superoxide dismutase enzymes signal transducer and activator of transcription tumor-associated macrophages therapy-induced senescence tumor necrosis factor alpha 12-O-tetradecanoylphorbol-13-acetate urokinase-type plasminogen activator vascular endothelial growth factor

Introduction Pterostilbene (PT) is a naturally occurring stilbenoid compound that originates from several natural plant sources. These sources include several types of grapes, peanuts, and blueberries, as well as some plants that are widely used in traditional medicine, such as Pterocarpus marsupium, Pterocarpus santalinus, and Vitis vinifera leaves, as well as the stem bark of Guibourtia tessmannii,1 a tree found in Africa that is commonly used in folk medicine.2 Pterostilbene is a dimethyl ether analog of resveratrol and has recently drawn much attention due to its health benefits as an antioxidant, anticancer, antidiabetic, and antihyperlipidemic agent.2 The anticancer effects of pterostilbene have been shown to inhibit cancer cell growth, and induce apoptosis, necrosis, autophagy, and senescence.2 In addition, pterostilbene inhibits adhesion, invasion, and metastasis of various cancer cells.2 Recently, pterostilbene has been identified as a CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1) inhibitor. Because CYP450 was thought to play a role in carcinogenesis, pterostilbene may reduce the risk of mutagenesis and cancer. Regarding antidiabetic activities, pterostilbene has been demonstrated to lower blood glucose levels and modulate PPARa (peroxisome proliferator-activated receptor a) activation signaling pathways. Other miscellaneous effects of pterostilbene include antiaging, analgesic, and neuroprotective effects. These health benefits associated with pterostilbene are mainly attributed to its antioxidant activity.1 Carcinogenesis is a pathological condition with multiple processes, starting with initiation, followed by promotion, and ultimately leading to progression.3 The administration of agents to prevent, block, or delay all stages of carcinogenesis can be defined as chemoprevention.4 Recent studies have indicated that natural stilbenes, which are a group of polyphenols characterized by the presence of a 1,2-diphenylethylene nucleus (Fig. 1), have the potential for the prevention and treatment of various diseases, including cancers.3 There are more than 400 stilbenes; among these, resveratrol is by far the most studied stilbene and has potent chemopreventive effects. Interestingly, pterostilbene, a natural analog of resveratrol, showed superior tumor inhibition activity because its bioavailability is 10 times better than resveratrol.2 Accordingly, this compound has awakened the interest of scientists for further chemopreventive studies. Pterostilbene has been reported to

FIG. 1 Structure of pterostilbene and resveratrol. Structure of (A) pterostilbene and (B) resveratrol.

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have chemopreventive effects in different types of cancers, such as bladder cancer, lung cancer, breast cancer, colon cancer, prostate cancer, or pancreatic cancer.4 The biological and specific mechanism of PT in cancer chemoprevention is still incompletely understood thus far. Therefore, this chapter will focus on the current understanding of the chemopreventive mechanisms of PT, including its molecular targets and signaling pathways in apoptosis, cell cycle arrest, autophagy, and senescence. In addition, the pharmacokinetics, bioavailability, and benefits in clinical trials will also be discussed.

Applications to other cancers or conditions Alterations in tumor metabolism could induce the production of lactate, nitric oxide (NO), ROS, and arachidonic acid metabolism products, thereby contributing to inflammatory responses.4 The inflammatory response is directly associated with pathogenesis, tumorigenesis, and regulation of the tumor microenvironment through immune cells. Considering the functions of the inflammatory microenvironment in carcinogenesis, it is expected that modulating inflammatory pathways may potentially enhance chemotherapy for various cancers. For instance, in patients with invasive bladder cancer, shortterm treatment with a COX-2 inhibitor increased apoptosis in tumor tissue. However, COX-2 inhibitors have been associated with increased cardiovascular risk.5 Alternatively, previous reports have indicated that the anticancer mechanism targeting the pathways of the inflammation mediators Stat3 and Stat5, which are constitutively over activated in various cancers.6, 7 In addition, there is evidence suggesting that ROS could induce activation of the NLRP3 inflammasome, which is related to carcinogenesis and highlights the therapeutic potential of inflammasomes in cancer therapy. This evidence addresses the role of ROS production and inflammation in tumorigenesis suggesting that they are potential chemopreventive targets in human malignancy. According to the previous studies, pterostilbene demonstrated potent chemopreventive effects in different processes of carcinogenesis (Fig. 6). In the initiation stage, pterostilbene inhibited ROS production, inflammation, and cell proliferation in chemical-induced carcinogenesis models. Autophagy induction is an important protective mechanism against tumorigenesis in the stage of initiation. Previous studies have indicated the potential autophagy-inducing effects of pterostilbene, which is a chemopreventive mechanism against various diseases, including cancers. In the promotion and progression stages of tumorigenesis, pterostilbene-induced apoptosis, necrosis, senescence, and inhibition of metastasis in numerous cancers. At the molecular level that apoptosis-related pathways such as caspase activation, inflammation-modulated signals including NFkB and Stat3, miRNAs, and lncRNAs; autophagy-mediated pathways such as LC3II, p62, and AMPK, and senescence-inducing mechanisms such as the p53; and ATM/Chk2 pathways are potential targets for pterostilbene when it exerts its chemopreventive effects (Fig. 6). Because many cancer cells are sensitive to apoptotic induction but become resistant through deregulation of apoptosis, there is growing interest in the anticancer therapy field to identify novel death mechanisms that may circumvent drug resistance. Thus, pterostilbene has become a promising chemopreventive agent for cancer therapy, including chemoresistant cells, according to its multitarget effects and safety. However, the role of ROS in pterostilbene-induced cell death mechanisms remains unclear and needs to be further investigated. It is now known that cancer is not a single disease, and the idea of a “monoshot” therapeutic strategy is misleading and often gains resistance. Pterostilbene is a natural phytochemical compound with antioxidant and anti-inflammatory effects and has been shown to modulate several cell death mechanisms in cancers through apoptosis, autophagy, senescence, and necrosis and downregulate inflammation, fibrosis, and metastasis in cancers. Beyond that, pterostilbene is also easily taken as part of a diet, and clinical trials have shown that PT is generally safe for use in humans at dosages up to 250 mg/day.2 In light of the safety profiles in humans and the beneficial effects of pterostilbene, it is suggested that pterostilbene can potentially be utilized as an effective chemopreventive agent.

Main text Pterostilbene (trans-3,5-dimethoxy-40 -hydroxystilbene; C16H16O3; MW, 256.296 g/mol) is a chemical classified as a benzylidene compound (stilbene) and is a naturally occurring phytoalexin identified in several plants of the genus Pterocarpus (of which Pterocarpus marsupium has been used for many years in the treatment of diabetes mellitus), in Vitis vinifera leaves, in some berries, and in grapes.1 Pterostilbene is a naturally occurring analog of the well-studied resveratrol, as shown in Fig. 1. Pterostilbene has similar pharmacological properties as resveratrol; however, it possesses several characteristics that are superior to those of resveratrol. Pterostilbene contains two methoxy groups and one hydroxyl group, whereas resveratrol has three hydroxyl groups. The two methoxy groups cause pterostilbene to be more lipophilic, increasing oral absorption and rendering a higher potential for cellular intake; therefore, the bioavailability of pterostilbene is increased, and it has a longer half-life (105 min) than resveratrol (14 min).4

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Pharmacokinetics of pterostilbene Oral administration of pterostilbene in male CD rats for 14 continuous days at 56 or 168 mg/kg/day showed 80% bioavailability. Pterostilbene exhibited an extensive tissue distribution with a Vss value (5.3 L/kg) that was greater than that of total body water (0.7 L/kg). The GI tract, liver, kidney, and urine processes high levels of phase II metabolites, and the GI tract may be the major site for the metabolism of pterostilbene.8 Plasma phase II metabolites are much higher than the parent compound. One study indicated that intravenous administration of pterostilbene (20 mg/kg) into rats resulted in the detection of a glucuronidated pterostilbene metabolite in both serum and urine.1 Using LC/APCI-MS/MS and LC/ESI/ MS/MS analyses, nine novel metabolites of pterostilbene from female C57BL/6 J mouse urine samples were collected 24 h after administration of 200 mg/kg pterostilbene through oral gavage.9 Pterostilbene can be metabolized in mice to generate mono-demethylated and mono-hydroxylated metabolites. Pterostilbene and its mono-demethylated and monohydroxylated metabolites are good substrates for glucuronidation and sulfation to form related phase II metabolites. Therefore, glucuronidation, sulfation, demethylation, and hydroxylation are the major biotransformation processes of pterostilbene in mice. Glucuronide and sulfate conjugates were reported to be the primary metabolites of pterostilbene, and sulfate conjugation was more extensive than glucuronidation of pterostilbene.10 It has been proposed that these conjugates could serve as storage pools for the parent drug; however, the biological activity and significance of the metabolites is unclear.10 Studies in rodents and in vitro studies with normal cells showed that pterostilbene administration is nontoxic. In mice fed trans-pterostilbene for 28 days at dosages up to 3000 mg/kg/body weight/day (500 times the estimated mean human intake), compared with the control, no significant toxic effect or adverse biochemical parameter was observed.11 In a clinical study, patients with hypercholesterolemia (cholesterol 200 mg/dL and/or baseline low-density lipoprotein cholesterol 100 mg/dL) administered pterostilbene alone (50 or 125 mg twice daily) or in combination with grape extracts (100 mg twice daily) for 6–8 weeks showed no adverse drug reaction based on biochemical analysis of hepatic, renal, or glucose markers.12 This indicated that PT is generally safe for humans at dosages up to 250 mg/day for 6–8 weeks. However, due to limited clinical studies, the safety of longer administration of pterostilbene in humans requires further investigation.

Oxidative stress and inflammation in cancer development Oxidative stress results from an imbalance between antioxidant mechanisms and the production and accumulation of ROS (reactive oxygen species) or RNS (reactive nitrogen species).13 Increased oxidative stress is not beneficial for cells; however, complete elimination of free radicals could also disrupt the normal functions of the body. ROS signaling plays an important role in biological processes, such as the regulation of the processes catalyzed by protein kinases, phosphatases, and other enzymes.14 Therefore, ROS signaling can initiate both the inhibition and activation of tumor formation. On the other hand, previous reports have indicated that almost all tumors showed an overproduction of intracellular ROS in cancer cells compared with their normal counterparts. Conversely, ROS formation stimulated by external agents and prooxidants can result in apoptosis and cancer cell death. Oxidative stress and inflammation are recognized as hallmarks of cancer. Numerous studies have provided evidence that ROS generation is thought to induce chronic inflammation leading to chemoresistance, angiogenesis, malignant transformation, and metastasis.15 Emerging evidence further indicates that the inflammasome plays a central role in the inflammation process related to various cancers.15 The inflammasome is a structure consisting of an assembly of one NOD-like receptor (NLR) protein (NLRP1, NLRP3, NLRP6, NLRC4, and NAIP5), an apoptosis-associated speck-like (ASC) adaptor protein, and caspase-1 protein. According to the two-hit model of inflammasome activation, the initial hit occurs when TLR is auto-phosphorylated by exposure to pathogen-associated molecular patterns (PAMPs) and/or damage-associated molecular patterns (DAMPs) resulting in NFkB (nuclear factor-kB) activation to promote the expression of NLRs and pro-IL-1b (interleukin-1b).16 The second hit assists the formation of inactive inflammasome complexes such as NLRP3, ASC, and caspase-1. Following activation, caspase-1 regulates the maturation of IL-1b and IL-18 or the inflammatorytriggered cell death called pyroptosis.15 Several pathways have been suggested as key mechanisms of inflammasome activation, including potassium ion efflux, calcium flux induced by stimuli such as ATP, mitochondrial dysfunction resulting in the production of ROS and NLRP3 complex formation. Then the cytosolic release of lysosomal cathepsin-B augments NLRP3 inflammasome activation.15 Increasing evidence suggests that inflammasome components, such as NLRP1, NLRP3, NLRP6, and IL-1b, have a role in tumorigenesis by modulating innate and adaptive immunity, apoptosis, differentiation, and gut microbiota.15 Conversely, it has also been suggested that inflammasome activation contributes to the inhibition of tumorigenesis specifically, depending on the tissue context. Therefore, the compounds that modulate the

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inflammatory process may be useful for chemoprevention and determining the potential therapeutic role of the modulation of inflammasome activities in tumorigenesis requires further investigation. The link between inflammation and cancer has two paradigms. First, inflammation-driven carcinogenesis through extrinsic pathways includes ROS that damage DNA and impair the DNA repair system, thereby initiating carcinogenesis. Second, excessive expression of pro-inflammatory mediators or chemokines such as IL-1b, IL-18, VEGF, FGF2, and Stat3 could perturb cell signaling pathways, thereby promoting cell growth, angiogenesis, and metastasis, as well as reducing susceptibility to the host immune response.17 With regard to pro-tumorigenic properties, inflammation-induced activation of various protein kinases, including JAK (Janus-activated kinase), Akt (active human protein kinases), and MAPK (mitogen-activated protein kinases), and induced aberrant activation of transcription factors, such as Stat (signal transducer and activator of transcription), NFkB, AP-1 (activator protein 1), and HIF-1a (hypoxia-inducible factor 1, alpha subunit), and NLRP3 inflammasomes have become potential targets for chemoprevention research.

Antioxidant and anti-inflammatory effects of pterostilbene The antioxidative effects of pterostilbene (with one hydroxyl group and two methoxy groups) were attributed to its unique structure that reduced extracellular ROS, whereas resveratrol (with three hydroxyl groups) neutralized ROS in whole blood and isolated lymphoblasts.18 This effect allowed the use of pterostilbene to target extracellular ROS species that are responsible for tissue damage during chronic inflammation.4 The free radical-scavenging property of pterostilbene has been investigated, and it was shown that pterostilbene’s peroxyl radical-scavenging activity appeared to be similar to that of resveratrol. The ability of pterostilbene to inhibit the oxidation of ABTS [2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] indicated that pterostilbene scavenged DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and ABAP [2,20 -azo-bis(2amidinopropane)]-derived peroxy radicals.1 The antioxidant ability of pterostilbene is most important because of the many health benefits of pterostilbene. For instance, pterostilbene has been reported to prevent oxLDL (oxidized low-density lipoprotein)-induced ROS, NFkB activation, p53 accumulation, apoptosis, and decreased mitochondrial membrane potential, thereby preventing apoptosis in HUVECs (human vascular endothelial cells).19 The antioxidative effects of the combination of pterostilbene and resveratrol were investigated in human erythrocytes in vitro. The results indicated that pterostilbene alone can protect erythrocyte membranes from lipid peroxidation and, when combined with resveratrol at a lower concentration, induce a synergistic protective effect against lipid peroxidation.20 The protective effect can explain, at least in part, the healthy benefits of these bioactive compounds when combined in the diet. In an in vivo cell model, pterostilbene has been reported to ameliorate the immediate inflammatory responses in TNFa (tumor necrosis factor alpha)-induced pancreatitis through downregulation of Stat3 and the secretion of lipase and the inflammatory cytokines IL-1b and IL-6.21 In another cell model, pterostilbene downregulated inflammatory iNOS and COX-2 gene expression in macrophages by inhibiting the activation of NFkB by interfering with the activation of PI3K/Akt/IKK (IkB kinase) and MAPK. Another study further indicated that pterostilbene can suppress the expression of several genes and their inflammatory products in LPS (lipopolysaccharide)-stimulated RAW264.7 macrophages and peritoneal macrophages from mice. The effect could be partly due to the diminution of NFkB activation by the proteasome, thereby suppressing activation of iNOS genes, and decreased the secretion of TNFa, IL-1b, IL-6, and NO levels. It has been suggested that pterostilbene may act as a proteasome inhibitor, thus indicating one of the mechanisms for its anti-inflammatory effects. Notably, pterostilbene was reported to inactivate the NLRP3/caspase-1 inflammasome in several models, including allergic contact dermatitis,22 amyloid-b-induced neuroinflammation,23 fructoseinduced podocyte oxidative stress and diabetes.24, 25 These reports have important implications for using pterostilbene in the development of an effective anti-inflammatory agent. The antioxidant and anti-inflammatory responses regulated by pterostilbene are shown in Fig. 2.

Chemopreventive mechanisms of pterostilbene in preclinical studies The chemopreventive effects of pterostilbene have been investigated in several models, including animal models and in vitro cancer cell lines, including bladder, breast, colon, liver, and gastric cancer cells and melanoma cell lines.21 Possible chemopreventive mechanisms of pterostilbene include cell cycle arrest, apoptosis, necrosis, autophagy, and senescence.21 Pterostilbene possesses potent antioxidant (low does) and prooxidant (high dose) properties in treated cells. The prooxidant effects of high-dose pterostilbene may induce cancer cell death through ROS generation. However, the mechanistic details underlying the protective effects of pterostilbene against cancer cells are still unclear and are worthy of further exploration. The current proposed anticancer mechanisms of pterostilbene are discussed in the following section.

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ROS Inflammation iNOS, COX-2 TNF-a, IL-6 NFkB, Stat3 MAPK, PI3K/Akt NLRP3 inflammasome

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Cancer Lipid peroxidation Pancreatitis Inflammatory-related diseases

Oxidative nitrosative stress FIG. 2 The antioxidant and anti-inflammatory responses regulated by pterostilbene. Pterostilbene scavenges extracellular ROS and eliminates intracellular oxidative/nitrosative stress. Pterostilbene inhibits the production of inflammatory cytokines TNFa and IL-6 and inhibits the expression of iNOS, COX-2, NFkB, Stat3, MAPK, PI3K/Akt, and NLRP3 inflammasome signaling pathways; thereby reducing the risk of inflammatory-related diseases such as cancer, lipid peroxidation, and pancreatitis.

Inhibiting inflammatory responses Numerous studies have been conducted to evaluate the cancer chemopreventive potential of pterostilbene by modulating inflammatory responses in some animal models. Using a mouse mammary organ culture model, carcinogen-induced preneoplastic lesions were significantly inhibited by pterostilbene. This may be partially due to the antioxidant activity of pterostilbene, which effectively scavenges peroxyl radicals and reduces singlet-oxygen-induced peroxidation.26 In the skin carcinogenesis model, pterostilbene significantly inhibited 7,12-dimethylbenz[a] anthracene (DMBA)/TPA (12-O-tetradecanoylphorbol-13-acetate)-induced skin tumor formation by suppressing TPA-induced activation of MAPK, PI3K/Akt, and AP-1, thereby downregulating iNOS and COX-2 expression.27 In colon cancer, pterostilbene was reported to be more potent than resveratrol in preventing azoxymethane (AOM)-induced colon tumorigenesis, and pterostilbene was shown to suppress colon tumorigenesis and cell proliferation through reduced the gene expression of Myc, cyclin D, NFkB p65 subunit, iNOS, COX-2, VEGF (vascular endothelial growth factor), PI3K/Akt and MMPs (matrix metalloproteinases), and downregulated the EGFR (epidermal growth factor receptor) signaling. Pterostilbene was also shown to induce apoptosis and Nrf2 (NF-E2 related factor 2)-mediated antioxidant signaling pathways in the mouse colon.28, 29 Pterostilbene reduced the colon tumor multiplicity of noninvasive adenocarcinomas, lowered proliferating cell nuclear antigen, and downregulated the expression of b-catenin and cyclin D1. Colon tumors from pterostilbene-fed animals showed reduced the expression of inflammatory markers, including TNFa, IL-1b, and IL-4, as well as nuclear staining for phospho-p65. In the in vitro models, pterostilbene reduced the activation of NFkB, JAK, MAPK, and the PI3K protein kinase cascade in HT29 cells, which seems to be a key pathway for eliciting the anti-inflammatory action of pterostilbene.30 Pterostilbene also inhibits urethan-induced lung carcinogenesis through the downregulation of NFkB, Stat3, ERK1/2, Akt/mTOR, and cell cycle pathways.4 Moreover, tumor-associated macrophages (TAMs) polarized to the M2 phenotype play key roles in tumor progression including lung cancer, through elevated MUC1 protein. Intriguingly, pterostilbene suppressed the self-renewal ability of M2-TAMs by downregulation MUC1, NFkB, CD133, b-catenin, and Sox2 expression.31 Pterostilbene also increased the expression of catalase, heme oxygenase1, glutathione peroxidase, and superoxide dismutase through the Nrf2 pathway and alleviated hydrogen peroxide-induced oxidative stress in the development of mouse embryos.32 These beneficial effects of pterostilbene make it a potential chemopreventive agent for inflammation-associated tumorigenesis. The chemopreventive effects of pterostilbene against chemical-induced carcinogenesis through inhibiting inflammation are shown in Fig. 3.

Inducing apoptosis in cancer cells It has been indicated that ROS formation stimulated by external agents and prooxidants can result in apoptosis and cancer cell death. Therefore, the anticancer effects of pterostilbene were suggested to be correlated with its prooxidant properties. Pterostilbene was found to induce apoptosis in bladder, breast, colon, gastric, liver, lung, pancreatic, and prostate cancers as well as leukemia and melanoma. Both intrinsic and extrinsic apoptosis pathways were identified in pterostilbene-induced apoptotic cell death. Pterostilbene increased ROS generation, which induces altered mitochondrial transmembrane potential, causing the release of cytochrome-c, followed by activation of the caspase cascade-triggered mitochondriadependent intrinsic pathways.4 Alternately, pterostilbene may act directly on the mitochondrial membrane. In several

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Peroxy radicals COX-1, COX-2 PGE2

MAPK (ERK1/2, p38, JNK) PI3K/Akt, NFkB, AP-1 iNOS, COX-2

TNFa, IL-4, IL-1b NFkB, c-Myc, Nrf2 iNOS, COX-2 Cyclin D1, b-catenin MMPs, EGFR, VEGF

EGFR, cell cycle, proliferation, Akt/mTOR, ERK1/2 NFkB, Stat3

DMBA induced breast cancer

(DMBA)/TPA induced skin cancer

AOM-induced colon cancer

Urethane-induced lung cancer

FIG. 3 The chemopreventive effects of pterostilbene against chemical-induced carcinogenesis. Pterostilbene inhibits carcinogenesis in DMBA induced breast and skin cancer, AOM-induced colon cancer, and urethane-induced lung cancer models through modulation of multiple pathways. Pterostilbene significantly inhibits inflammatory pathways including NFkB, iNOS, COX-2, and Stat3 pathways. In addition, Akt/mTOR, ERK1/2, and cell cycle regulatory pathways were inhibited by pterostilbene, leading to the induction of apoptosis and autophagy in cancer cells.

cancer cell lines, the downregulation of PI3K/Akt/mTOR, phospho-Stat3, ERK1/2, Bcl-2, c-FLIPS/L, Bcl-xL, survivin, and XIAP and the activation of p53, JNK, AMPK (AMP-activated protein kinase), Bax, Bid, Bad, cytochrome c, Smac/Diablo, and caspases as well as cytosolic Ca2+ overload, were involved in the pterostilbene-induced intrinsic apoptotic pathway. Pterostilbene also induced extrinsic death pathways in leukemia and gastric cancer cells through Fas, ROS, endoplasmic reticulum (ER) stress, ERK 1/2, and p38/C/EBP-homologous protein (CHOP) signaling pathways, leading to the expression of DR4 and DR5.33, 34 The apoptosis effects of pterostilbene also involved ER stress. In esophageal cancer cells, pterostilbene treatment-induced apoptosis via increased expression of ER stress-related molecules including GRP78, ATF6, p-PERK, p-eIF2a, and CHOP.35 In general, one of the important anticancer mechanisms of pterostilbene is apoptosis induction in various cancer cells. The apoptotic mechanisms regulated by pterostilbene are shown in Fig. 4.

Pterostilbene induces autophagy in cancer cells Autophagy (or self-eating) was first described by Christian de Duve in 1963 as a lysosome-mediated degradation process for nonessential or damaged cellular components.36 Autophagy is a degradation process involving sequestration of parts of

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Bcl-2

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Bax

Cytochrome c Caspases Smac/Diablo

GRP78, ATF6, p-PERK, CHOP p-eIF2a

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Caspase 8

Extrinsic pathways

FIG. 4 Apoptosis-inducing effects of pterostilbene. Apoptosis induction by pterostilbene occurs through the activation of mitochondria-dependent intrinsic pathways, including ROS generation, loss of mitochondria membrane potential, released cytochrome c and Smac/Diablo, and activation of caspases. Pterostilbene induces activation of Fas-mediated extrinsic pathways. Pterostilbene inhibits the activation of Akt and Stat3, which are correlated with apoptosis inhibition.

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the cytoplasm in double-membrane vesicles (autophagosomes) that fuse with lysosomes, forming autolysosomes where cytoplasmic material is hydrolyzed, and the resulting amino acids and other macromolecular precursors can be recycled.16 Autophagy is ubiquitous in eukaryotic cells and plays an important role in the development and diverse pathophysiological conditions, including providing protection against neurodegeneration, infections, and tumor development.16 The center of autophagy activation is the formation of the autophagosome by a series of steps, including phagophore elongation and maturation. These processes are controlled by a set of proteins called Atg (autophagy-related) proteins and require two ubiquitin-like conjugation systems: Atg5-Atg12 conjugate and Atg8 (a homolog of human LC3) lipidation and the type III PI3K-Beclin1 complex. The type III PI3K-Beclin 1 complex is negatively regulated by interactions with Bcl-2 family proteins. Signaling pathways that regulate autophagy include the PI3K/Akt/mTOR and MEK/ERK1/2 signaling pathways.16 ERK1/2 phosphorylates the Ga-interacting protein, which accelerates GTP hydrolysis by the Gai3 protein, resulting in the induction of autophagy.37 In addition, AMPK activation then inhibits mTOR, thereby activating autophagy.16 It has also been suggested that autophagy may provide a useful way to prevent cancer development, limit tumor progression, and enhance the efficacy of cancer treatments. Importantly, recent studies have also indicated that autophagy acts as a negative regulator of inflammasome activation through the deregulation of NLRP3 or pro-IL-1b, thereby restraining the inflammation process.16 Therefore, extensive attention has been paid to the role of autophagy in cancer development and therapy. Studies regarding pterostilbene-induced autophagy in cancer cells remain limited. Pterostilbene can induce autophagy at a low concentration in vascular endothelial cells via a rapid elevation in intracellular Ca2+ concentration and subsequent AMPK activation, which in turn inhibits mTOR. This cytoprotective autophagy helped to remove accumulated toxic oxLDLs and inhibit apoptosis in HUVECs.19 Therefore, it has been proposed that pterostilbene could be a potential lead compound for developing a class of autophagy regulators for the treatment of autophagy-related diseases. Accordingly, the pterostilbene-mediated chemopreventive effects were a result of the inhibition of EGFR and its downstream pathways, leading to slowed cell cycle progression, and of the induction of apoptosis and autophagy during urethane-induced lung tumorigenesis.38 Another study conducted by Chen et al. indicated that pterostilbene effectively inhibits the growth of both sensitive and chemoresistant bladder cancer cells by inducing apoptosis, necrosis, autophagy, and cell cycle arrest.6 The author further indicated that pterostilbene-induced autophagy at an earlier stage and apoptosis occurred at a later stage. Pterostilbene inhibited the Akt/mTOR/P70S6K pathway and activated the ERK1/2 pathway, suggesting that these changes mediate pterostilbene-induced autophagy. However, pterostilbene-induced autophagy alone was not sufficient to induce cell death. After a latency period, pterostilbene induced cell death via apoptosis. In addition, inhibition of autophagy by inhibitors may increase the sensitivity of cells to death signals. Consistent with this study, pterostilbene was reported to induce protective autophagy in breast cancer cells, multiple myeloma cells, acute lymphoblastic leukemia cells and suggesting that the combination of autophagy inhibitors with pterostilbene could serve as a new and promising strategy for the treatment of cancers.4 In contrast, pterostilbene and its derivative ANK-199 were reported to induce autophagic cell death in oral cancer cells.4 All of these studies have strongly indicated the autophagy-inducing effects of pterostilbene. Accordingly, pterostilbene could exert its chemopreventive effects in various diseases through autophagy induction. Notably, pterostilbene could induce protective or pro-death autophagy in cancer cells dependent on cell type or treatment conditions (Fig. 5). The exact mechanisms require further investigation. Therefore, the preventive strategy should be carefully formulated to enhance the anticancer effects of pterostilbene when different types of autophagy could be induced by pterostilbene in cancer cells.

Inhibits inflammation Pterostilbene

AMPK ERK1/2

Autophagy mTOR P70S6K

Atg5, LC3II p62

Prevents autophagydefective diseases: neurodegeneration Pro-death in oral cancer cells Pro-survival in cancer cells: combined with autophagy inhibitor to enhance chemotherapeutic effects

FIG. 5 Chemopreventive effects of pterostilbene by inducing autophagy. Pterostilbene significantly induces the activation of ERK1/2 and AMPK pathways resulted in autophagy induction in cells. Autophagy induced by pterostilbene is an important regulator of the downregulation of inflammasome activation and prevents autophagy-defective diseases. With regard to cancer therapy, pterostilbene induces autophagic cell death in oral cancer cells. In contrast, pterostilbene induces pro-survival autophagy thereby combination with autophagy inhibitor could enhance chemosensitivity of pterostilbene.

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Pterostilbene induces cell cycle arrest in cancer cells Pterostilbene can induce both cytotoxic and cytostatic mechanisms in many cancer cells. Pterostilbene was reported to block cell cycle progression at the G0/G1 phase in AGS gastric cancer, prostate cancer, pancreatic cancer, and breast cancer cells with similar mechanisms.39 In AGS cancer cells, pterostilbene decreased the expression of cell cycle regulatory proteins, such as pRb, cyclin A and cyclin E, and upregulated the expression of p53, p21, p27, and p16.40 Similar results were observed in p53 wild-type LNCaP prostate cancer cells in which pterostilbene-induced G0/G1 arrest by inducing p53 expression, AMPK activation, and further upregulating p21 expression.41 Additionally, pterostilbene inhibits androgenand estrogen-mediated pathways and therefore may contribute to its anti-prostate cancer efficacy.42 In breast cancer cells, pterostilbene induced apoptosis, cell cycle arrest through the downregulation of cyclin D1, and Wnt signaling.39 Interestingly, treatment with pterostilbene at a lower dose induced S phase arrest in MCF7 breast cancer and MIA PaCa-2 pancreatic cancer cells, whereas a higher dose of pterostilbene induced the accumulation of cells in the G0/G1 phase. In addition, pterostilbene caused an increase in S phase cells in HL60 myeloid leukemia cells but had no significant effect on the cell cycle distribution of K562, another leukemia cell line.34 In HeLa cells, pterostilbene downregulates the HPV oncoprotein E6, induces caspase-3 activation, upregulates p53 protein levels, and induces S phase arrest. Pterostilbene treatment also altered bladder cancer cell cycle distribution by increasing the G0/G1 phase and decreasing the G2/M phase. S phase arrest occurs at lower concentrations of pterostilbene, whereas higher doses of pterostilbene-induced G0/G1 arrest, followed by a shift to S phase arrest at 48 and 72 h. The G0/G1 arrest may be associated with the time-dependent decrease in cell cycle regulatory cyclin proteins and phosphorylation of Rb.6 Therefore, pterostilbene-induced cell cycle arrest likely contributed to overall growth suppression in cancer cells. Interestingly, pterostilbene treatment-induced S phase arrest was the dominant effect in various cancer cells, suggesting that the specific mechanisms regarding DNA synthesis could be altered by pterostilbene.43 The effects of pterostilbene on cell cycle distribution depend on different cancer types and treatment doses. The overall chemopreventive effects of pterostilbene is shown in Fig. 6.

Pterostilbene induces senescence in cancer cells Telomerase inhibition and senescence induction in lung cancer cells are novel anticancer mechanisms of pterostilbene.44 Telomerase is a ribonucleoprotein complex consisting of two subunits, human telomerase reverse transcriptase (hTERT) and the small nuclear human telomerase RNA component. hTERT is the major catalytic component of telomerase that provides stability and the compensating telomere (tandem repetitions of hexanucleotide TTAGGG) sequence by using an RNA template.2 In normal cells, telomeres progressively shorten with advanced aging then trigger DNA damage signaling leading to replicative senescence, inhibition of proliferation, or cell death. This is a protective mechanism in normal cells to prevent further proliferation of cells that may harbor genetic alterations.2 However, more than 80% of human tumors or tumor-derived cancer cells overexpress telomerase or activate hTERT.2 Telomerase activity is also critical for cancer cell development. Therefore, the development of telomerase inhibitors as anticancer agents could be a reasonable and feasible chemopreventive strategy. In Chen’s study, pterostilbene was reported to inhibit telomerase activity through the downregulation of hTERT expression preferentially in p53-positive lung cancer cells. After inhibition of hTERT activity and expression, pterostilbene induced the DNA damage response and subsequent ATM/Chk2/p53 activation, leading to permanent S phase arrest and senescence.44 Using molecular docking studies, Tippani et al. first confirmed the interaction between PT and the active site of telomerase.45 Similarly, Daniel et al. indicated that pterostilbene-induced Chemopreventive effects of pterostilbene in carcinogenesis

Initiation

Anti-oxidation Inhibits inflammation Inflammasome inactivation Apoptosis Autophagy Inhibits proliferation

Promotion

Induces ROS Suppresses M2-TAM Apoptosis Autophagy Inhibits proliferation Cell cycle arrest

Progression

Senescence Telomerase inhibition Inhibits invasion and metastasis Regulation of miRNA/lncRNAs

FIG. 6 Chemopreventive effects of pterostilbene in carcinogenesis. In the initiation stage, pterostilbene inhibits ROS production, inflammation, and cell proliferation in chemical-induced carcinogenesis models. Apoptosis and autophagy induction are important protective mechanisms against tumorigenesis in the stage of initiation. In the promotion and progression stages, pterostilbene induces apoptosis, necrosis, cell cycle arrest, senescence, and inhibition of telomerase activity, and suppresses metastasis in cancers. Therefore, pterostilbene is an attractive and safe natural product for chemoprevention.

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cell cycle arrest and reduced hTERT expression in MCF-7 and MDA-MB 231 cells.2 Therefore, previous studies indicated that pterostilbene inhibited hTERT expression and activity in cancer cells, leading to cell cycle arrest and senescence. In addition to telomere shortening, cancer senescence can be triggered by various other stimuli, including ROS production, DNA damage response, nutrient depletion, oncogene activation, which is called oncogene-induced senescence (OIS), and anticancer therapy, such as chemotherapeutic agents or irradiation-induced senescence called therapy-induced senescence (TIS).2 The characteristics of senescence are flat and enlarged cell shape, positive staining of senescenceassociated b-galactosidase (SA-b-gal) activity, accumulation of lysosomes, and chromatin remodeling accompanied by the formation of senescence-associated heterochromatin foci and irreversible cell cycle arrest.44 The major pathways controlling senescence include the activation of the p53/p21, p16/RB, ATM/Chk2, and ATR/Chk1 pathways.2 Compared to cytotoxic anticancer agents, agents that promote senescence can achieve the anticancer effects at far lower doses, thereby reducing the side effects of anticancer therapy. In addition, senescent cells could be eliminated by immune cells by positively or negatively regulating the immune response by secreting cytokines, chemokines, extracellular matrix proteases, and growth factors, which are termed the senescence-associated secretory phenotype.2 Therefore, growing evidence has suggested that TIS could be a promising and safe strategy to induce cytostasis in cancer treatment.2 In this manner, several phytochemicals, including pterostilbene, were reported to induce senescence in cancer cells. Chen et al. indicated that pterostilbene-induced senescence partially occurred via a p53-dependent mechanism, triggering inhibition of telomerase activity, and ATM/Chk2 activation in lung cancer cells. In addition, the activation of the ATM/Chk2/p53 pathway may act as feedback regulation to inhibit hTERT expression, increasing DNA damage, and senescence in lung cancer cells.44 This suggests that pterostilbene has the potential to induce senescence at lower concentrations in cancer cells through various pathways including telomerase inhibition, DNA damage response, ATM/Chk2/p53 activation, and replicative stress in cancer cells.

Pterostilbene inhibits invasion and metastasis in cancer cells Pterostilbene inhibits cancer growth by inducing apoptosis and cell cycle arrest and also inhibits invasion and metastasis. Pterostilbene regulates multiple signaling pathways that control metastasis and invasion, as demonstrated by studies conducted in breast cancer, colon cancer, liver cancer, and melanoma.4 In breast cancer, the dominant active form of b-catenin could reverse the inhibitory effects of pterostilbene on growth, indicating that Wnt signaling is important for the growth inhibition by pterostilbene.39 Previous studies also suggested that pterostilbene protects against TPA-mediated metastasis of liver cancer via downregulation of the EGF, VEGF, EGFR, PKC, PI3K, MAPK, AP-1, and NFkB pathways, followed by the suppression of MMP-9 expression.46 Pterostilbene could suppress HRG-b1 (heregulin-b1)-mediated cell invasion, motility, and cell transformation of MCF-7 human breast carcinoma through the downregulation of MMP-9 activity and growth inhibition. Pterostilbene was also shown to downregulate p38, the downstream target for HRG-b1.47 In prostate cancer, pterostilbene inhibited metastatic inducers MMP-9, AMACR (alpha-methylacyl-CoA racemase), and metastasisassociated protein 1 (MTA1), suggesting that pterostilbene could be effective in the treatment of prostate cancer.48, 49 Similar results were observed in hepatocellular carcinoma in which pterostilbene exerted its anticancer and anti-metastatic effects by regulating the levels of the MTA1/HDAC1/NuRD complex, promoting PTEN acetylation and apoptosis.50 In a lung cancer metastasis model, pterostilbene significantly prevented lung colonization and metastasis of LLC cells and reduced established tumor growth in mouse lungs through AKT activation, ERK inactivation, and suppressed polyfibronectin assembly in lung cancer cells.51 The downregulation of urokinase-type plasminogen activator (u-PA), NFkB, SP-1, and CREB signaling pathways also resulted in metastasis inhibition in oral cancer and breast cancer cells after treatment with pterostilbene.52, 53 Interestingly, a previous study indicated that pterostilbene could penetrate the bloodbrain barrier and suppress breast cancer metastasis to the brain by targeting c-Met signaling.54 As such, these results reveal that pterostilbene is a novel, effective, anti-metastatic agent that functions by downregulating MMP-9 gene expression, fibronectin assembly, and u-PA and MTA-regulated metastatic signaling pathways.

Chemopreventive effects of pterostilbene by regulation of microRNAs MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression in cells by affecting the stability and transcriptional activities of their target mRNA by binding to the 30 -untranslated regions of the mRNA.55 miRNAs have potential roles in regulating cancer development or inhibition. For instance, miR-19a forms part of the miR-17-92 cluster and is overexpressed in various cancers.56 Therefore, the regulation of miRNAs could be a potential strategy for the development of anticancer agents. In hepatocellular carcinoma, pterostilbene treatment downregulated miR-19a and induced PTEN/Akt pathway regulation, leading to proliferation inhibition, S phase arrest, apoptosis induction, and reduced cell invasion.56

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Similarly, pterostilbene downregulated miR-17-5p and miR-106a-5p both in tumors and in systemic circulation and then rescued PTEN mRNA and protein levels, leading to reduced prostate cancer growth in vivo.57 In breast cancer cells, pterostilbene suppressed tumor growth and metastasis by reducing Src/FAK signaling, which is negatively correlated with miR-205.58 Similarly, pterostilbene suppressed glioma stem cells by negatively modulating GRP78 signaling with an increase in miR-205.59 Moreover, pterostilbene suppressed the generation of cancer stem cells and metastatic potential under the influence of M2 TAMs via modulating epithelial-mesenchymal transition (EMT)-associated signaling pathways, specifically the NFkB/miR-488 circuit.60 Previous studies also indicated that pterostilbene regulated long noncoding RNAs (lncRNAs) in MCF7 cells. Pterostilbene increased the expression of the lncRNAs MEG3, TUG1, H19, and DICER1-AS1 but reduced the expression of LINC01121, PTTG3P, and HOTAIR. Furthermore, apoptosis induction, ER stress, autophagy, and reduced EMT and proliferation occurred after pterostilbene treatment.4 All these studies indicated that pterostilbene regulates the expression of miRNAs and/or lncRNAs in cancer cells that are largely correlated with its anticancer and anti-inflammatory effects. Therefore, the regulation of miRNAs/lncRNAs could be a novel chemopreventive mechanism of pterostilbene and the intensive mechanisms warrant further investigation.

Summary points l

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Pterostilbene is a naturally occurring stilbenoid compound that originates from several natural plant sources. These sources include grapes, peanuts, and blueberries as well as some plants widely used in traditional medicine, such as Pterocarpus marsupium, Pterocarpus santalinus, Vitis vinifera leaves, and the stem bark of Guibourtia tessmannii. Pterostilbene is a dimethyl ether analog of resveratrol and has many health benefits such as antioxidant, anticancer, antidiabetic, and antihyperlipidemic effects. The antioxidant effects make it an effective chemopreventive agent for many cancers. At the molecular level, pterostilbene modulates various signaling pathways, including NFkB/Stat3, the NLRP3 inflammasome, AMPK, PI3K/AKT, p53/ATM/Chk2, telomerase activity leading to cell death, downregulation of inflammation and inhibition of metastasis in cancer cells. This chapter discusses the chemopreventive effects and mechanisms of pterostilbene through necrosis, apoptosis, autophagy, cell cycle arrest, senescence, inhibition of telomerase activity, invasion, and metastasis, and modulation of the expression of miRNAs, making it a potential agent for the treatment of different types of cancers.

References 1. Remsberg CM, Yanez JA, Ohgami Y, Vega-Villa KR, Rimando AM, Davies NM. Pharmacometrics of pterostilbene: preclinical pharmacokinetics and metabolism, anticancer, antiinflammatory, antioxidant and analgesic activity. Phytother Res 2008;22(2):169–79. 2. Lee YH, Chen YY, Yeh YL, Wang YJ, Chen RJ. Stilbene compounds inhibit tumor growth by the induction of cellular senescence and the inhibition of telomerase activity. Int J Mol Sci 2019;20(11):E2716. 3. Sirerol JA, Rodriguez ML, Mena S, Asensi MA, Estrela JM, Ortega AL. Role of natural stilbenes in the prevention of cancer. Oxidative Med Cell Longev 2016;2016:3128951. 4. Chen RJ, Kuo HC, Cheng LH, Lee YH, Chang WT, Wang BJ, et al. Apoptotic and nonapoptotic activities of pterostilbene against cancer. Int J Mol Sci 2018;19(1):E287. 5. Zhu Z, Shen Z, Xu C. Inflammatory pathways as promising targets to increase chemotherapy response in bladder cancer. Mediat Inflamm 2012;2012:528690. 6. Chen RJ, Ho CT, Wang YJ. Pterostilbene induces autophagy and apoptosis in sensitive and chemoresistant human bladder cancer cells. Mol Nutr Food Res 2010;54(12):1819–32. 7. Loh CY, Arya A, Naema AF, Wong WF, Sethi G, Looi CY. Signal transducer and activator of transcription (STATs) proteins in cancer and inflammation: functions and therapeutic implication. Front Oncol 2019;9:48. 8. Kuhnle G, Spencer JP, Chowrimootoo G, Schroeter H, Debnam ES, Srai SK, et al. Resveratrol is absorbed in the small intestine as resveratrol glucuronide. Biochem Biophys Res Commun 2000;272(1):212–7. 9. Shao X, Chen X, Badmaev V, Ho CT, Sang S. Structural identification of mouse urinary metabolites of pterostilbene using liquid chromatography/ tandem mass spectrometry. Rapid Commun Mass Spectrom 2010;24(12):1770–8. 10. Kapetanovic IM, Muzzio M, Huang Z, Thompson TN, McCormick DL. Pharmacokinetics, oral bioavailability, and metabolic profile of resveratrol and its dimethylether analog, pterostilbene, in rats. Cancer Chemother Pharmacol 2011;68(3):593–601. 11. Ruiz MJ, Fernandez M, Pico Y, Manes J, Asensi M, Carda C, et al. Dietary administration of high doses of pterostilbene and quercetin to mice is not toxic. J Agric Food Chem 2009;57(8):3180–6. 12. Riche DM, McEwen CL, Riche KD, Sherman JJ, Wofford MR, Deschamp D, et al. Analysis of safety from a human clinical trial with pterostilbene. J Toxicol 2013;2013:463595. 13. Da Costa LA, Badawi A, El-Sohemy A. Nutrigenetics and modulation of oxidative stress. Ann Nutr Metab 2012;60(Suppl. 3):27–36.

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14. Afanas’ev I. Reactive oxygen species signaling in cancer: comparison with aging. Aging Dis 2011;2(3):219–30. 15. Zhiyu W, Wang N, Wang Q, Peng C, Zhang J, Liu P, et al. The inflammasome: an emerging therapeutic oncotarget for cancer prevention. Oncotarget 2016;7(31):50766–80. 16. Chen RJ, Lee YH, Yeh YL, Wu WS, Ho CT, Li CY, et al. Autophagy-inducing effect of pterostilbene: a prospective therapeutic/preventive option for skin diseases. J Food Drug Anal 2017;25(1):125–33. 17. Kundu JK, Surh YJ. Emerging avenues linking inflammation and cancer. Free Radic Biol Med 2012;52(9):2013–37. 18. Perecko T, Jancinova V, Drabikova K, Nosal R, Harmatha J. Structure-efficiency relationship in derivatives of stilbene. Comparison of resveratrol, pinosylvin and pterostilbene. Neuro Endocrinol Lett 2008;29(5):802–5. 19. Zhang L, Zhou G, Song W, Tan X, Guo Y, Zhou B, et al. Pterostilbene protects vascular endothelial cells against oxidized low-density lipoproteininduced apoptosis in vitro and in vivo. Apoptosis 2012;17(1):25–36. 20. Mikstacka R, Rimando AM, Ignatowicz E. Antioxidant effect of trans-resveratrol, pterostilbene, quercetin and their combinations in human erythrocytes in vitro. Plant Foods Hum Nutr 2010;65(1):57–63. 21. McCormack D, McDonald D, McFadden D. Pterostilbene ameliorates tumor necrosis factor alpha-induced pancreatitis in vitro. J Surg Res 2012;178(1): 28–32. 22. Wang BJ, Chiu HW, Lee YL, Li CY, Wang YJ, Lee YH. Pterostilbene attenuates hexavalent chromium-induced allergic contact dermatitis by preventing cell apoptosis and inhibiting IL-1beta-related NLRP3 inflammasome activation. J Clin Med 2018;7(12):E489. 23. Li Q, Chen L, Liu X, Cao Y, Bai Y, Qi F. Pterostilbene inhibits amyloid-beta-induced neuroinflammation in a microglia cell line by inactivating the NLRP3/caspase-1 inflammasome pathway. J Cell Biochem 2018;119(8):7053–62. 24. Kosuru R, Kandula V, Rai U, Prakash S, Xia Z, Singh S. Pterostilbene decreases cardiac oxidative stress and inflammation via activation of AMPK/ Nrf2/HO-1 pathway in fructose-fed diabetic rats. Cardiovasc Drugs Ther 2018;32(2):147–63. 25. Wang W, Ding XQ, Gu TT, Song L, Li JM, Xue QC, et al. (2015). Pterostilbene and allopurinol reduce fructose-induced podocyte oxidative stress and inflammation via microRNA-377. Free Radic Biol Med 2015;83:214–26. 26. Rimando AM, Cuendet M, Desmarchelier C, Mehta RG, Pezzuto JM, Duke SO. Cancer chemopreventive and antioxidant activities of pterostilbene, a naturally occurring analogue of resveratrol. J Agric Food Chem 2002;50(12):3453–7. 27. Tsai ML, Lai CS, Chang YH, Chen WJ, Ho CT, Pan MH. Pterostilbene, a natural analogue of resveratrol, potently inhibits 7,12-dimethylbenz[a] anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin carcinogenesis. Food Funct 2012;3(11):1185–94. 28. Chiou YS, Tsai ML, Nagabhushanam K, Wang YJ, Wu CH, Ho CT, et al. Pterostilbene is more potent than resveratrol in preventing azoxymethane (AOM)-induced colon tumorigenesis via activation of the NF-E2-related factor 2 (Nrf2)-mediated antioxidant signaling pathway. J Agric Food Chem 2011;59(6):2725–33. 29. Chiou YS, Tsai ML, Wang YJ, Cheng AC, Lai WM, Badmaev V, et al. Pterostilbene inhibits colorectal aberrant crypt foci (ACF) and colon carcinogenesis via suppression of multiple signal transduction pathways in azoxymethane-treated mice. J Agric Food Chem 2010;58(15):8833–41. 30. Paul S, Rimando AM, Lee HJ, Ji Y, Reddy BS, Suh N. Anti-inflammatory action of pterostilbene is mediated through the p38 mitogen-activated protein kinase pathway in colon cancer cells. Cancer Prev Res 2009;2(7):650–7. 31. Huang WC, Chan ML, Chen MJ, Tsai TH, Chen YJ. Modulation of macrophage polarization and lung cancer cell stemness by MUC1 and development of a related small-molecule inhibitor pterostilbene. Oncotarget 2016;7(26):39363–75. 32. Ullah O, Li Z, Ali I, Xu L, Liu H, Shah SZA, et al. Pterostilbene alleviates hydrogen peroxide-induced oxidative stress via nuclear factor erythroid 2 like 2 pathway in mouse preimplantation embryos. J Reprod Dev 2019;65(1):73–81. 33. Hung CM, Liu LC, Ho CT, Lin YC, Way TD. Pterostilbene enhances TRAIL-induced apoptosis through the induction of death receptors and downregulation of cell survival proteins in TRAIL-resistance triple negative breast cancer cells. J Agric Food Chem 2017;65(51):11179–91. 34. Tolomeo M, Grimaudo S, Di Cristina A, Roberti M, Pizzirani D, Meli M, et al. Pterostilbene and 30 -hydroxypterostilbene are effective apoptosisinducing agents in MDR and BCR-ABL-expressing leukemia cells. Int J Biochem Cell Biol 2005;37(8):1709–26. 35. Feng Y, Yang Y, Fan C, Di S, Hu W, Jiang S, et al. Pterostilbene inhibits the growth of human esophageal cancer cells by regulating endoplasmic reticulum stress. Cell Physiol Biochem 2016;38(3):1226–44. 36. De Duve C, Wattiaux R. Functions of lysosomes. Annu Rev Physiol 1966;28:435–92. 37. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science 2000;5497:1717–21. 38. Chen RJ, Tsai SJ, Ho CT, Pan MH, Ho YS, Wu CH, et al. Chemopreventive effects of pterostilbene on urethane-induced lung carcinogenesis in mice via the inhibition of EGFR-mediated pathways and the induction of apoptosis and autophagy. J Agric Food Chem 2012;60(46):11533–41. 39. Wang Y, Ding L, Wang X, Zhang J, Han W, Feng L, et al. Pterostilbene simultaneously induces apoptosis, cell cycle arrest and cyto-protective autophagy in breast cancer cells. Am J Transl Res 2012;4(1):44–51. 40. Pan MH, Chang YH, Badmaev V, Nagabhushanam K, Ho CT. Pterostilbene induces apoptosis and cell cycle arrest in human gastric carcinoma cells. J Agric Food Chem 2007;55(19):7777–85. 41. Lin VC, Tsai YC, Lin JN, Fan LL, Pan MH, Ho CT, et al. Activation of AMPK by pterostilbene suppresses lipogenesis and cell-cycle progression in p53 positive and negative human prostate cancer cells. J Agric Food Chem 2012;60(25):6399–407. 42. Wang TT, Schoene NW, Kim YS, Mizuno CS, Rimando AM. Differential effects of resveratrol and its naturally occurring methylether analogs on cell cycle and apoptosis in human androgen-responsive LNCaP cancer cells. Mol Nutr Food Res 2010;54(3):335–44. 43. Chatterjee K, AlSharif D, Mazza C, Syar P, Al Sharif M, Fata JE. Resveratrol and pterostilbene exhibit anticancer properties involving the downregulation of HPV oncoprotein E6 in cervical cancer cells. Nutrients 2018;10(2):E243.

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44. Chen RJ, Wu PH, Ho CT, Way TD, Pan MH, Chen HM, et al. P53-dependent downregulation of hTERT protein expression and telomerase activity induces senescence in lung cancer cells as a result of pterostilbene treatment. Cell Death Dis 2017;8(8) e2985. 45. Tippani R, Prakhya LJ, Porika M, Sirisha K, Abbagani S, Thammidala C. Pterostilbene as a potential novel telomerase inhibitor: molecular docking studies and its in vitro evaluation. Curr Pharm Biotechnol 2014;14(12):1027–35. 46. Pan MH, Chiou YS, Chen WJ, Wang JM, Badmaev V, Ho CT. Pterostilbene inhibited tumor invasion via suppressing multiple signal transduction pathways in human hepatocellular carcinoma cells. Carcinogenesis 2009;30(7):1234–42. 47. Pan MH, Lin YT, Lin CL, Wei CS, Ho CT, Chen WJ. Suppression of heregulin-beta1/HER2-modulated invasive and aggressive phenotype of breast carcinoma by pterostilbene via inhibition of matrix metalloproteinase-9, p38 kinase cascade and Akt activation. Evid Based Complement Alternat Med 2011;2011:562187. 48. Chakraborty A, Gupta N, Ghosh K, Roy P. In vitro evaluation of the cytotoxic, anti-proliferative and anti-oxidant properties of pterostilbene isolated from Pterocarpus marsupium. Toxicol in Vitro 2010;24(4):1215–28. 49. Kumar A, D’Silva M, Dholakia K, Levenson AS. In vitro anticancer properties of table grape powder extract (GPE) in prostate cancer. Nutrients 2018;10(11):E1804. 50. Qian YY, Liu ZS, Yan HJ, Yuan YF, Levenson AS, Li K. Pterostilbene inhibits MTA1/HDAC1 complex leading to PTEN acetylation in hepatocellular carcinoma. Biomed Pharmacother 2018;101:852–9. 51. Wang YJ, Lin JF, Cheng LH, Chang WT, Kao YH, Chang MM, et al. Pterostilbene prevents AKT-ERK axis-mediated polymerization of surface fibronectin on suspended lung cancer cells independently of apoptosis and suppresses metastasis. J Hematol Oncol 2017;10(1):72. 52. Ko HS, Kim JS, Cho SM, Lee HJ, Ahn KS, Kim SH, et al. Urokinase-type plasminogen activator expression and Rac1/WAVE-2/Arp2/3 pathway are blocked by pterostilbene to suppress cell migration and invasion in MDA-MB-231 cells. Bioorg Med Chem Lett 2014;24(4):1176–9. 53. Lin CW, Chou YE, Chiou HL, Chen MK, Yang WE, Hsieh MJ, et al. Pterostilbene suppresses oral cancer cell invasion by inhibiting MMP-2 expression. Expert Opin Ther Targets 2014;18(10):1109–20. 54. Xing F, Liu Y, Sharma S, Wu K, Chan MD, Lo HW, et al. Activation of the c-Met pathway mobilizes an inflammatory network in the brain microenvironment to promote brain metastasis of breast cancer. Cancer Res 2016;76(17):4970–80. 55. Filipowicz W, Bhattacharyya SN, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 2008;9(2):102–14. 56. Qian YY, Liu ZS, Zhang Z, Levenson AS, Li K. Pterostilbene increases PTEN expression through the targeted downregulation of microRNA-19a in hepatocellular carcinoma. Mol Med Rep 2018;17(4):5193–201. 57. Dhar S, Kumar A, Rimando AM, Zhang X, Levenson AS. Resveratrol and pterostilbene epigenetically restore PTEN expression by targeting oncomiRs of the miR-17 family in prostate cancer. Oncotarget 2015;6(29):27214–26. 58. Su CM, Lee WH, Wu AT, Lin YK, Wang LS, Wu CH, et al. Pterostilbene inhibits triple-negative breast cancer metastasis via inducing microRNA-205 expression and negatively modulates epithelial-to-mesenchymal transition. J Nutr Biochem 2015;26(6):675–85. 59. Huynh TT, Lin CM, Lee WH, Wu AT, Lin YK, Lin YF, et al. Pterostilbene suppressed irradiation-resistant glioma stem cells by modulating GRP78/ miR-205 axis. J Nutr Biochem 2015;26(5):466–75. 60. Mak KK, Wu AT, Lee WH, Chang TC, Chiou JF, Wang LS, et al. Pterostilbene, a bioactive component of blueberries, suppresses the generation of breast cancer stem cells within tumor microenvironment and metastasis via modulating NF-kappaB/microRNA 448 circuit. Mol Nutr Food Res 2013;57(7):1123–34.

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Chapter 41

Resveratrol, reactive oxygen species, and mesothelioma Saime Batırel Faculty of Medicine, Department of Medical Biochemistry, Marmara University, Istanbul, Turkey

List of abbreviations AMPK AP-1 Bak Bax Bcl-2 Bcl-xL Bid ERK Keap1 MAPK Mcl-1 MMPs MPM mTOR NF-kB Nrf2 PGC-1a PI3K PTEN ROS SIRTs SOD Sp1 STAT-3 TNF-a

AMP-activated protein kinase activator protein-1 Bcl-2 homologous antagonist/killer Bcl-2-associated X protein B cell lymphoma-2 B-cell lymphoma-extra large BH3 interacting-domain death agonist extracellular-signal-regulated kinase Kelch-like ECH-associated protein 1 mitogen-activated protein kinase myeloid cell leukemia 1 matrix metalloproteinases malignant pleural mesothelioma mammalian target of rapamycin nuclear factor kappa-B nuclear factor erythroid 2 like 2 proliferator-activated receptor gamma coactivator-1 alpha phosphoinositide 3-kinase phosphatase and tensin homolog reactive oxygen species sirtuins superoxide dismutase specificity protein 1 signal transducer and activator of transcription-3 tumor necrosis factor-alpha

Introduction It was shown that natural compounds in plant-based foods exhibit a wide variety of positive biological effects. An especially inverse relationship between the intake of them by diet and incidence of cancer development drew big attention. It was known that these phytochemicals mainly exert their anticancer potential through their effects on generation of reactive oxygen species (ROS) and/or antioxidant mechanisms of the cells.1 The anticarcinogenic properties of resveratrol, as a natural phytoalexin, have been known for more than 20 years. Its chemopreventive and chemotherapeutic effects on different cancer types were evaluated in in vitro and in vivo studies.2 Malignant pleural mesothelioma (MPM) is a rare but very aggressive and fatal tumor of mesothelial cells in the pleura. Despite multimodality therapy, MPM cells are very resistant to conventional treatment. Researchers try to find new effective treatment agents that would cope with the antiapoptotic characteristics of MPM cells. Additionally, as most

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00041-9 © 2021 Elsevier Inc. All rights reserved.

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of the MPM patients have an asbestos exposure history which causes oxidative stress,3 antioxidant agents such as resveratrol might contribute to the treatment. In light of these backgrounds, anticarcinogenic, prooxidant, and antioxidant effects of resveratrol make it a good candidate in the treatment of MPM. Here we summarize the studies related to the role of resveratrol on MPM.

Applications to other cancers or conditions Here we discussed the effects of resveratrol on malignant pleural mesothelioma (MPM) as a natural anticarcinogenic agent. At the same time, since 1997 its anticarcinogenic properties were shown in a wide variety of other cancer types such as colon, breast, prostate, and lung. It was shown that resveratrol affects carcinogenesis at all three stages (initiation, promotion, and progression).2 The combination treatments of resveratrol and conventional therapeutics also attract significant attention because resveratrol can decrease the side effects and the effective dosages of these therapeutic agents.2 The ability of resveratrol to inhibit cancer cell proliferation and induce cell death contributes to the chemotherapeutic and chemoprotective properties of this agent. It also inhibits angiogenesis by decreasing the VEGF pathway. The invasion and metastasis processes required for cancer progression can be interrupted by resveratrol. In particular, the inhibition of matrix metalloproteinases (MMPs) expression is an important step for this effect. Many studies showed evidences that resveratrol also has chemopreventive potential. The main mechanisms under this effect include the pathways related to antioxidants and anti-inflammatory effects and pro-oncogenes.4 In many in vitro and in vivo studies, the signaling pathways which take a role in carcinogenesis including MEK/ERK, STAT, and PI3K/AKT/mTOR were evaluated in order to explain the anticarcinogenic properties of resveratrol in different type of cancers.5 On the other hand, besides all this exciting data, the poor oral viability of resveratrol limits its clinical use as a chemotherapeutic and chemopreventive agent.

Resveratrol Resveratrol (3,5,40 -trihydroxystilibene) is a natural phytoalexin and was first isolated from the root of white opuntum. Since then, it was found in a wide variety of plants, such as grapes, pines, legumes, nuts, berries, soybeans, and pomegranates.2, 6 The structure of resveratrol consists of two phenolic rings linked by an ethylene bridge. Resveratrol naturally exist in two isomeric forms, trans and cis. The trans form is the more abundant and stable and biologically active form. It is converted to the cis form by exposure to sunlight or UV radiation7, 8 (Fig. 1). Resveratrol is rapidly absorbed into the body via the gastrointestinal system after oral intake and rapidly metabolized by the liver. Unfortunately, the systemic bioavailability of resveratrol is less than 1%,7 while the trans form has a relatively higher bioavailability when compared with the cis form.9 In order to increase the bioavailability, many derivatives of resveratrol have been synthesized such as the methoxylated, hydroxylated, and halogenated forms.8 Resveratrol is synthesized in plants in response to infections, stress, injury, and exposure to UV.10 It reveals a wide range of biological activities such as antiaging, anti-inflammatory, anti-glycation, neuroprotective, anticancer, and antioxidant effects11 (Fig. 2).

FIG. 1 Structures of trans- and cis-resveratrol.

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FIG. 2 Most common biological activities of resveratrol.

With all these exciting effects, the poor bioavailability of resveratrol limits the use of this agent on cancer patients.12 In a human study, the mean average plasma concentration of the parent resveratrol compound reached 0.55 nmol/mL, with the consumption of 5.0 g resveratrol orally.13 However, in vitro studies showed that higher levels than this concentration are required for the anticarcinogenic effects of resveratrol. On the other hand, some in vivo studies showed that low doses can cause chemopreventive effects in cancer patients. This contradiction was explained with two hypotheses. One of them is that the metabolites of resveratrol also contribute to its anticancer effects. The other is that conjugated resveratrol is hydrolyzed and regenerates the parent compound.4

Antioxidant effects of resveratrol Many studies have proved that resveratrol is a powerful antioxidant with direct and indirect mechanisms (Fig. 3). Direct antioxidant mechanisms of resveratrol include scavenging free radicals and inhibition of ROS production. These mechanisms are associated with the presence of three hydroxyl groups in the structure of resveratrol. ROS generation is decreased by resveratrol through its metal chelation activity.11 Transition metals such as iron and copper promote the formation of ROS by the Fenton reaction. Resveratrol binds the metal ions and decreases the generation of hydroxyl radicals by the Fenton reaction. Additionally, treatment with resveratrol reduces ROS formation from the NADPH oxidase system. In terms of the indirect antioxidant defense mechanisms of resveratrol, it replenishes glutathione levels, which removes ROS, and acts as a cofactor of antioxidant enzymes. Many studies showed that resveratrol enhances the expression and activity of endogenous antioxidant enzymes such as catalase, glutathione peroxidase, and superoxide dismutase (SOD).7 The antioxidant effects of resveratrol are generated via the pathways triggered by Nuclear factor erythroid 2 like 2 (Nrf2) and sirtuins (SIRTs).14 Nrf2 is a transcription factor which plays a role in antioxidant response, protection from toxic and carcinogenic substances. In physiologic states, it is found in inactive form which is bound to Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm and nuclear translocation of Nrf2 is prevented. After stimulation by oxidative conditions, Nrf2 splits from Keap1 and translocates into the nucleus, which leads to the expression of antioxidant enzymes. It was known that resveratrol inactivates Keap1, and activates Nrf2 subsequently.7, 14 Resveratrol enhances mitochondrial biogenesis and modulates the expression of mitochondrial proteins through three main factors, proliferator-activated receptor gamma coactivator 1a (PGC-1a), SIRT1, and AMP-activated protein kinase (AMPK).14 Sirtuin type 1 (SIRT1) is involved in many biological functions including cell survival.7 It regulates the activity

FIG. 3 Direct and indirect antioxidant mechanisms of resveratrol.

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of PGC-1a. In basal conditions, PGC1a is in an acetylated inactive form. SIRT1 activates PGC-1a via removal of the acetyl group in response to oxidative stress, which results in the expression of antioxidant enzymes. The published data shows that resveratrol stimulates SIRT1, which is mediated by AMPK.7, 14

Malignant pleural mesothelioma Malignant mesothelioma is a tumor which originates from mesothelial cells lining the pleural and peritoneal cavities. Mesothelial cells can be differentiated into epithelial, sarcomatoid, or biphasic (mixed) histologic subtypes of MPM, of which epithelioid is the most prevalent.3 Even if it is a rare tumor, in countries where asbestos was widely used in the past, the incidence of MPM is increasing. Besides asbestos exposure, the other possible etiologies are simian virus 40 (SV40) infection and genetic predisposition.15 Despite multimodality therapy, including aggressive surgery, adjuvant radiation, and chemotherapy, the prognosis of mesothelioma is very poor. The prognosis depends on the histologic subtype. Sarcomatoid type has the worst prognosis with 4 months median survival, while epithelioid and biphasic subtypes have 14 and 10 months, respectively.16 Surgical resection is the main treatment for MPM. However, many patients are unable to undergo surgery since they are diagnosed at advanced stages of the disease. A combination of pemetrexed and cisplatin or raltitrexed and cisplatin is used for standard chemotherapy treatment.17 However, the effectiveness of these agents is low because of the resistance of MPM cells to apoptosis.18

Asbestos and malignant pleural mesothelioma In all, 80% of MPM patients have asbestos fiber exposure. Asbestos refers to naturally occurring silicate minerals. There are six common types of asbestos fibers: crocidolite, amosite, anthophyllite, tremolite, actinolite, and chrysotile. The first five of them are amphibole types and have short, straight, and stiff fibers with more durable characteristics. Chrysotile, also called white asbestos, is a serpentine type and has long, curly, and pliable fibers.3, 15 There is a long latency period of 30–50 years between asbestos exposure and the development of MPM. Most patients diagnosed with MPM are in their 60s.15 The mechanism why asbestos causes mesothelioma is not clarified. Researchers focus on the increased production of ROS induced by asbestos. Asbestos causes oxidative stress with various mechanisms. This can occur directly or indirectly. The iron in asbestos directly promotes the formation of hydroxyl radical (%) from hydrogen peroxide via the Fenton reaction. Asbestos also stimulates ROS generation through the iron-mediated Haber-Weiss reaction.18 Indirectly, chronic inflammation stimulated by asbestos also causes increased release of ROS by inflammatory, lung epithelial, and mesothelial cells.19 After exposure, asbestos fibers are internalized by mesothelial cells with integrins and other receptors and causes prolonged local and pleural inflammation, which induces ROS generation.18 ROS stimulates apoptosis and subsequent compensatory hyperplasia, and alters the cell cycle of mesothelial or epithelial cells. Additionally, it is well known that oxidative stress contributes to all three stages of carcinogenesis: initiation, promotion, and progression through mutations, DNA damage, DNA cross-links, and base modifications.20 The amount of fiber toxicity depends on the biodurability, surface reactivity, genetic properties of the patient and dose, size, and diameter of fibers.19 It has been shown that long fibers cause much greater inflammatory responses than shorter fibers. Long-term asbestos exposure triggers chronic inflammation and leads to an increase of ROS production by inflammatory cells. This condition alters immune cells and reduces tumor immunity and subsequently oncogenic transformation of those cells.18 ROS induces mesothelioma development via different signal pathways. Exposure of mesothelial cells to ROS increases the activation of matrix metalloproteinases (MMPs) enzymes which lyse the extracellular matrix of normal tissue. This proteolysis facilitates the local spread of pleural malignant mesothelioma. Because of that, increased MMP-2 levels have been associated with poor prognosis.18 Additionally, subsequent contact with asbestos leads macrophages to produce tumor necrosis factor-alpha (TNF-a). Binding with its receptor on mesothelial cells, TNF-a activates nuclear factor kappa-B (NF-kB pathway) which plays an important role in the development of MPM.21 Asbestos fibers also induce the autophosphorylation and activity of extracellular-signal-regulated kinase (ERK) in the mitogen-activated protein kinase (MAPK) cascade of mesothelial cells. It was found that this pathway is more activated in mesothelioma cells than in nonneoplastic mesothelial cells. The MAPK signaling cascade activates activator protein 1 (AP-1). This transcription factor is redoxsensitive and associated with the development of cell proliferation and tumor promotion18 (Fig. 4).

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FIG. 4 Molecular mechanisms of asbestos-induced mesothelioma.

Antiapoptotic effects of resveratrol on MPM cells Many natural phytochemicals were investigated in terms of their anticancer properties. Inhibiting survival, activating apoptosis and synergistic effects with conventional chemotherapy and radiotherapy effects make them candidates for treatment of cancer. In 1997, Jang et al. observed for the first time that resveratrol inhibits carcinogenesis in mouse skin cancer.22 Since then, resveratrol is well defined as a chemopreventive and chemotherapeutic agent in a wide variety of cancer types. It was revealed that the anticancer properties of resveratrol arise from its antioxidative, anti-inflammatory, antimutagenic, and antiproliferative effects.6 It is well known that mesothelioma cells are resistant to apoptosis, which is a very important mechanism that eliminates the cancer cells from the body. This resistance frustrates the treatment period of the disease. A cell can go to apoptosis with two mechanisms, extrinsic or intrinsic pathways. The intrinsic pathway, known as the mitochondrial apoptotic pathway, is regulated by B cell lymphoma-2 (Bcl-2) family member proteins. Antiapoptotic family members contain Bcl-2, Bcl-xL, and Mcl-1, while Bax, Bak, Bid, and Bim are proapoptotic protein members.23 It is well known that overexpression of antiapoptotic proteins in cancer cells relates to chemoresistance. It was also shown that Mcl-1 and Bcl-xL expressions were overexpressed in most mesothelioma cells.24, 25 Because of that, agents targeting antiapoptotic proteins came into prominence in MPM treatment. Accumulated data indicated that resveratrol induces apoptosis in a wide variety of cancer types such as skin, nasopharyngeal, lung, bladder, breast, and colon cancers, as well as leukemia, glioma, and mesothelioma cells.4, 26, 27 Apoptotic properties of resveratrol are mediated by many cell signaling pathways. One of the mechanisms of apoptotic activity of resveratrol is induction of generation of ROS in cancer cells.27, 28 ROS stimulates the intrinsic apoptosis pathway, which is regulated by mitochondria. After the stimulation, proapoptotic Bax or Bak proteins are expressed. These proteins lead to an increasing outer mitochondrial membrane permeability and decreasing mitochondrial membrane potential. Subsequently proapoptotic factors such as cytochrome c are released from the mitochondria into the cytosol where the classical caspase pathway is activated.29 It was observed that after treatment of biphasic MPM, cells with resveratrol, proapoptotic regulator proteins, Bid, and Bim were activated, while Bcl-xL is downregulated30 (Fig. 5).

Chemoprotective effects of resveratrol on MPM cells In combination with chemotherapeutic agents, resveratrol sensitizes cancer cells to the apoptotic effect of the chemotherapeutics, decreasing drug resistance and also enhancing the therapeutic efficacy of these agents. Meanwhile, its ability to reduce side effects such as cardiotoxicity, hepatotoxicity, and renal toxicity has been shown in a wide variety of studies.31 The mechanisms by which resveratrol overcomes the drug resistance include regulating apoptosis, cell cycle, and ROS generation. Antiapoptotic factors, such as Bcl-xL, are important for the prevention of apoptosis, and upregulation of these proteins contributes to drug resistance and poor prognosis. Because Bcl-xL is overexpressed in MPM cells,32 downregulation of this gene is defined as a potential target for mesothelioma treatment.3 It has been observed that when

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FIG. 5 Molecular basis of chemotherapeutic effects of resveratrol.

translation of the Bcl-xL gene is inhibited, mesothelioma cells are chemosensitized to cisplatin.33 In another study, the combination treatment of resveratrol and cisplatin exerted a synergistically induced apoptosis effect through a ROSdependent mechanism in mesothelioma cells. The treatment increased ROS generation and impaired mitochondrial activity with an increase in the Bax/Bcl-2 ratio.34 The same researchers also showed that the combination treatment with resveratrol and clofarabine simultaneously targets Mcl-1 and Bcl-xL proteins, leading to stronger inhibition of cell proliferation, promoting apoptosis, and blocking cell cycle progression in epithelioid MPM cells.35 Furthermore, in another study, exposure to biphasic MPM cells to the combined treatment of resveratrol and clofarabine caused a downregulation of Mcl-1 protein which leads to enhanced caspase-3/7 activity. On the other hand, healthy mesothelial cells exhibited an upregulation of this gene.36 Specificity Protein 1 (Sp1) is a transcription factor. Besides its functions in cell differentiation, cell growth, and immune responses, Sp1 is involved in apoptosis.37 Because its overexpression is negatively correlated with prognosis of the cancers, it was shown as a target for cancer therapy.38 In a study, resveratrol induced apoptosis of biphasic MPM cells via suppression of the expression of Sp1 protein in a dose- and time-dependent manner. In the same study, resveratrol also inhibited tumor growth of these cells implanted in nude mice by suppressing expression of this protein.30

Effects of resveratrol on cell cycle of MPM cells Furthermore, in a previous study, it was observed that resveratrol regulates the cell cycle which is altered in MPM. This effect of resveratrol is performed by modulation of the expression of Sp1 regulatory proteins such as p21, p27, cyclin D1, which have a role in cell survival and the cell cycle. Inhibition of the cell cycle by resveratrol in this study occurred by inducing sub-G1 arrest in biphasic MPM cells.30 In another study, the combination treatment of resveratrol and clofarabine caused a synergistic inhibition in biphasic MPM cell proliferation and downregulation of Sp1. Additionally, Sp1-dependent gene products, c-Met, cyclin D1, and p21, were downregulated.39 Cyclin D1, one of the regulators of the cell cycle, is essential for the G1 to S phase transition in the cycle, and is established as a proto-oncogene.40 C-met is also responsible for cell division, through inhibition of receptor tyrosine kinases. The majority of studies have observed that overexpression of Cyclin D and c-Met is associated with recurrence and poor prognosis of cancers, including MPM.41–44 The inhibitory effect of resveratrol on the expression of cyclin D1 in epithelioid MPM cells was observed in a previous study.27 p21 and p27 are inhibitors of the cell cycle, which are controlled by p53, a tumor suppressor gene. High dose phytoestrogens such as resveratrol have been shown to stimulate the expression of p21, p27, and p53.45 p53 has a crucial role in cell survival, cell cycle, and apoptosis. It induces cell cycle arrest and apoptosis under stress such as increased ROS production. It also helps the DNA repairing pathway.46 Inactivation of the p53 gene is a frequent mutation in various human cancers, including MPM.47 It was shown that resveratrol increases expression of the p53 protein in epithelioid MPM cells.27

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Signaling pathways in anticancer effects of resveratrol Inducing cell apoptosis and cell cycle arrest by resveratrol occurs through inhibition of the PI3K/AKT/mTOR pathway, which is more activated in mesothelioma cells than in nonneoplastic mesothelial cells.48 The activity of this pathway is redox-regulated. Activation of PI3K causes activation of AKT, which promotes cell survival and inhibits apoptosis through activation of proapoptotic proteins. AKT also directly activates mTOR which is related to decreased survival of patients with malignant peritoneal mesothelioma.49 Moreover, PTEN is responsible for AKT activation and is inhibited by hydrogen peroxide reversibly. In some MPM cell lines, homozygous deletion of PTEN was observed, which results in the activation of the PI3-kinase/Akt pathway.50 It was proved that the PI3K/AKT/mTOR pathway is important in the chemoresistance of MPM.51 In a study, the combination treatment of clofarabine and resveratrol inhibited Akt phosphorylation in biphasic MPM cells, while activation of Akt was not detected in normal mesothelial cells.39 This data suggests that resveratrol plays a role in overcoming chemoresistance in MPM through inhibition of Akt. Resveratrol also copes with chemoresistance by inhibiting the signal transducer and activator of transcription 3 (STAT-3). Combined treatments of chemotherapeutics and resveratrol resulted in decreased cell survival through inhibition of STAT3 proteins.52 Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and Nuclear factor-kB (NF-kB) are other signaling proteins that have roles in the process of oxidative stress and development of cancer.53 Nrf2 controls cell homeostasis in response to oxidative stress. Low oxidative stress induces Nrf2 and synthesis of antioxidative enzymes is increased. Nrf2 is defined as a good protein because of its ability to protect cells from carcinogens via the antioxidant system. On the other hand, prolonged activation of Nrf2 gives cancer cells a growth advantage because of the induction of the antioxidant system. This situation increases resistance to chemotherapeutic drugs. Therefore, when Nrf2 expression is suppressed, cancer cell death is maximized by chemotherapeutic drugs.54 In a previous study, the combination treatment with resveratrol and clofarabine synergistically decreased the cell viability of biphasic MPM cells through the downregulation of Nrf2.55 NF-kB is another transcription factor which has a role in cell proliferation, apoptosis, and inflammation. In physiological conditions, it binds to its inhibitory protein, IkB. The pathway is activated by increased ROS generation as seen in asbestos exposure. After activation, IkB is degraded and NFkB is activated.56 Since NF-kB inhibits apoptosis, the increased NF-kB levels cause the cancer cells to become more chemotherapyresistant.54 Resveratrol eliminates antiapoptotic proteins expression in cancer cells through the suppression of the NF-kB pathway. With suppression of this transcription factor, resveratrol enables chemosensitization of cancer cells.8

Chemopreventive properties of resveratrol Besides the chemotherapeutic, chemoprotective, and synergistic effects, previous studies have demonstrated that resveratrol also shows chemopreventive properties in the development of cancer. Resveratrol protects DNA, lipids, and proteins from oxidative damage and induces DNA repair7 (Fig. 6). These properties of resveratrol lead to a cytoprotective effect on

FIG. 6 Chemopreventive potential of resveratrol.

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normal cells such as neurons, brain tissue, renal and retinal pigment epithelium, cardiomyocytes, and keratinocytes. However, as discussed above, resveratrol has the ability to induce apoptosis in cancer cells. These opposite effects can occur simultaneously because of the higher intracellular ROS levels and inefficiency of antioxidative mechanisms in the cancer cells.57 It is also related to having different targets of resveratrol in cancer and healthy cells.11 This dual effect makes resveratrol a potential agent for cancer prevention. However, the chemopreventive effects of this agent should be investigated and proved with more clinical trials, especially in mesothelioma.

Dual effects of resveratrol on cancer cells Many studies showed that the anticarcinogenic effects of resveratrol have a dose, time, and cell-type-dependent manner.11 While lower doses (0.1–1.0 mg/mL) of resveratrol increase the expression of proteins related to cell survival and proliferation proteins, higher levels (10.0–100.0 mg/mL) stimulate cell death in cancer cells through inhibition of synthesis of nucleic acids and proteins.8, 58 This dual effect of resveratrol is the result of having both antioxidant and prooxidant effects.8 Although resveratrol decreases membrane potential at high concentrations, it positively affects mitochondria function at lower ranges in cells which give rise to its antioxidant properties.7 Resveratrol exerts chemotherapeutic effects through ROS accumulation in cancer cells which induces apoptosis at higher levels.8 Conventional therapeutic agents such as chemotherapeutic drugs and radiotherapy also exert their effects by increasing ROS levels in cancer cells. However, because resveratrol shows both oxidative and antioxidative effects, administration of resveratrol in combination with chemotherapeutics may enhance the antioxidative capacity of the cells and generate unwanted effects that increase the survival of cancer cells and the drug resistance. At that point, a time and concentration-dependent manner of resveratrol effects becomes more important during treatment with this agent. One of the difficulties of MPM treatment is that MPM cells are resistant to oxidant effects of chemotherapeutic agents. One of the reasons why mesothelioma cells are more resistant to oxidants than normal mesothelial cells is upregulation of the antioxidant enzymes. It is believed that increased antioxidant enzymes might be caused by increased asbestos-induced ROS levels in mesothelioma.18 Because of that, the roles of resveratrol on the antioxidant system of MPM cells drew attention. It was assumed that resveratrol might affect the antioxidant enzymes of MPM cells with a cell-type manner. In a study with biphasic MPM cells, treatment of resveratrol did not affect intracellular ROS levels but increased SOD-2 expression26 while in another study with epithelioid MPM cells, resveratrol increased the level of ROS but did not change the expressions of antioxidant enzymes.27

Conclusion Resveratrol possesses antitumor effects and is a possible candidate for treatment and prevention of MPM. However, the low bioavailability and dual effects of resveratrol prevent the use of this natural product in clinical trials. Taking together all the pros and cons of resveratrol on the treatment of MPM, more in vitro and in vivo studies should be performed before putting resveratrol into the guidelines for treatment of cancer.

Summary points l

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Resveratrol (3,5,40 -trihydroxystilibene), a natural phytoalexin, has many biological activities including anticancer and antioxidant effects. Resveratrol has both antioxidant and prooxidant effects on cells. Malignant mesothelioma, a tumor originating from mesothelial cells, is an aggressive and fatal cancer because of the resistance of MPM cells to apoptosis. Asbestos exposure is the best known etiology for MPM and causes the disease through increased ROS generation. Resveratrol shows chemotherapeutic, chemoprotective effects on MPM cells. The anticarcinogenic effects of resveratrol have a dose, time, and cell-type-dependent manner.

References 1. Chikara S, Nagaprashantha LD, Singhal J, Horne D, Awasthi S, Singhal SS. Oxidative stress and dietary phytochemicals: role in cancer chemoprevention and treatment. Cancer Lett 2018;413:122–34. https://doi.org/10.1016/j.canlet.2017.11.002. 2. Catalgol B, Batirel S, Taga Y, Ozer NK. Resveratrol: French paradox revisited. Front Pharmacol 2012;3:141. https://doi.org/10.3389/ fphar.2012.00141.

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3. Rascoe PA, Jupiter D, Cao X, Littlejohn JE, Smythe WR. Molecular pathogenesis of malignant mesothelioma. Expert Rev Mol Med 2012;14:e12. https://doi.org/10.1017/erm.2012.6. 4. Elshaer M, Chen Y, Wang XJ, Tang X. Resveratrol: an overview of its anti-cancer mechanisms. Life Sci 2018;207:340–9. https://doi.org/10.1016/j. lfs.2018.06.028. 5. Ko JH, Sethi G, Um JY, Shanmugam MK, Arfuso F, Kumar AP, et al. The role of resveratrol in cancer therapy. Int J Mol Sci 2017;18(12). https://doi. org/10.3390/ijms18122589. 6. Dybkowska E, Sadowska A, S´widerski F, Rakowska R, Wysocka K. The occurrence of resveratrol in foodstuffs and its potential for supporting cancer prevention and treatment. A review. Rocz Panstw Zakl Hig 2018;69(1):5–14. 7. Truong VL, Jun M, Jeong WS. Role of resveratrol in regulation of cellular defense systems against oxidative stress. Biofactors 2018;44(1):36–49. https://doi.org/10.1002/biof.1399. 8. Salehi B, Mishra AP, Nigam M, Sener B, Kilic M, Sharifi-Rad M, et al. Resveratrol: a double-edged sword in health benefits. Biomedicine 2018;6(3): E91. https://doi.org/10.3390/biomedicines6030091. 9. Wenzel E, Somoza V. Metabolism and bioavailability of trans-resveratrol. Mol Nutr Food Res 2005;49(5):472–81. 10. Hasan M, Bae H. An overview of stress-induced resveratrol synthesis in grapes: perspectives for resveratrol-enriched grape products. Molecules 2017;22(2):E294. https://doi.org/10.3390/molecules22020294. 11. Galiniak S, Aebisher D, Bartusik-Aebisher D. Health benefits of resveratrol administration. Acta Biochim Pol 2019;66(1):13–21. https://doi.org/ 10.18388/abp.2018_2749. 12. Singh AP, Singh R, Verma SS, Rai V, Kaschula CH, Maiti P, et al. Health benefits of resveratrol: evidence from clinical studies. Med Res Rev 2019; 39(5):1851–91. https://doi.org/10.1002/med.21565. 13. Brown VA, Patel KR, Viskaduraki M, Crowell JA, Perloff M, Booth TD, et al. Repeat dose study of the cancer chemopreventive agent resveratrol in healthy volunteers: safety, pharmacokinetics, and effect on the insulin-like growth factor axis. Cancer Res 2010;70(22):9003–11. https://doi.org/ 10.1158/0008-5472.CAN-10-2364. 14. Gibellini L, Bianchini E, De Biasi S, Nasi M, Cossarizza A, Pinti M. Natural compounds modulating mitochondrial functions. Evid Based Complement Alternat Med 2015;527209. https://doi.org/10.1155/2015/527209. 15. Carbone M, Adusumilli PS, Alexander Jr HR, Baas P, Bardelli F, Bononi A, et al. Mesothelioma: scientific clues for prevention, diagnosis, and therapy. CA Cancer J Clin 2019;1–28. https://doi.org/10.3322/caac.21572. 16. Meyerhoff RR, Yang CF, Speicher PJ, Gulack BC, Hartwig MG, D’Amico TA, et al. Impact of mesothelioma histologic subtype on outcomes in the surveillance, epidemiology, and end results database. J Surg Res 2015;196(1):23–32. https://doi.org/10.1016/j.jss.2015.01.043. 17. Berzenji L, Van Schil P. Multimodality treatment of malignant pleural mesothelioma. F1000Res 2018;22:7. https://doi.org/10.12688/ f1000research.15796.1. 18. Benedetti S, Nuvoli B, Catalani S, Galati R. Reactive oxygen species a double-edged sword for mesothelioma. Oncotarget 2015;6(19):16848–65. Review, 26078352. 19. Liu G, Cheresh P, Kamp DW. Molecular basis of asbestos-induced lung disease. Annu Rev Pathol 2013;8:161–87. https://doi.org/10.1146/annurevpathol-020712-163942. 20. Gorrini C, Harris IS, Mak TW. Modulation of oxidative stress as an anticancer strategy. Nat Rev Drug Discov 2013;12(12):931–47. https://doi.org/ 10.1038/nrd4002. 21. Yang H, Bocchetta M, Kroczynska B, Elmishad AG, Chen Y, Liu Z, et al. TNF-a inhibits asbestos-induced cytotoxicity via a NF-kB-dependent pathway, a possible mechanism for asbestos-induced oncogenesis. Proc Natl Acad Sci U S A 2006;103(27):10397–402. https://doi.org/10.1073/ pnas.0604008103. 22. Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CW, et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 1997;275:218–20. 23. Kang MH, Reynolds CP. Bcl-2 inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy. Clin Cancer Res 2009;15(4):1126–32. https://doi.org/10.1158/1078-0432.CCR-08-0144. 24. Soini Y, Kinnula V, Kaarteenaho-Wiik R, Kurttila E, Linnainmaa K, P€a€akk€o P. Apoptosis and expression of apoptosis regulating proteins bcl-2, mcl-1, bcl-X, and bax in malignant mesothelioma. Clin Cancer Res 1999;5(11):3508–15. 25. O’Kane SL, Pound RJ, Campbell A, Chaudhuri N, Lind MJ, Cawkwell L. Expression of bcl-2 family members in malignant pleural mesothelioma. Acta Oncol 2006;45(4):449–53. 26. Batirel S, Mutlu-Altundag E, Kurt E, Batirel HF. Resveratrol induces apoptosis through oxidative stress in biphasic malignant pleural mesothelioma cells. Turk J Biochem 2016;41(S5). https://doi.org/10.1515/tjb-2016-frontmatters. 27. Batirel S, Mutlu-Altundag E, Toplayici S, Corek C, Batirel HF. Resveratrol induces cell cycle arrest and apoptosis in epithelioid malignant pleural mesothelioma cells. Turk J Biochem 2018;43(2):197–204. https://doi.org/10.1515/tjb-2017-0083. 28. Wang D, Gao Z, Zhang X. Resveratrol induces apoptosis in murine prostate cancer cells via hypoxia-inducible factor 1-alpha (HIF-1a)/reactive oxygen species (ROS)/P53 signaling. Med Sci Monit 2018;24:8970–6. https://doi.org/10.12659/MSM.913290. 29. Farsinejad S, Gheisary Z, Ebrahimi Samani S, Alizadeh AM. Mitochondrial targeted peptides for cancer therapy. Tumour Biol 2015;36(8):5715–25. https://doi.org/10.1007/s13277-015-3719-1. 30. Lee KA, Lee YJ, Ban JO, Lee YJ, Lee SH, Cho MK, et al. The flavonoid resveratrol suppresses growth of human malignant pleural mesothelioma cells through direct inhibition of specificity protein 1. Int J Mol Med 2012;30(1):21–7. https://doi.org/10.3892/ijmm.2012.978. 31. Xiao Q, Zhu W, Feng W, Lee SS, Leung AW, Shen J, et al. A review of resveratrol as a potent chemoprotective and synergistic agent in cancer chemotherapy. Front Pharmacol 2019;9:1534. https://doi.org/10.3389/fphar.2018.01534.

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32. Cao X, Littlejohn J, Rodarte C, Zhang L, Martino B, Rascoe P, et al. Up-regulation of Bcl-xl by hepatocyte growth factor in human mesothelioma cells involves ETS transcription factors. Am J Pathol 2009;175(5):2207–16. https://doi.org/10.2353/ajpath.2009.090070. 33. Littlejohn JE, Cao X, Miller SD, Ozvaran MK, Jupiter D, Zhang L, et al. Bcl-xL antisense oligonucleotide and cisplatin combination therapy extends survival in SCID mice with established mesothelioma xenografts. Int J Cancer 2008;123(1):202–8. https://doi.org/10.1002/ijc.23452. 34. Lee YJ, Lee GJ, Yi SS, Heo SH, Park CR, Nam HS, et al. Cisplatin and resveratrol induce apoptosis and autophagy following oxidative stress in malignant mesothelioma cells. Food Chem Toxicol 2016;97:96–107. https://doi.org/10.1016/j.fct.2016.08.033. 35. Lee YJ, Hwang IS, Lee YJ, Lee CH, Kim SH, Nam HS, et al. Knockdown of Bcl-xL enhances growth-inhibiting and apoptosis-inducing effects of resveratrol and clofarabine in malignant mesothelioma H-2452 cells. J Korean Med Sci 2014;29(11):1464–72. https://doi.org/10.3346/ jkms.2014.29.11.1464. 36. Lee YJ, Lee YJ, Lee SH. Resveratrol and clofarabine induces a preferential apoptosis-activating effect on malignant mesothelioma cells by Mcl-1 down-regulation and caspase-3 activation. BMB Rep 2015;48(3):166–71. 37. Beishline K, Azizkhan-Clifford J. Sp1 and the ’hallmarks of cancer’. FEBS J 2015;282(2):224–58. https://doi.org/10.1111/febs.13148. 38. Guan H, Cai J, Zhang N, Wu J, Yuan J, Li J, et al. Sp1 is upregulated in human glioma, promotes MMP-2-mediated cell invasion and predicts poor clinical outcome. Int J Cancer 2012;130(3):593–601. https://doi.org/10.1002/ijc.26049. 39. Lee YJ, Lee YJ, Im JH, Won SY, Kim YB, Cho MK, et al. Synergistic anti-cancer effects of resveratrol and chemotherapeutic agent clofarabine against human malignant mesothelioma MSTO-211H cells. Food Chem Toxicol 2013;52:61–8. https://doi.org/10.1016/j.fct.2012.10.060. 40. Tobin NP, Bergh J. Analysis of cyclin D1 in breast cancer: a call to arms. Curr Breast Cancer Rep 2012;4(3):171–3. 41. Michalides R, van Veelen N, Hart A, Loftus B, Wientjens E, Balm A. Overexpression of cyclin D1 correlates with recurrence in a group of forty-seven operable squamous cell carcinomas of the head and neck. Cancer Res 1995;55(5):975–8. 42. Maulik G, Shrikhande A, Kijima T, Ma PC, Morrison PT, Salgia R. Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition. Cytokine Growth Factor Rev 2002;13(1):41–59. 43. Christensen JG, Burrows J, Salgia R. c-Met as a target for human cancer and characterization of inhibitors for therapeutic intervention. Cancer Lett 2005;225(1):1–26. 44. Jagadeeswaran R, Ma PC, Seiwert TY, Jagadeeswaran S, Zumba O, Nallasura V, et al. Functional analysis of c-Met/hepatocyte growth factor pathway in malignant pleural mesothelioma. Cancer Res 2006;66(1):352–61. 45. Bilal I, Chowdhury A, Davidson J, Whitehead S. Phytoestrogens and prevention of breast cancer: the contentious debate. World J Clin Oncol 2014;5 (4):705–12. https://doi.org/10.5306/wjco.v5.i4.705. 46. Ko LJ, Prives C. p53: puzzle and paradigm. Genes Dev 1996;10(9):1054–72. 47. Hylebos M, Van Camp G, van Meerbeeck JP, Op de Beeck K. The genetic landscape of malignant pleural mesothelioma: results from massively parallel sequencing. J Thorac Oncol 2016;11(10):1615–26. https://doi.org/10.1016/j.jtho.2016.05.020. 48. Zhou S, Liu L, Li H, Eilers G, Kuang Y, Shi S, et al. Multipoint targeting of the PI3K/mTOR pathway in mesothelioma. Br J Cancer 2014;110 (10):2479–88. https://doi.org/10.1038/bjc.2014.220. 49. Varghese S, Chen Z, Bartlett DL, Pingpank JF, Libutti SK, Steinberg SM, et al. Activation of the phosphoinositide-3-kinase and mammalian target of rapamycin signaling pathways are associated with shortened survival in patients with malignant peritoneal mesothelioma. Cancer 2011;117 (2):361–71. https://doi.org/10.1002/cncr.25555. 50. Sekido Y. Genomic abnormalities and signal transduction dysregulation in malignant mesothelioma cells. Cancer Sci 2010;101(1):1–6. https://doi. org/10.1111/j.1349-7006.2009.01336.x. 51. Wilson SM, Barbone D, Yang TM, Jablons DM, Bueno R, Sugarbaker DJ, et al. mTOR mediates survival signals in malignant mesothelioma grown as tumor fragment spheroids. Am J Respir Cell Mol Biol 2008;39(5):576–83. https://doi.org/10.1165/rcmb.2007-0460OC. 52. Bhardwaj A, Sethi G, Vadhan-Raj S, Bueso-Ramos C, Takada Y, Gaur U, et al. Resveratrol inhibits proliferation, induces apoptosis, and overcomes chemoresistance through down-regulation of STAT3 and nuclear factor-kappaB-regulated antiapoptotic and cell survival gene products in human multiple myeloma cells. Blood 2007;109(6):2293–302. 53. Wardyn JD, Ponsford AH, Sanderson CM. Dissecting molecular cross-talk between Nrf2 and NF-kB response pathways. Biochem Soc Trans 2015;43 (4):621–6. https://doi.org/10.1042/BST20150014. 54. Bellezza I, Mierla AL, Minelli A. Nrf2 and NF-kB and their concerted modulation in cancer pathogenesis and progression. Cancers (Basel) 2010;2 (2):483–97. https://doi.org/10.3390/cancers2020483. 55. Lee YJ, Im JH, Lee DM, Park JS, Won SY, Cho MK, et al. Synergistic inhibition of mesothelioma cell growth by the combination of clofarabine and resveratrol involves Nrf2 downregulation. BMB Rep 2012;45(11):647–52. 56. Morgan MJ, Liu ZG. Crosstalk of reactive oxygen species and NF-kB signaling. Cell Res 2011;21(1):103–15. https://doi.org/10.1038/cr.2010.178. 57. Grabacka MM, Gawin M, Pierzchalska M. Phytochemical modulators of mitochondria: the search for chemopreventive agents and supportive therapeutics. Pharmaceuticals (Basel) 2014;7(9):913–42. https://doi.org/10.3390/ph7090913. 58. Szende B, Tyiha´k E, Kira´ly-Veghely Z. Dose-dependent effect of resveratrol on proliferation and apoptosis in endothelial and tumor cell cultures. Exp Mol Med 2000;32(2):88–92.

Chapter 42

Exercise, selenium, and cancer cells Mahdieh Molanouri Shamsia and Zuhair Mohammad Hassanb a

Physical Education & Sport Sciences Department, Faculty of Humanities, Tarbiat Modares University, Tehran, Iran, b Department of Immunology,

School of Medical Sciences, Tarbiat Modares University, Tehran, Iran

List of abbreviations AMPK DNA GPx HIF-1a IL-15 IL-6 IL-8 LIF NK OSM Se TAZ TNF-a tRNA TrxR VEGF YAP

AMP-activated protein kinase deoxyribonucleic acid glutathione peroxidase hypoxia-inducible factor-1 alpha interleukin-15 interleukin-6 interleukin-8 leukemia inhibitory factor natural killer oncostatin M selenium transcriptional coactivator with PDZ-binding motif tumor necrosis factor alpha transfer ribonucleic acid thioredoxin reductases vascular endothelial growth factor yes-associated protein 1

Introduction Physical exercise in order to provide skeletal muscle energy demands the activation of some metabolic pathways that in long-term exercise can induce metabolic adjustment in different tissues.1 Exercise activities can be used as a coadjuvant in chemotherapy and radiotherapy in cancer treatments. It can be effective in improving physical and psychological complications of cancer and related therapies such as fitness, body composition, physical function, fatigue, sleep quality, and health-related quality of life.2, 3 Recent studies also confirm the effects of exercise in tumor incidence, progression, and metastasis. Also, the effects of exercise activity on hypoxia, cell metabolism, and antitumor immune phenotype have been observed in cancer cells.4, 5 It is suggested that exercise with different type, frequency, duration, and intensity can influence cancer cells in different manners.6 Nutritional supplements, especially antioxidants such as selenium (Se), with consideration to antioxidant, antiinflammatory, and antimetastatic effects have been considered as anticancer and chemopreventive interventions in cancer.7 Despite the positive effects of selenium, some studies have shown toxic and prooxidant effects of selenium in cancer patients.8, 9 Besides the effects of physical exercise on cancer cells, it may probably be able to control the destructive effects of selenium supplementation by metabolic reprogramming of cancer cells. Some exercise factors such as myokines can be possible mechanisms for the induction of these metabolic reprogramming effects. In this chapter, we will highlight the role of physical exercise and selenium in the prevention and treatment of cancer, and discuss possible mechanisms for modulating the effects of exercise during selenium supplementation.

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00042-0 © 2021 Elsevier Inc. All rights reserved.

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Selenium and human health Selenium is classified as an essential trace element and dietary micronutrient that has been found in suboptimum levels in most populations of the world. In humans, selenium has nutritional functionality as selenoproteins that can act through transfer ribonucleic acid (tRNA)-mediated incorporation of selenocysteine.10, 11 Also, Selenium acts as selenomethionine with replacing sulfur in methionine and can bind with certain proteins as selenium-binding proteins.12 One of the important members of the selenoproteins family is glutathione peroxidase (GPx) that acts as an antioxidant enzyme. This enzyme catalyzes the oxidation of reduced glutathione and allows for the reduction of hydrogen peroxide to water. So, it can prevent lipid peroxidation and cellular damage. Also, these enzymes can regulate deoxyribonucleic acid (DNA) transcription and cell proliferation.13, 14 Thioredoxin reductase (TrxR) is another selenoenzyme that acts as an antioxidant and is activated in all tissues. This selenoenzyme is responsible for degrading peroxides and hydroperoxides outside cell membranes in order to prevent cell death, DNA damage, and tissue atrophy.15 TrxR as an enzyme in the thioredoxin system can have an important role in many cellular functions such as redox control of transcription factors, synthesis of deoxyribonucleotides, cell growth, and protection against oxidative stress.16 Selenoprotein also exists in some other enzyme systems including iodothyronine deiodinases, selenophosphate synthases as well as selenoprotein P and selenoprotein W.17 Also, selenium can have many actions on the endocrine system by expression of selenoproteins that are involved in thyroid hormone regulation. Moreover, the interaction between selenium status, sex hormones, and thyroid metabolism was proposed in some studies.18, 19 Selenium has a significant role in the insulin signaling cascade, the expression of lipogenic enzymes, and in carbohydrate metabolism in the liver.20, 21 Development of insulin resistance has been related to changes in selenium levels and dietary supplementation with Se decreased fasting serum insulin concentrations.22 In addition, immune stimulatory effects of Se supplementation were observed in T cell proliferation, natural killer cell activity, cytotoxic lymphocyte, and innate immune cells.10, 23 Also, the effects of selenium on human blood leukocytes and the anti-inflammatory effects of this element have been observed in some studies.24 On the other hand, human selenium toxicity was observed in some studies. A U-shaped association between serum selenium and all-cause mortality was proposed. It seems that both low and high physiological ranges of selenoproteins can cause harmful effects.25 Also, physiological side effects, such as muscle atrophy, depressive symptoms, coronary artery risk, etc., were observed in high doses.26, 27 Some other studies proposed that selenium supplementation with consideration to its metabolic effects can be associated with an increased risk of diabetes.27 Using selenium as a dietary supplement has been proposed in recent years. In the following section, we will discuss the possible effects of selenium in cancer prevention and treatment.

Selenium and cancer Selenium supplementation has been suggested to protect against several types of cancer such as lung, bladder, colorectal, liver, esophageal, gastric cardia, thyroid, breast, and prostate.28 Supplementation with 50 mg daily selenium for about 8 years had decreased 35% incidence of hepatocellular carcinoma.29 Similar results were proposed in a meta-analysis in 16 studies on lung cancer, in which the results showed that higher selenium exposure defined as serum Se in >100 mg/L or intake >55 mg/day decreased the risk of lung cancer exposure.30 A follow-up study in China showed inverse relations between serum levels of selenium and death from esophageal squamous cell carcinoma and gastric cardia cancer in 1103 subjects.31 Antioxidant effects, interaction with some metals that were associated with an increase in the risk of cancer, and reversing the expression of genes implicated in carcinogenesis are some mechanisms that have been proposed for preventive effects of selenium in cancer.32 However, Vinceti et al. performed a meta-analysis that included 83 studies in different countries. They concluded that in different cancers selenium supplementation did not affect overall cancer incidence.28 This group considered genetic background, nutritional status, and effects of various forms of selenium as potential positive effects of selenium in some studies. In addition to the possible effects of selenium on cancer prevention, the effects of Se supplementation in the management of different types of cancers have been assessed in many studies. Selenium levels are decreased in some different cancer types, and it is important for cancer patients to maintain Se status in a certain range, especially for GPx activity.33 Also, the effects of selenium supplementation as a potential adjuvant in cancer have been mentioned when trying to decrease the side effects of radiation and chemotherapy treatments.34 The exact mechanisms of Se that cause the anticancer effects are not clear. Some possible mechanisms can be improving the antioxidant defense systems, reduction of oxidative stress, limiting DNA damage, induction of apoptosis, cell cycle arrest and DNA repair genes, inhibition of protein kinase C activity and cell growth, shift toward anticancer immunity, and Se effects on estrogen- and androgen-receptor expression.35

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Moreover, the potential effects of selenium as an antiangiogenesis or anti-metastasis agent have been proposed in some studies. Selenium supplementation can be effective on vascular endothelial growth factor (VEGF) as a factor for angiogenesis and downregulate some genes that are included in metastasis such as osteopontin and collagen.36 It is proposed that the therapeutic effects of selenium will only be seen in high dietary intake and supra-nutritional supplementation.35 Lubinski et al. showed that the optimum level of selenium that can help the outcome of laryngeal cancer patients is above 70mg/L37. Some studies have proposed that these doses are close to the toxic levels. Kenfield et al. showed that a high dose of selenium supplement after a diagnosis of nonmetastatic prostate cancer may increase the risk of prostate cancer mortality.38 It is proposed that although antioxidants in high doses have shown beneficial effects as a treatment strategy for a cancer situation, they are also implicated with increasing the cancer cell progression, especially in advanced stages.39 However, it seems that a redox state can be effective on the pharmacological and toxicity effects of selenium.17 Physical exercise can change the plasma and tissues redox status in an intensity-dependent manner. Gueritat et al. showed that a combination of exercise training and antioxidants could induce synergistic effects through oxidative stress modulation in prostate cancer progression.40 We also have shown that a combination of exercise training and selenium nanoparticles could prevent physiological side effects of selenium and decreased tumor volume in 4T1 breast cancer mouse model.41 Whether and how exercise training can help prevention and treatment of cancer besides some supplements such as selenium is something we will discuss in the following sections.

Physical exercise and cancer Exercise training with proper intensity and duration is associated with a lower risk of various cancers such as colon, endometrial in overweight and obese women, breast, prostate, gastroesophageal, ovarian, renal, lung, and pancreatic.3 Although it is not possible to draw a complete conclusion about the intensity, type, frequency, and duration of physical activity across the life span, it is clear that higher intensity exercise activities seem to be superior to light activities such as occupational, transport, and household.42 Also, various secondary complications, such as physical and psychological issues, have been reported for cancer treatments. Lifestyle changes such as exercise and dietary supplements appear to be effective in reducing the risk of secondary complications.42 Various evidences have shown that physical exercises were effective on bone loss and diseases, muscle loss and weight imbalance, cachexia, peripheral neuropathy, lymphedema, pain, fatigue, sleep disorders, depression, anxiety, quality of life, and self-esteem in cancer patients.43 Also, it is suggested that exercise training has a potential role in reducing chemotoxicity throughout a wide range of cancers.44 Various mechanisms have been controversial regarding the effects of physical exercise on tumor growth. Some studies have shown that physical exercise can control tumor growth and its intrinsic factors through interplay with systemic factors. Tumor growth depends on some extrinsic factors during physical exercise, which include changes in tumor blood flow, shear stress, pH regulation, heat production, sympathetic activation, and endocrine effects. All these factors are influenced by the duration, type, frequency, and intensity of the exercise. Changes in tumor tissue blood flow and increased intra-tumor perfusion following exercise have been observed in some cancers such as breast, prostate, and lymphoma.5 Increased blood flow to tumor tissue can increase access to systemic therapies and greater infiltration of immune cells. Also, it is shown that vascular normalization with increased protein levels in some angiogenesis factors such as hypoxia-inducible factor-1 alpha (HIF-1a) and possibly VEGF in the tumor microenvironment following exercise training may be effective in suppressing metastasis (Fig. 1).45 FIG. 1 Effects of physical exercise on tumor microenvironment. Systemic exerciseinduced responses can influence the tumor microenvironment and probably can control tumor development. These systemic responses are dependent on the duration, type, frequency, and intensity of the physical exercise.

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Identification and eradication of tumor cells through immune factors is one of the body’s major weapons to fight the pressures of cancer. Immune regulation at the systemic immune system and host immunity plays a major role in this process. Changes in natural killer (NK) cells and cytotoxic T cells in the tumor microenvironment have been associated with improved patient status.46 In recent years, changes in NK cells and macrophages, in particular, have been approved in terms of the number and enhanced cytotoxicity in the innate immune system following exercise activities in cancer.5 Exercise-induced epigenetic changes such as histone acetylation and DNA methylation have been approved as potential mechanisms in NK cell activation following physical exercise. It is suggested that epigenetic changes with exercise are dose dependent and low to moderate doses could not induce epigenetic changes in NK cells.47 Also, the number and mobilization of NK cells were dependent on changes in catecholamine concentrations. Since catecholamine concentrations are intensity dependent, it seems that exercise-induced NK cells are intensity dependent too.48 Other factors that are associated with changes in catecholamine concentrations following acute exercise are inflammatory cytokines. Changes in some inflammatory cytokines such as interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-a) following acute physical exercise have been associated with reduced tumor growth in clone and breast cancers.49, 50 Also, physical exercise with different intensity had various effects on the pro- and antioxidant balance. Acute physical exercise can induce oxidative stress responses based on force and metabolic demands. Although chronic inflammation and increased levels of oxidative stress can be effective in triggering cancer formation and development, cancer adjuvants such as chemotherapy and radiation therapy with exacerbated production of oxidative stress and inflammatory cytokines are effective in inducing cell death in the tumor cells.51 So, acute high-intensity physical exercise can be effective as coadjuvants alongside current treatments such as chemotherapy and radiotherapy for inducing acute oxidative stress, inflammatory cytokines, and immune responses. On the other hand, cancer has been described as a chronic inflammatory disease. In fact, tumor cells upregulate some inflammatory cytokines that show immunosuppressive effects. The modulatory effects of exercise on host immune responses can decrease inflammatory responses. Also, exercise training can modulate inflammation with metabolic improvements in some tissues such as fat and skeletal muscle. Skeletal muscle as an endocrine organ produces and releases some cytokines and proteins, called myokines, that can be mediated by the anti-inflammatory and metabolic effects of exercise on other tissues.52 Also, cancer cells metabolism as an essential aspect of tumor growth can be affected by physical exercise. Based on the Wahlberg effect, tumors prefer using an accelerated aerobic glycolytic to provide its energy.5 So, a change in tumor metabolism toward oxidative phosphorylation by alterations in some factors such as AMP-activated protein kinase (AMPK) that undergo endurance type exercise activities can be effective in controlling tumor growth.5 Also, Hofmann suggested that high intensity anaerobic exercise can inhibit glycolysis in tumor. So, its effects on tumor metabolism are stronger than moderate intensity aerobic exercise.6 Exercise can be effective on angiogenesis, metastasis, apoptosis, oxidative balance, epigenetic, antitumor immunity, and metabolic pathways in the tumor microenvironment. With consideration to common mechanisms that are mentioned for Se and physical exercise in cancer, maybe they can synergistically induce additive effects. Also, the combined effects of nutrition therapy and exercise prescription should always be considered to manage treatment-related side effects and complications. Selenium showed therapeutic effects in high dietary intake and supra-nutritional supplementation that can be toxic and show physiological side effects. Whether the exercise can be effective in controlling the toxic effects of selenium in cancer is controversial.

Exercise and selenium: Possible metabolic reprogramming in cancer cells In recent years, research in the field of tumor metabolism has revealed a network of metabolic regulators beyond that of the Wahlberg effect. While metabolic reprogramming used to consider how and why cancer cells preferentially utilize glucose via aerobic glycolysis, now we know that cancer cells use different nutrient sources and metabolic regulation of the tumor microenvironment is related to some factors such as host immune responses and nutrient availability.53 So, some interventions such as physical exercise that can induce the regulation of metabolism in different tissues and change host immune cells and some metabolites may be involved in metabolic reprogramming in cancer cells (Fig. 2). The effects of selenium in different forms and doses on metabolism are controversial. It is proposed that Selenium can accelerate glucose metabolism with activation of insulin signaling, glycolytic pathway, and pyruvate metabolism.20, 21 On the other hand, physical exercise, especially of higher intensity, is effective in inhibiting glycolysis and inducing glycolysisdependent mitochondrial dysfunction in tumor cells. Changes in some circulating metabolites such as lactate following high-intensity exercise are part of the potential regulators in tumor cells. It is proposed that increased systemic lactate levels following high-intensity exercise can inhibit glycolysis and net lactate production in tumor cells and improve antitumor

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FIG. 2 Metabolic reprogramming effects of exercise in tumor cells. The possible effects of exercise on tumor metabolic pathways, cachexia treatment and decreasing chronic inflammation through myokines, catecholamine, metabolites, and immune responses can induce metabolic reprogramming in tumor cells.

immunity.6, 54 Also, acute exercise-induced catecholamines can activate some metabolic pathways such as the Hippo tumor suppressor that inhibits the tumor metabolism. The Hippo pathway inhibits nuclear transcriptional coactivators YAP and TAZ that have a regulatory role on glucose metabolism, fatty acid metabolism, mevalonate metabolism, and glutamine metabolism in cancer cells.55, 56 In addition, cancer cells for proliferation, survival, and metastasis induce metabolic changes that lead to cancer cachexia. Cachexia syndrome is associated with body weight loss, inflammation, skeletal muscle, and fat wasting to provide external nutrient sources for cancer cells. We have shown that selenium nanoparticles in 4T1 tumor bearing mice induce inflammation, skeletal muscle atrophy, decreases in food intake, marked weight loss, and lead to cachexia situation.41 Physical exercise can stimulate metabolic pathways that in chronic ways as exercise training can optimize metabolic function in different tissues. Our studies showed that a combination of selenium and aerobic interval exercise training can prevent skeletal muscle atrophy and subsequently cachexia in cancer.41, 57 The limitation of nutrient sources for cancer cells with metabolic adjustment in other tissues such as skeletal muscle and fat following exercise can be one of the main ways for metabolic reprogramming in cancer cells. Also, skeletal muscle myokines have important roles in the interaction between skeletal muscle and different tissues. The possible interaction between skeletal muscles, liver, and fat tissues can modulate metabolism and body composition in cancer. Moreover, the possible cross talk between skeletal muscle and tumor tissue during physical exercise through myokines can affect metabolic pathways in cancer cells. The effects of myokines on the metabolic pathway in tumor cells can be one of the mechanisms for activation of antitumor immune responses. Increased levels of muscle IL-6 following exercise have been shown to be effective in greater secretion of NK cells and a reduction in tumor volume.58 Also, leukemia inhibitory factor (LIF) as a myokine could activate Hippo and YAP signaling.59 The regulatory effects of exercise through myokines on tumor metabolism may be another mechanism for regulating effects of physical exercise during selenium supplementation. Increased levels of some myokines such as OSM and interleukin-15 (IL-15) in the skeletal muscle of tumor-bearing mice were associated with antitumor immune responses in mammary carcinoma following exercise training and selenium combination in our studies.41, 57 In another way, despite the positive effects of acute high-intensity physical exercise on tumor cells, this kind of exercise can be associated with increasing some inflammatory and stress factors. If the acute effects of acute high-intensity exercise are not properly restored, it can chronically increase inflammatory and oxidative stress and lead to faster tumor growth.60 Chronic increased levels of stress factors can upregulate metabolic pathways and be involved in Warburg effects in cancer

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cells.61 Selenium supplementation has shown antioxidant effects following intense exercise activity in some studies.62, 63 So, selenium as an antioxidant can be effective in modulation of stress factors following high-intensity exercise in cancer. It seems that physical exercise can induce metabolic reprogramming in cancer cells. Also, cancer cachexia prevention and metabolic adjustment following exercise training in other tissues such as skeletal muscle and fat can limit nutrient sources for cancer cells. These metabolic effects can induce metabolic reprogramming effects in cancer cells during selenium supplementation and provoke antitumor immune responses. Also, the antioxidant effects of selenium have probably beneficial effects against exercise-induced oxidative damage. However, the combination effects of exercise training with antioxidants, such as selenium, on both health and disease conditions are not clear until now. Further studies in this area, especially clinical trials, may be useful for appropriate recommendations to cancer patients. Evaluation of different doses of selenium supplementation and exercise trainings with different type, intensity, and duration can also be effective in finding effective prescriptions in different cancers.

Applications to other cancers or conditions In this chapter, we reviewed the effects of a combination of physical exercise and selenium supplementation on cancer cells. We showed that physical exercise, in addition to its effects on the physical and psychological complications of cancer and related therapies, can be effective on the tumor microenvironment. Exercise can modulate systemic and host metabolic pathways through some possible mechanisms such as myokines, metabolites, immune responses, and catecholamines. We showed that selenium in high doses can be toxic and may increase the risk of cancer mortality in breast cancer. Exercise can modulate selenium’s side effects with metabolic reprogramming in cancer cells. We showed that combining exercise training and selenium supplementation could be a strategy for managing tumor volume and stimulating host and splenocytes antitumor immune responses in breast cancer. Also, exercise training not only prevented selenium side effects such as skeletal muscle atrophy, chronic inflammation, and anorexia, but also could prevent a cachexia situation in tumor-bearing mice. Our evidences have approved effects of selenium and exercise combination on breast cancer. Kenfield et al. showed that a high dose of selenium supplementation after diagnosis of nonmetastatic prostate cancer may increase the risk of prostate cancer mortality.38 Exercise probably can induce metabolic reprogramming in other cancers such as prostate during selenium supplementation. The combination effects of exercise training with antioxidants such as selenium on both health and disease conditions are not clear until now. Further studies in this area, especially clinical trials, may be useful for appropriate recommendations to cancer patients. Evaluation of different doses of selenium supplementation and exercise trainings can also be effective in finding effective prescriptions in different cancers.

Summary points l l l l l

l l l

Physical exercise affects metabolism and antitumor immune phenotype in cancer cells. The type, frequency, duration, and intensity of physical exercise can be effective on metabolic responses in cancer cells. Antioxidant, anti-inflammatory, and antimetastatic effects of selenium were observed in cancer. Selenium becomes a prooxidant and is toxic at high doses in cancer. Prevention of cancer cachexia and metabolic adjustment following exercise training can limit nutrient sources for cancer cells. Exercise probably modulates effects of selenium supplementation by metabolic reprogramming in cancer. Myokines as exercise factors act as possible mechanisms for metabolic reprogramming in cancer. Selenium as an antioxidant decreases stress factors following high-intensity exercise.

References 1. Caspersen CJ, Powell KE, Christenson GM. Physical activity, exercise, and physical fitness: definitions and distinctions for health-related research. Public Health Rep 1985;100:126–31. 2. Shim UJ, Kim HJ, Oh SC, Lee S, Choi SW. Exercise during adjuvant treatment for colorectal cancer: treatment completion, treatment-related toxicities, body composition, and serum level of adipokines. Cancer Manag Res 2019;11:5403–12. 3. Ruiz-Casado A, Martı´n-Ruiz A, P
erez LM, Provencio M, Fiuza-Luces C, Lucia A. Exercise and the hallmarks of cancer. Trends Cancer 2017;3 (6):423–41. 4. Ashcraft KA, Warner AB, Jones LW, Dewhirst MW. Exercise as adjunct therapy in cancer. Semin Radiat Oncol 2019;29(1):16–24. 5. Pedersen L, Christensen JF, Hojman P. Effects of exercise on tumor physiology and metabolism. Cancer J 2015;21(2):111–6. 6. Hofmann P. Cancer and exercise: warburg hypothesis, tumour metabolism and high-intensity anaerobic exercise. Sports (Basel) 2018;6(1):E10. 7. Whanger PD. Selenium and its relationship to cancer: an update. Br J Nutr 2004;91(1):11–28.

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8. Drake EN. Cancer chemoprevention: selenium as a prooxidant, not an antioxidant. Med Hypotheses 2006;67(2):318–22. 9. Zoidis E, Seremelis I, Kontopoulos N, Danezis GP. Selenium-dependent antioxidant enzymes: actions and properties of selenoproteins. Antioxidants (Basel) 2018;7(5):E66. 10. Avery JC, Hoffmann PR. Selenium, selenoproteins, and immunity. Nutrients 2018;10(9):E1203. 11. Mix H, Lobanov AV, Gladyshev VN. SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes. Nucleic Acids Res 2007;35(2):414–23. 12. Behne D, Kyriakopoulos A. Mammalian selenium-containing proteins. Annu Rev Nutr 2001;21:453–73. 13. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: biochemical role as a component of glutathione peroxidase. Science 1973;179(4073):588–90. 14. Rayman MP. Selenium in cancer prevention: a review of the evidence and mechanism of action. Proc Nutr Soc 2005;64(4):527–42. 15. Maiorino M, Thomas JP, Girotti AW, Ursini F. Reactivity of phospholipid hydroperoxide glutathione peroxidase with membrane and lipoprotein lipid hydroperoxides. Free Radic Res Commun 1991;12–13(Pt. 1):131–5. 16. Karlenius TC, Tonissen KF. Thioredoxin and cancer: a role for thioredoxin in all states of tumor oxygenation. Cancers (Basel) 2010;2(2):209–32. 17. Hawkes WC, Alkan Z. Regulation of redox signaling by selenoproteins. Biol Trace Elem Res 2010;134(3):235–51. 18. Larsen PR, Zavacki AM. The role of the iodothyronine deiodinases in the physiology and pathophysiology of thyroid hormone action. Eur Thyroid J 2012;1(4):232–42. 19. Zagrodzki P, Ratajczak R. Selenium status, sex hormones, and thyroid function in young women. J Trace Elem Med Biol 2008;22(4):296–304. 20. Wang N, Tan HY, Li S, Xu Y, Guo W, Feng Y. Supplementation of micronutrient selenium in metabolic diseases: its role as an antioxidant. Oxidative Med Cell Longev 2017;2017:7478523. 21. Chen H, Qiu Q, Zou C, Dou L, Liang J. Regulation of hepatic carbohydrate metabolism by Selenium during diabetes. Chem Biol Interact 2015;232:1–6. 22. Jacobs ET, Lance P, Mandarino LJ, Ellis NA, Chow HS, Foote J, et al. Selenium supplementation and insulin resistance in a randomized, clinical trial. BMJ Open Diabetes Res Care 2019;7(1):e000613. 23. Rayman MP. Selenium and human health. Lancet 2012;379(9822):1256–68. 24. Bentley-Hewitt KL, Chen RK, Lill RE, Hedderley DI, Herath TD, Matich AJ, et al. Consumption of selenium-enriched broccoli increases cytokine production in human peripheral blood mononuclear cells stimulated ex vivo, a preliminary human intervention study. Mol Nutr Food Res 2014;58 (12):2350–7. 25. Cold F, Winther KH, Pastor-Barriuso R, Rayman MP, Guallar E, Nybo M, et al. Randomised controlled trial of the effect of long term selenium supplementation on plasmacholesterol in an elderly Danish population. Br J Nutr 2015;114(11):1807–18. 26. Morris JS, Crane SB. Selenium toxicity from a misformulated dietary supplement, adverse health effects, and the temporal response in the nail biologic monitor. Nutrients 2013;5(4):1024–57. 27. Colangelo LA, He K, Whooley MA, Daviglus ML, Morris S, Liu K. Selenium exposure and depressive symptoms: the coronary artery risk development in young adults trace element study. Neurotoxicology 2014;41:167–74. 28. Vinceti M, Filippini T, Del Giovane C, Dennert G, Zwahlen M, Brinkman M, et al. Selenium for preventing cancer. Cochrane Database Syst Rev 2018;1:CD005195. 29. Yu SY, Zhu YJ, Li WG, Huang QS, Huang CZ, Zhang QN, et al. A preliminary report on the intervention trials of primary liver cancer in high-risk populations with nutritional supplementation of selenium in China. Biol Trace Elem Res 1991;29(3):289–94. 30. Zhuo H, Smith AH, Steinmaus C. Selenium and lung cancer: a quantitative analysis of heterogeneity in the current epidemiological literature. Cancer Epidemiol Biomark Prev 2004;13(5):771–8. 31. Wei WQ, Abnet CC, Qiao YL, Dawsey SM, Dong ZW, Sun XD, et al. Prospective study of serum selenium concentrations and esophageal and gastric cardia cancer, heart disease, stroke, and total death. Am J Clin Nutr 2004;79(1):80–5. 32. Bj€ ornstedt M, Fernandes AP. Selenium in the prevention of human cancers. EPMA J 2010;1(3):389–95. 33. Jiao Y, Wang Y, Guo S, Wang G. Glutathione peroxidases as oncotargets. Oncotarget 2017;8(45):80093–102. 34. Muecke R, Micke O, Schomburg L, Kisters K, Buentzel J, Huebner J, et al. Selenium supplementation in radiotherapy patients: do we need to measure selenium levels in serum or blood regularly prior radiotherapy? Radiat Oncol 2014;9:289. 35. Tinggi U. Selenium: its role as antioxidant in human health. Environ Health Prev Med 2008;13(2):102–8. 36. Chen YC, Prabhu KS, Mastro AM. Is selenium a potential treatment for cancer metastasis? Nutrients 2013;5(4):1149–68. 37. Lubi
nski J, Marciniak W, Muszynska M, Jaworowska E, Sulikowski M, Jakubowska A, et al. Serum selenium levels and the risk of progression of laryngeal cancer. PLoS One 2018;13(1):e0184873. 38. Kenfield SA, Van Blarigan EL, DuPre N, Stampfer MJ, Giovannucci EL, Chan JM. Selenium supplementation and prostate cancer mortality. J Natl Cancer Inst 2014;107(1):360. 39. Sarangarajan R, Meera S, Rukkumani R, Sankar P, Anuradha G. Antioxidants: friend or foe? Asian Pac J Trop Med 2017;10(12):1111–6. 40. Gueritat J, Lefeuvre-Orfila L, Vincent S, Cretual A, Ravanat JL, Gratas-Delamarche A, et al. Exercise training combined with antioxidant supplementation prevents the antiproliferative activity of their single treatment in prostate cancer through inhibition of redox adaptation. Free Radic Biol Med 2014;77:95–105. 41. Molanouri Shamsi M, Chekachak S, Soudi S, Quinn LS, Ranjbar K, Chenari J, et al. Combined effect of aerobic interval training and selenium nanoparticles on expression of IL-15 and IL-10/TNF-a ratio in skeletal muscle of 4T1 breast cancer mice with cachexia. Cytokine 2017;90:100–8. 42. Oruc¸ Z, Kaplan MA. Effect of exercise on colorectal cancer prevention and treatment. World J Gastrointest Oncol 2019;11(5):348–66.

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43. Ferioli M, Zauli G, Martelli AM, Vitale M, McCubrey JA, Ultimo S, et al. Impact of physical exercise in cancer survivors during and after antineoplastic treatments. Oncotarget 2018;9(17):14005–34. 44. Schumann M, Schulz H, Hackney AC, Bloch W. Feasibility of high intensity interval training with hyperoxia vs. intermittent hyperoxia and hypoxia in cancer patients undergoing chemotherapy—study protocol of a randomized controlled trial. Contemp Clin Trials Commun 2017;8:213–7. 45. Amitani M, Asakawa A, Amitani H, Inui A. Control of food intake and muscle wasting in cachexia. Int J Biochem Cell Biol 2013;45(10):2179–85. 46. Hojman P, Gehl J, Christensen JF, Pedersen BK. Molecular mechanisms linking exercise to cancer prevention and treatment. Cell Metab 2018;27 (1):10–21. 47. Schenk A, Bloch W, Zimmer P. Natural killer cells—an epigenetic perspective of development and regulation. Int J Mol Sci 2016;17(3):326. 48. Idorn M, Hojman P. Exercise-dependent regulation of NK cells in cancer protection. Trends Mol Med 2016;22(7):565–77. 49. Devin JL, Hill MM, Mourtzakis M, Quadrilatero J, Jenkins DG, Skinner TL. Acute high intensity interval exercise reduces colon cancer cell growth. J Physiol 2019;597(8):2177–84. 50. Dethlefsen C, Lillelund C, Midtgaard J, Andersen C, Pedersen BK, Christensen JF, et al. Exercise regulates breast cancer cell viability: systemic training adaptations versus acute exercise responses. Breast Cancer Res Treat 2016;159(3):469–79. 51. Yang H, Villani RM, Wang H, Simpson MJ, Roberts MS, Tang M, et al. The role of cellular reactive oxygen species in cancer chemotherapy. J Exp Clin Cancer Res 2018;37(1):266. 52. Leal LG, Lopes MA, Batista Jr ML. Physical exercise-induced myokines and muscle-adipose tissue crosstalk: a review of current knowledge and the implications for health and metabolic diseases. Front Physiol 2018;9:1307. 53. Sullivan MR, Danai LV, Lewis CA, Chan SH, Gui DY, Kunchok T, et al. Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability. elife 2019;8:e44235. 54. Romero-Garcia S, Moreno-Altamirano MM, Prado-Garcia H, Sa´nchez-Garcı´a FJ. Lactate contribution to the tumor microenvironment: mechanisms, effects on immune cells and therapeutic relevance. Front Immunol 2016;7:52. 55. Dethlefsen C, Hansen LS, Lillelund C, Andersen C, Gehl J, Christensen JF, et al. Exercise-induced catecholamines activate the hippo tumor suppressor pathway to reduce risks of breast cancer development. Cancer Res 2017;77(18):4894–904. 56. Zhang X, Zhao H, Li Y, Xia D, Yang L, Ma Y, et al. The role of YAP/TAZ activity in cancer metabolic reprogramming. Mol Cancer 2018;17(1):134. 57. Molanouri Shamsi M, Chekachak S, Soudi S, Gharakhanlou R, Quinn LS, Ranjbar K, et al. Effects of exercise training and supplementation with selenium nanoparticle on T-helper 1 and 2 and cytokine levels in tumor tissue of mice bearing the 4 T1 mammary carcinoma. Nutrition 2019;57:141–7. 58. Pedersen L, Idorn M, Olofsson GH, Lauenborg B, Nookaew I, Hansen RH, et al. Voluntary running suppresses tumor growth through epinephrine- and IL-6-Dependent NK cell mobilization and redistribution. Cell Metab 2016;23:554–62. 59. Hergovich A. YAP-Hippo signalling downstream of leukemia inhibitory factor receptor: implications for breast cancer. Breast Cancer Res 2012;14 (6):326. 60. Sa´ez Mdel C, Barriga C, Garcı´a JJ, Rodrı´guez AB, Ortega E. Exercise induced stress enhances mammary tumor growth in rats: beneficial effect of the horrmone melatonin. Mol Cell Biochem 2006;294(1–2):19–24. 61. Fiaschi T, Chiarugi P. Oxidative stress, tumor microenvironment, and metabolic reprogramming: a diabolic liaison. Int J Cell Biol 2012;2012:762825. 62. Akil M, Gurbuz U, Bicer M, Halifeoglu I, Baltaci AK, Mogulkoc R. Selenium prevents lipid peroxidation in liver and lung tissues of rats in acute swimming exercise. Bratisl Lek Listy 2015;116(4):233–5. 63. White SH, Johnson SE, Bobel JM, Warren LK. Dietary selenium and prolonged exercise alter gene expression and activity of antioxidant enzymes in equine skeletal muscle. J Anim Sci 2016;94(7):2867–78.

Chapter 43

Silybum marianum, antioxidant activity, and cancer patients Sepideh Elyasi Department of Clinical Pharmacy, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

List of abbreviations SM ROS NOX4 Nrf2 LDL AP1 GSH TNF EGFR SOD ERK RSK MAPK NSCLC TPA MCD MMP TRAMP IGF NO DMH RT-PCR DNMT PTEN mTOR ALL RT

silymarin reactive oxygen species NADPH oxidase 4 nuclear factor erythroid 2 low-density lipoprotein activator protein 1 glutathione tumor necrosis factor epidermal growth factor receptor superoxide dismutase extracellular-signal-regulated kinase ribosomal S6 kinase mitogen-activated protein kinase nonsmall cell lung cancer 12-O-tetradecanoyl phorbol-13-acetate mast cell density matrix metallopeptidases transgenic adenocarcinoma of the mouse prostate insulin-like growth factor nitric oxide 1,2-dimethylhydrazine reverse transcriptase polymerase chain reaction DNA methyltransferase phosphatase and tensin homolog mammalian target of rapamycin acute lymphoblastic leukemia radiotherapy

Introduction Silymarin, an antioxidant flavonoid complex derived from the herb milk thistle (Silybum marianum), has been used for long time in the treatment of liver diseases. It is a member of the Asteraceae family (Compositae) native to a limited area of the Mediterranean, but grown for centuries through Europe and North and South America; so naturalized also on these continents.1, 2 Silymarin (SM) is composed of several isomer flavonolignans: silybin (also spelled silybinin or silibinin), isosilybin, silychristin, silydianin, and dehydrosilibinin. Silybin is the key component and is frequently proposed as the substance responsible for SM biological activity.2–4 These properties seem to have resulted from their ability to scavenge free radicals, reactive oxygen species (ROS), and to chelate metal ions. Silymarin is often used in the treatment of liver diseases as it protects liver cells by stabilizing the membrane permeability through inhibiting lipid peroxidation and preventing liver glutathione depletion.5, 6 Moreover, silymarin shows different mechanisms in preventing the coronary vascular diseases by increasing enzymatic antioxidants, mitochondrial enzymes, and expression of nuclear factor erythroid Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00043-2 © 2021 Elsevier Inc. All rights reserved.

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2 (Nrf2) and by decreasing lipid peroxidation, expressions of NADPH oxidase 4 (NOX4), low-density lipoprotein (LDL), total cholesterol, and triglyceride level in the blood, thus preventing cardiac dysfunction and dyslipidemia.7 Silymarin shows antidepressant like activity perhaps by alleviating oxidative stress or by modulating corticosterone response, and antioxidant defense system in hippocampus and cerebral cortex in mice.8 It also has strong antidiabetic activity through reducing oxidative stress and inflammatory response, partially by inhibiting hepatic NADPH oxidase expression and the NF-kB and activator protein 1 (AP1) signaling pathways.9, 10 Silymarin has a hydroxyl group at C5 and a carbonyl group at C4, which may form a chelate with ferrous iron. This chelation can increase its antioxidant activity to the level of most active scavengers, possibly by site-specific scavenging. The free hydroxyl groups at C5 and C7 on the silymarin structure may also favor the inhibition of lipid peroxidation by reacting with peroxy radicals. This ability of silymarin leads to a noticeable elevation of the cellular antioxidant defense machinery by ameliorating the harmful effects of free radical reaction and by increasing glutathione (GSH) content, which is vital for preserving the ferrous state.6 The activity against a variety of cancers has sparked much interest from drug discovery world. Silymarin and silibinin have most of the characteristics of an ideal cancer chemopreventive agent; they are nontoxic to normal cells, selectively inhibit the growth of cancer cells, and are biologically available following its oral administration.11, 12 In the following, we briefly review the use of silymarin in various cancers (Table 1).

Skin cancer It is proposed that about 2–3 million cases of nonmelanoma and 0.132 million cases of melanoma skin cancer occur each year all over the world. Reactive oxygen species have been shown to stimulate transcription factors like AP-1 and NF-kB which result in cell proliferation and cell death and finally in skin cancer. Silymarin inhibits tumor necrosis factor (TNF)-a, mitogenic, and cell survival and also epidermal growth factor receptor (EGFR)-mediated mitogenic signaling pathway.12, 16 It induces apoptosis and downregulates expression of numerous pro-inflammatory cytokines. Moreover, it inhibits transactivation of androgen receptor and activates cellular check points and causes G1 and/or G2-M cell cycle arrest.12 However, its low deposition to skin after oral administration restricts its use in the prevention and treatment of skin cancer. As cancer cells develop in the epidermis of the skin, topical delivery of silymarin can be useful in achieving adequate concentration of drug in skin layers. Low uptake because of the barrier function of the stratum corneum is a main concern in topical delivery of silymarin. In a newly published study, silymarin-loaded nanostructured lipid carriers were prepared for epidermal drug deposition enhancement in topical applications in melanoma cell line and albino mice as ex vivo and in vivo TABLE 1 Summary of the chemopreventive effect of silymarin against various cancer sites cancer sites. Cancer type

References

Skin cancer

Katiyar et al.13, Deep and Agarwal12, Yu et al.14, Lee et al.15, Vaid et al.16, and Ng et al.17

Larynx and lung cancer

Bang et al.18, Cheung et al.19, and Mateen et al.20

Breast cancer

Kim et al.21, Wang et al.22, Tamaki et al.23, Kim et al.24, Noh et al.25, Dunnick et al.26, Dastpeyman et al.27, and Oh et al.28

Hepatic and pancreatic cancers

Ramakrishnan et al.29, Brandon-Warner et al.30, Nambiar et al.31, and Bousserouel et al.32

Ovarian cancer

Cho et al.33

Prostate cancer

Deep and Agarwal12, Verschoyle et al.34, Raina et al.35, Mokhtari et al.36, Wu et al.37, Flaig et al.38, Vidlar et al.39, Kavitha et al.40, Lu et al.41, and Ting et al.42

Colorectal cancer

Colombo et al.43, Kauntz et al.44, Toyoda-Hokaiwado et al.45, Yan et al.46, Kauntz et al.47, Kauntz et al.48, Karim et al.49, and Raina et al.50

Kidney and bladder cancer

Tyagi et al.51, Tyagi et al.52, and Kaur et al.53

Cervical cancer

Garcia-Maceira and Mateo54 and Yu et al.55

Leukemia

Ladas et al.56

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models, respectively. The volume of large tumors significantly reduced from 5.02 to 3.05 mm3, levels of IL-1a and TNF-a were significantly low, and levels of superoxide dismutase (SOD), catalase, and GSH significantly increased in the group of mice treated with silymarin-NLC gel. Furthermore, in skin treated with placebo and conventional gels, a basosquamous carcinoma and squamous cell carcinoma were noticed, respectively.16, 17 Yu et al. proposed that silibinin inhibited the cell growth in a dose- and time-dependent manner in human epidermoid carcinoma A431 cells. At a high dose (400 mM) it induced apoptosis through both the intrinsic and extrinsic apoptotic pathways via the generation of ROS and nitric oxide (NO). Silibinin treatment (50 mg/kg) daily for 4 days reversed dermal and epidermal autophagy levels from UVB irradiation-induced improper autophagy intervention, restored the balance between cell survival and death, and protected skin against damage through mediation of p53 activation in dermal and epidermal cells.14 Lee et al. showed that treatment of melanoma cells with silybin reduced the phosphorylation of extracellular-signal-regulated kinase (ERK)-1/2 and ribosomal S6 kinase (RSK)-2, which are controlled by upstream kinases such as mitogen-activated protein kinase (MAPK)1/2. The obstruction of MAPK1/2-ERK1/2-RSK2 signaling by silybin resulted in a reduced activation of NF-kB, activator protein-1, and STAT3, which are transcriptional regulators of a variety of proliferative genes in melanomas. It attenuated the growth of melanoma xenograft tumors in nude mice. Silybin inhibited the kinase activity of MAPK-1/2 and RSK-2 in melanoma cells. Cell cycle arrest was induced at the G1 phase and inhibited melanoma cell growth in vitro and in vivo. It was inferred that silybin suppresses melanoma growth by directly targeting MAPK- and RSK-mediated signaling pathways.15 In studies evaluating the protective effect of silymarin during tumor promotion stage, its application before each UVB exposure resulted in a prolonged latency period by an extra 3 weeks before the onset of the first tumor and in reduced tumor incidence, multiplicity, and tumor volume throughout the treatment course. A much more obvious effect of silymarin was observed in studies involving complete carcinogenesis by UVB. Topical application of silymarin for 14 days before UVB exposure as a tumor initiator and also during UVB-induced tumor promotion both delayed the latency period by 9 weeks and resulted in extremely significant protection against both tumor incidence and tumor multiplicity throughout the treatment period. Moreover, silymarin treatment significantly reduced the occurrence of sunburn and the number of apoptotic cells formed after UVB exposure. Furthermore, silymarin treatment inhibited UVB-caused cutaneous edema. The prevention of UVB-induced immunosuppression and oxidative stress by silymarin may be associated with the prevention of photocarcinogenesis in mice.12, 13 In a study by Vaid and Katiyar, the antioxidant, antiinflammatory, and immunomodulatory properties of silymarin were proposed for its protective role in UV-induced skin cancer. It is effective on the microenvironment constituents by disrupting tumor growth, angiogenesis, inflammation, and metastasis via targeting the signaling molecules for epithelialto-mesenchymal transition, proteases activation, adhesion, motility, invasiveness, etc.11, 16

Larynx and lung cancer Silibinin has been shown to inhibit tumor cell growth and stimulate apoptotic cell death in both small cell lung carcinoma cells [(SCLC) SHP-77] and nonsmall cell lung carcinoma cells [(NSCLC) A-549] in a dose- and time-dependent manner. Silymarin inhibited cell invasion via the reduction of matrix metallopeptidases (MMP)s and urokinase-type plasminogen activator, and through inactivation of both PI3K-Akt and MAPK signaling pathways. In an in vivo mouse model of urethane-induced lung tumor, the administration of silibinin inhibited tumor progression by reducing the cell proliferation, modulating cyclin expression, and suppressing expression of angiogenic growth factors like vascular endothelial growth factor and tumor-promoting enzymes (such as inducible nitric oxide synthase and cyclooxgenase-2).19 Bang et al. demonstrated that silibinin induces apoptosis of laryngeal squamous carcinoma SNU-46 cells by decreasing survivin (the protein inhibitor of apoptosis) expression in a dose- and time-dependent manner.18 Mateen et al. observed that silibinin in combination with HDAC inhibitor (Trichostatin A) or DNMT inhibitor (50 -aza-deoxycytidine) significantly restored E-cadherin levels in NSCLC cells. Treatment of NSCLC cells, with basal E-cadherin levels, by silibinin further increased their expression and inhibited migratory and invasive potential. Besides, silibinin with or without the inhibitors downregulated the expression of Zeb1, known to be a major transcriptional repressor of E-cadherin.20

Breast cancer Breast cancer accounts for 23% and 14% of the total cancer cases and deaths, respectively. Moreover, it is the most frequently diagnosed cancer and the leading cause of cancer-related death among women all over the world.57 Kim et al. described the downregulating effect of silibinin on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 and COX-2 expression in the human breast cancer MCF-7 and MDA-MB231 cells, probably by the obstruction of NF-kB,

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PI3K/Akt, and ERK1/2 signaling.21 Wang et al. observed that silibinin has a sustained O2-production capability in MCF-7 cells. It was correlated to the apoptosis enhancement ability of exogenous SOD.22 Tamaki et al. assessed the inhibitory effects of several herbal extracts and isoflavonoids, such as S. marianum, on intestinal breast cancer resistance protein (BCRP). It weakly inhibited BCRP-mediated transport of the xenobiotic methotrexate.23 Kim et al. stated the augmentation of TPA induced G2/M phase arrest in breast cancer cells and p21 expression by silibinin. It seems that silibinin has an additive effect on TPA-induced growth arrest through the PI-3-kinase/Akt-dependent pathway.21 Kim et al. also explained that CD44 and MMP-9 expression was reduced by silibinin treatment in a dose-dependent manner in human breast cancer SKBR3 and BT474 cells. Moreover, silibinin inhibited the phosphorylation of EGF receptor and the downstream signaling molecule ERK1/2.24 Noh et al. found a dose- and time-dependent reduction in viability in MMT assay, Western blot and flow cytometry in MCF-7 cells, which was correlated to increased p53 expression and induction of apoptosis. The combination of silibinin and UVB resulted in an additive effect on apoptosis in MCF-7 cells.25 Dunnick et al. considered the effect of S. marianum extract in rodents and described a reduction in mammary gland tumors. The radical-scavenging and antioxidant properties of silibinin and allied flavonolignans were proposed as the mechanisms of this protection.26 A dosedependent inhibition of Cdc42 and D4-GDI mRNAs expression by RT-PCR in MDA-MB-231 cells was shown by Dastpeyman et al. Moreover, silibinin effectively modulated b1-integrin signaling pathway and RAF1 function in an indirect manner.27 Oh et al. reported that TPA-induced MCF7 cell migration and MMP-9 expression was significantly diminished and phosphorylation of MEK and ERK was inhibited by silibinin.28

Hepatic and pancreatic cancers Nambiar et al. examined in vitro and in vivo effects of silibinin against both primary and advanced stages of human pancreatic carcinoma cells. Silibinin (25–100 mM) treatment for 24–72 h resulted in a dose- and time-dependent cell growth inhibition in BxPC-3 cells (27%–77%) and PANC-1 cells (22%–45%). Feeding nude mice with silibinin (0.5%, w/w in AIN-93M diet for 7 weeks) inhibited BxPC-3 and PANC-1 tumor xenograft growth and tumor volume and weight were significantly reduced.31 Ramakrishnan et al. reported that silymarin treatment inhibited the abnormal increase in mast cell density (MCD) (associated with neoplasm) and downregulated the expressions of MMP-2 and MMP-9 (involved in invasion and angiogenesis). Grafting of the cancer cells into mouse liver, followed by oral administration of silibinin (700 mg/kg) for 4 weeks, limited tumor growth, by downregulation of MMP-7, MMP-9, and IL1b and the upregulation of TRAIL, DR5, and caspase-3 activation.29 Bousserouel et al. stated the silibinin-mediated activation of TRAIL death receptor apoptotic signaling pathway both in in vitro and in hepatocarcinoma grafts in mice. Silibinin activated the extrinsic apoptotic pathway in Hep55.1C cells, demonstrated the upregulation of TRAIL and death receptor 5 transcripts and also the activation of caspase-3 and -8.32 Brandon-Warner et al. confirmed that silibinin inhibits in vitro cytochrome p4502E1 induction, ethanol metabolism, and ROS generation in hepatocellular carcinoma HCC cells in vitro.30

Ovarian cancer Cho et al. examined the effect of silibinin in vitro and in vivo on tumor growth in human ovarian cancer cells. Silibinin increased ROS production inside the cancer cells and compromised their viability. Oral administration of silibinin to animals with subcutaneous A2780 cells reduced tumor volume. It may induce a decrease in Ki-67-positive cells, an increase in transferase-mediated dUTP nick end labeling-positive cells, activation of caspase-3, and inhibition of p-ERK and p-Akt.33

Prostate cancer Prostate cancer is the second most common cancer in men worldwide, with an estimated 1,100,000 new cases and 307,000 deaths in 2012.58 Ting et al. stated that silibinin is capable of modulating cell signaling, proliferation, apoptosis, epithelialmesenchymal transition, invasion, metastasis, and angiogenesis. Thus, it might be developed as a prostate cancer chemopreventive agent.42 Silibinin or silipide (silibinin formulated with phospholipids) when given in diet delayed tumor development in transgenic adenocarcinoma of the mouse prostate (TRAMP) model, plasma levels of insulin-like growth factor (IGF)-1 (involved in cell transformation and proliferation) decreased by 36%, and tumor size reduced by 31%.34 Raina et al. also showed that the mice fed with 1% silibinin-enriched diet for 8–15 weeks presented less severe lesions in TRAMP model. This effect may be mediated by antiproliferative effect and also inhibition of angiogenesis. Reduced expressions of platelet endothelial cell adhesion molecule-1/CD-31, VEGF and associated receptors, HIF-1a, and iNO synthase were associated

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with the lowered tumorigenicity. Metastasis to distant organs was also diminished in silibinin-fed mice, which was associated with a decreased expression of MMPs, mesenchymal markers snail-1, and fibronectin in the prostatic tissue and retaining of epithelial characteristics.35 Mokhtari et al. stated that when PC-3 cells were incubated with silibinin, it resulted in a dose- and time-dependent inhibition of viability, motility, and adhesion of highly metastatic cells.36 Wu et al. found that silibinin treatment results in the upregulation of cytokeratin-18 and downregulation of vimentin and MMP2 in bone metastatic prostate ARCaPM cell line. Also, it inhibited NF-kB p50 translocation via the upregulation of IkB-a protein. Besides, it downregulated the expression of two major epithelial-mesenchymal transition regulators ZEB1 and SLUG, as cancer progression markers.37 Kavitha et al. observed the effect of silibinin on bone metastatic prostate cancer PCA cells-induced osteoclastogenesis employing human PC3MM2, PC3, and C4-2B and murine macrophage RAW264.7 cells. It inhibited PCA cell-induced osteoclast activity and differentiation in RAW264.7 cells by modulating the expression of several cytokines (IGF-1, TGF-b, TNF-a, I-TAC, M-CSF, G-CSF, GM-CSF) that are important in osteoclastogenesis, at 30–90 mM dose. In RAW264.7 cells, silibinin decreased the RANKL-induced expression and nuclear localization of NFATc1, which is proposed as the master regulator of osteoclastogenesis. Also, the DNA binding activity of NFATc1 and its regulators NF-kB and AP1, and the protein expression of osteoclast specific markers (TRAP, OSCAR, and cathepsin K) were decreased. Silibinin also reduced the expression of osteomimicry biomarkers (RANKL, Runx2, osteocalcin, and PTHrP) in cell culture (PC3 and C4-2B cells) and/or in PC3 tumors.40 Lu et al. found that silibinin inhibits LRP6 promoter activity and decreases LRP6 mRNA levels in prostate cancer PC-3 and DU-145 cells.41 Deep et al. evaluated different isomers of silybin (silybin A, silybin B, isosilybin A, and isosilybin B) for the management of advanced prostate cancer. Ingestion of these flavonolignans (50 and 100 mg/kg) successfully inhibited the growth of advanced human prostate cancer PCA DU145 xenografts. Immunohistochemical assays discovered that these isomers selectively inhibit tumor angiogenesis biomarkers (CD31 and nestin) and signaling molecules regulating angiogenesis (VEGF, VEGFR1, VEGFR2, phosphoAkt, and HIF-1a).12 Limited numbers of human studies have also been performed in this field. In a study by Flaig et al., high-dose oral silybin-phytosome achieves high blood concentrations transiently, but low levels of silibinin are found in prostate tissue. It may be due to its short half-life, the brief duration of therapy in this study, or an active process removing silibinin from the prostate.38 Vidlar et al. in clinical human study determined whether a daily administration of a silymarin and selenium combination for 6 months would modify basic clinical chemistry and oxidative stress markers, and also the quality of life score in men after radical prostatectomy. A total of 37 participants, 2–3 months after the surgery, were randomly assigned to receive 570 mg/d of silymarin and 240 mg/d of selenium as selenomethionine or placebo for 6 month. This intervention improved the quality of life score, reduced LDL, and total cholesterol while increasing the serum selenium levels. Also, it reduced the markers of lipid metabolism linked with prostate cancer progression.39

Colorectal cancer Colorectal cancer is the most common gastrointestinal cancer and the third leading cause of cancer death after lung and breast cancers.59Toyoda-Hokaiwado et al. explored the preventive effects of silymarin against carcinogenicity and genotoxicity induced by 1,2-dimethylhydrazine (DMH) injection followed by oral DSS in the colon of F344 gpt delta transgenic rats. Animals were fed diets containing silymarin for 4 weeks, from 1 week before DMH injection and samples were collected at 4, 20, and 32 weeks after the DMH treatment. Silymarin (100–500 ppm) dose dependently suppressed the tumor formation. Similarly, it considerably reduced (20%) the mutant frequency in the colon.45 In a study by Colombo et al., silymarin-doxorubicin and silymarin-paclitaxel treatments were evaluated on two colon carcinoma LoVo and the multidrug-resistant isogenic LoVo/DX cell lines. Pretreatment with low dose of silymarin was synergistically effective with both doxorubicin and paclitaxel in LoVo cells, while high dose of silymarin showed additive effect with both the chemotherapeutics. But, silymarin favorably affected the uptake and cell cycle effects of the chemotherapeutics only in LoVo cell.43 Kauntz et al. showed that silibinin prompted apoptosis in primary tumor SW480 and their derived metastatic SW620 cells showed by DNA fragmentation and activation of caspase-3. The expression of TRAIL and death receptors at the cell surface in SW480 as well as in SW620 cells were also enhanced with silymarin. The protein Bid was cleaved in SW480 cells, representative of a cross-talk between extrinsic and intrinsic apoptotic pathway. Besides, silibinin activated the intrinsic apoptotic pathway in both cell lines.44 Yan et al. evaluated the effect of a blend of polyphenon E (green tea) and siliphos (main component silibinin) on the growth of subcutaneous colon cancer CT-26 in several murine models. After daily administration for 7–9 days before the procedure and for 7–21 days postoperatively, the blend significantly inhibited tumor mass, number and size of hepatic metastases, and rate of proliferation and apoptosis.46 Kauntz et al. used azoxymethane-induced colon carcinogenesis model in rats. One week after the genotoxic agent injection, the rats received 300 mg silibinin/kg body weight daily by gavage for 7 weeks. Silibinin-treated rats showed a twofold reduction in the

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number of azoxymethane-induced hyperproliferative crypts and aberrant crypt foci in the colon. Silibinin induced apoptosis via the downregulation of the antiapoptotic protein Bcl-2 and upregulation of the proapoptotic protein Bax. Furthermore, this treatment significantly reduced the genetic expression of biomarkers of the inflammation (IL1b, TNFa) and their downstream target MMP7. Silibinin successfully checked colon carcinogenesis by antiinflammation and eventual apoptosis. Moreover, this research team investigated the effect of silibinin and TRAIL in SW480 and SW620 cells by means of reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry and showed synergistic induction of cell death in both the cell lines, exhibited upregulation of death receptor 4 and 5. Synergistic activation of caspase-3, -8, and -9 by silibinin and TRAIL was shown by colorimetric assays. Silibinin and TRAIL potentiated activation of the mitochondrial apoptotic pathway and downregulated the antiapoptotic proteins Mcl-1 and XIAP.47 Karim et al. proposed that silibinin effectively targeted Cdk4 pathway in Apc /+ mice and resulted in the hypophosphorylation of the retinoblastoma protein as a tumor suppression protein, inhibited cell growth, and induced apoptosis. Silibinin blocked the development of intestinal adenomas in the mice by 52%.49 Raina et al. stated that silibinin induces oxidative stress in SW480 cells as ROS generation is accompanied by the disruption of mitochondrial transmembrane potential and cytochrome c release. These modifications activate procaspase 3, resulting in apoptosis.50 Kauntz et al. found that silibinin could significantly inhibit DNA methyltransferase (DNMT) activity in both primary adenocarcinoma cells SW480 and their derived metastatic cells SW620 cells. Selective targeting of the inhibitors and high safety of silibinin held promise for cancer inhibition.48

Kidney and bladder cancer Intraperitoneal administration of the nephrotoxic agents to mice induced noticeable oxidative stress in kidney, presented by high renal metallothionein expression, depletion of glutathione content, and enhanced production of aldehyde products. Kaur et al. declared that feeding mice with 0.5% and 1% silymarin diet lowered oxidative stress and inflammation by promoting metallothionein expression, reducing NF-kB activation, and decreasing the expression of pro-inflammatory mediators. Moreover, silymarin also suppressed the induced hyperproliferation in kidney, improving renal ornithine decarboxylase activity and DNA synthesis.53 In human bladder transitional-cell papilloma RT4 cells, silibinin treatment decreased protein and mRNA levels of survivin, a member of apoptosis protein gene family inhibitors which is deregulated in bladder cancer.51 Silibinin similarly stimulated apoptotic death, presented by propidium iodide/annexin V flow cytometry and caspase-9,-3 and PARP cleavages. Another study on RT4 cells also confirmed their response to silibinin by both the intrinsic and extrinsic apoptotic pathways. Silibinin was found to activate p53 and caspase-2, leading to mitochondrial permeabilization, cytochrome c release, and cleavage of the cyclin-dependent kinase inhibitor, Cip1/p21.52 Another study found that silibinin-induced cell cycle arrest and apoptosis were related to the regulation of the CDKI-CDK-cyclin cascade, and caspase and PARP cleavages.19

Cervical cancer Yu et al. stated that silymarin induced apoptosis of cervical cancer C-33A cells through the modulation of Bcl-2 family proteins and activation of caspase 3. Besides, it inhibited the phosphorylation of Akt by increasing the expression of phosphatase and tensin homolog (PTEN). Also, silymarin significantly inhibited the expression of MMP-9 in C-33A cells.55 Garcia-Maceira and Mateo found that silibinin inhibits the accumulation and transcriptional activity of hypoxia-induced HIF-1a protein in HeLa cells. The decrease was connected with strong dephosphorylation of mammalian target of rapamycin (mTOR) and its effectors ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein-1, a pathway known to regulate HIF-1a expression at the translational level. Also, silibinin reduced hypoxia-induced vascular VEGF release by HeLa cells, and this effect was potentiated by the PI3K/Akt inhibitor LY294002.54

Leukemia Ladas et al. performed a double-blind, randomized study in children with acute lymphoblastic leukemia to investigate the therapeutic feasibility of S. marianum. The patients received it for 28 days. Biochemical analysis showed significantly lower aspartate amino transferase enzyme in the milk thistle group. Chemotherapy doses were reduced in 61% of the milk thistle group compared with 72% of the placebo group. A modest synergistic effect was also reported in an in vitro study. Furthermore, it did not reduce the effects of chemotherapy agents normally used for the treatment of leukemia.56

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Antimetastatic effect Metastasis is the final and most life-threatening stage of cancer progression with high morbidity and mortality. Silibinin has revealed outstanding antimetastatic efficacy against various cancers in different preclinical models. Silibinin antimetastatic efficacy has been described through pleiotropic mechanisms including the inhibition of epithelial-to-mesenchymal transition in cancer cells including the loss of intercellular junctions, disruption of the tumor basement membrane, activation and rearrangement of the cytoskeleton resulting in increased motility, and the release of cells from parent epithelial tissue.11

Radiotherapy- and chemotherapy-induced adverse reaction management (Table 2) Hepatoprotectant Hepatotoxicity is a common toxicity which can result in chemotherapy dose reductions or withdrawal in adult and pediatric cancer patients. Chemotherapy agents that could produce hepatotoxicity include dactinomycin, daunorubicin, docetaxel, gemcitabine, imatinib, 6-mercaptopurine, methotrexate, and oxaliplatin. S. marianum is administered to lots of patients for chemotherapy-induced hepatotoxicity prevention. The rationale is to support the liver which is a multifunctional organ, including responding to increased metabolic demands caused by tumor growth, assisting in metabolizing products produced during a tumor killing process or reduced by chemotherapy and radiation, and helping in the processing of chemotherapeutic agents in cancer patients. But, some oncologists are against milk thistle’s prescription in hepatocellular cancer because of a theoretical concern about S. marianum’s ability to stimulate hepatocyte regeneration; it may stimulate the growth of the tumor. One case report described the silymarin administration in a 34-year-old woman with promyelocytic leukemia. The patient received 800 mg/d silymarin during her methotrexate and 6-mercaptopurine maintenance therapy. During the 4 months of treatment with silymarin, her liver enzyme levels remained normal, and no treatment discontinuation became necessary. A randomized, double-blind study was also accomplished on 50 children with grade 2 or higher acute lymphoblastic leukemia (ALL) in which they received a milk thistle supplement (Siliphos, Thorne Research, Dover, Idaho) (5.1 mg/kg/d) or placebo for 28 days. Their mean aspartate aminotransferase (AST; P < 0.05) decreased significantly and a trend toward a significant reduction in alanine aminotransferase (ALT; P < 0.07) was reported. More children in the intervention group experienced a more than 50% reduction in total bilirubin after day 28 in comparison with placebo (P ¼ 0.0069).56, 60 Cisplatin is a powerful chemotherapeutic agent used for the treatment of various cancers. While its nephrotoxicity has been accepted as the most important dose-limiting adverse reaction, much is unknown about cisplatin-induced hepatotoxicity, as usually happens at high doses and is not considered as a dose-limiting toxicity. Oxidative stress is one of the most important mechanisms involved in cisplatin-induced toxicity. The mitochondrion is the primary target for cisplatin-induced oxidative stress, resulting in the loss of mitochondrial protein-SH, inhibition of calcium uptake, and a reduction in the mitochondrial membrane potential. It is shown that pretreatment with silymarin can improve the SOD, GSHPx, GSH activities, and serum NO level.2 Doxorubicin has been extensively used over the past several decades to treat patients with several cancers, including hepatocellular carcinoma. Hepatotoxicity is a quite common adverse effect observed in patients with cancers other than liver who are treated with doxorubicin. Silymarin reduced, delayed, or prevented its toxic effects which are usually associated with hydroxyl radical production, act as an antioxidant-limiting oxidative stress, protected the integrity of the genome, and antagonized apopotic and necrotic cell death while increasing the antiapoptotic Bcl-xL protein levels and

TABLE 2 Summery of chemoprotective and radioprotective effect of silymarin. Protected organ

References

Liver

Nencini et al.2, Greenlee et al.60, Ladas et al.56, and Patel et al.61

Kidney

Greenlee et al.60 and Shahbazi et al.62

Heart

Greenlee et al.60 and Zholobenko and Modriansky63

Mucocutaneous

Elyasi et al.64, Elyasi et al.65, and Karbasforooshan et al.66

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minimizing the leakage of proapoptotic cytochrome c from liver mitochondria. These findings show the protective actions of silymarin in liver, and raise the possibility to have protection in other organs during doxorubicin treatment including the heart.61

Kidney protectant Even today, despite of the good supportive care, about 10%–30% of treated patients with cisplatin experience serum creatinine increment and drug-induced nephrotoxicity. Numerous in vitro and in vivo studies have considered the promising protective effects of silymarin flavonolignans and flavonoids against cisplatin and ifosfamide-induced nephrotoxicity. Silymarin-treated animals and cell cultures showed low cisplatin-induced nephrotoxicity. Cisplatin-related nephrotoxicity is mediated somewhat by inflammatory and oxidative stress, so antiinflammatory and antioxidant agents like silymarin have been thought to prevent cisplatin nephrotoxicity.60, 62 However, in a pilot, randomized, double-blinded, placebocontrolled clinical trial, oral silymarin 420 mg/d in three divided doses starting 24–48 h before the cisplatin infusion and continuing till the end of three 21-day cisplatin-containing chemotherapy courses was not significantly effective in reducing nephrotoxicity incidence and the level of urinary magnesium and potassium wasting.62 Moreover, it is proposed that S. marianum may protect the kidney from radiation damage. Silymarin had significant preventive effects on urea and creatinine serum level increment in an animal model, when administered orally before exposure to gamma radiation.60

Cardioprotectant Silymarin and its constituents protected rat heart microsomes and mitochondria against doxorubicin-induced lipid peroxidation, so it seems that silymarin may prevent doxorubicin-mediated cardiotoxicity.60 It has emerged from animal models that silymarin can protect the heart against ischemia reperfusion injury, perhaps by preconditioning. The evidence seems to indicate that the ingredients of silymarin reduce the activity of both the Erk/MEK and IP3K/Akt pro-survival pathways, whose activation is central to ACh, bradykinin, and ouabain preconditioning. At the same time, it is important that stimulation of estrogen receptors, inhibition of MMPs, phosphodiesterases, and mitochondrial ROS generation by silymarin’s components could help preconditioning. Additionally the antiinflammatory properties of certain components may play a role in protecting tissue. While major silybin is the usual suspect for these effects, other minor components of the extract have also been shown to possess an important cardioprotective activity.63

Mucocutaneous protection Hand-foot syndrome is a common dose-limiting adverse reaction of capecitabine in more than half of patient with gastrointestinal cancers. Based on a pilot, randomized, double-blinded, placebo-controlled clinical trial, prophylactic administration of silymarin 1% gel after 9 weeks of application could significantly reduce the hand-foot syndrome and delays its occurrence. Antioxidant properties of silymarin as well as the antiinflammatory and immunomodulatory properties could be its proposed mechanisms of actions.64 Mucositis is a frequent severe complication of radiation therapy in patient with head and neck cancer. Unscheduled dose reductions or treatment interruptions because of severe mucositis may potentially compromise the efficacy of therapy and result in diminished quality of life. Based on a pilot, randomized, double-blinded, placebo-controlled clinical trial, prophylactic administration of conventional form of silymarin tablets 420 mg daily in three divided doses starting at the first day of radiotherapy for 6 weeks could significantly reduce the severity of radiotherapy-induced mucositis and delay its occurrence, by modulating its antioxidant and immunomodulatory effect.65 More than 80% of patients with breast cancer undergoing postsurgical radiotherapy (RT) will develop radiodermatitis and approximately 10% of these patients show grade 3 lesions. Side effects may reduce the patient’s compliance and can be limiting factors to follow RT protocols. Therefore, there is a high need for more effective prophylactic treatments. Based on a pilot, randomized, double-blinded, placebo-controlled clinical trial, prophylactic administration of silymarin 1% gel after 5 weeks of application could significantly reduce the severity of radiodermatitis and delay its occurrence.66

Silymarin administration and dosing Unprocessed milk thistle seeds usually contain 4%–6% silymarin. Many commercial preparations are standardized to contain 70%–80% silymarin content using its primary flavonolignan silibinin as the reference. Clinicians generally choose

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to use silymarin standardized products, generally at a dose of 140 mg 3 times per day. The published data on silymarin use in children focus on intravenous doses of 20–50 mg/kg body weight for mushroom poisoning.60 Absorption of silymarin after oral administration is relatively low and peak plasma concentrations are attained in 6 h in animal and humans.7 Considering the short half-life of silibinin, it is critical to be sure that the adequate amounts of the agent remain in the circulation and target organ tissues for its anticancer effect occurrence. The clinical use of the parenteral form of Silibinin (silibinin dihemisuccinate) is limited by its short half-life. An attractive way for more effective therapy may be the formation of silibinin-phosphatidylcholine complexes to increase silibinin lipid solubility.19

Silymarin adverse reactions and drug interactions As a therapeutic agent, silymarin is well tolerated and mostly free of adverse effects and few negative drug interactions have been reported.2 Occasional side effects include mild gastrointestinal disturbance, nausea, diarrhea, and headache reported in clinical trials.7, 60 The available data indicate that milk thistle has few side effects associated with doses less than 5 g/d. One trial supports an increased incidence of adverse effects with doses greater than 10 g/d.60 In humans, the consumption of 13 g of oral silibinin-phytosome daily, in three divided doses, appeared to be well tolerated in patients with advanced prostate cancer and is the recommended phase II dose.19 Silymarin has been used in pregnant women with intrahepatic cholestasis at doses of 560 mg/d for 16 days with no observed toxicity to the patient or the fetus.60 Silymarin is a CYP2D8 substrate and inhibits CYP3A4 and CYP1A1 activity in vitro, yet in vivo interactions have not been corroborated. The effects of silymarin on CYP450 isozymes and aldoketoreductases in animals remain unclear.61

Summary points l

l l

l

l

This chapter focuses on silymarin indications as an adjuvant to chemotherapy agents for cancer treatment and also for the management of chemotherapy- and radiotherapy-induced adverse reactions. Silymarin is the active component of S. marianum, and belongs to Asteraceae family. It is famous for its hepatoprotective effects which are mainly mediated with its antioxidant and antiinflammatory effects. Different animal and human studies suggest that silymarin and its active component, silybin, work as antioxidant, free radicals scavenger, and inhibitor of lipid peroxidation activity. Based on available data and studies, it seems that most of silymarin effects in oncology indications are mediated by its antioxidant property.

References 1. Morazzoni P, Bombardelli E. Silybum marianum (Carduus marianus). Fitoterapia 1995;66:3–42. 2. Nencini C, Giorgi G, Micheli L. Protective effect of silymarin on oxidative stress in rat brain. Phytomedicine 2007;14(2–3):129–35. 3. Ding T, Tian S, Zhang Z, Gu D, Chen Y, Shi Y, et al. Determination of active component in silymarin by RP-LC and LC/MS. J Pharm Biomed Anal 2001;26(1):155–61. 4. Karimi G, Vahabzadeh M, Lari P, Rashedinia M, Moshiri M. “Silymarin”, a promising pharmacological agent for treatment of diseases. Iran J Basic Med Sci 2011;14(4):308–17. 5. Feher J, Lengyel G. Silymarin in the prevention and treatment of liver diseases and primary liver cancer. Curr Pharm Biotechnol 2012;13(1):210–7. 6. Mansour HH, Hafez HF, Fahmy NM. Silymarin modulates cisplatin-induced oxidative stress and hepatotoxicity in rats. J Biochem Mol Biol 2006;39 (6):656–61. 7. Taleb A, Ahmad KA, Ihsan AU, Qu J, Lin N, Hezam K, et al. Antioxidant effects and mechanism of silymarin in oxidative stress induced cardiovascular diseases. Biomed Pharmacother 2018;102:689–98. 8. Thakare VN, Dhakane VD, Patel BM. Potential antidepressant-like activity of silymarin in the acute restraint stress in mice: modulation of corticosterone and oxidative stress response in cerebral cortex and hippocampus. Pharmacol Rep 2016;68(5):1020–7. 9. Feng B, Meng R, Huang B, Shen S, Bi Y, Zhu D. Silymarin alleviates hepatic oxidative stress and protects against metabolic disorders in high-fat dietfed mice. Free Radic Res 2016;50(3):314–27. 10. Huang WY, Cai YZ, Zhang Y. Natural phenolic compounds from medicinal herbs and dietary plants: potential use for cancer prevention. Nutr Cancer 2010;62(1):1–20. 11. Patel S. A promising CAM therapeutic for multiple cancers: milk thistle (Silybum). In: Emerging bioresources with nutraceutical and pharmaceutical prospects. applied environmental science and engineering for a sustainable future. Cham: Springer; 2015. 12. Deep G, Agarwal R. Chemopreventive efficacy of silymarin in skin and prostate cancer. Integr Cancer Ther 2007;6(2):130–45.

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13. Katiyar SK. Treatment of silymarin, a plant flavonoid, prevents ultraviolet light-induced immune suppression and oxidative stress in mouse skin. Int J Oncol 2002;21(6):1213–22. 14. Yu Y, Fan SM, Yuan SJ, Tashiro S, Onodera S, Ikejima T. Nitric oxide (NO) generation but not ROS plays a major role in silibinin-induced autophagic and apoptotic death in human epidermoid carcinoma A431 cells. Free Radic Res 2012;46:1346–60. 15. Lee MH, Huang Z, Kim DJ, Kim SH, Kim MO, Lee SY, et al. Direct targeting of MEK1/2 and RSK2 by silybin induces cell cycle arrest and inhibits melanoma cell growth. Cancer Prev Res (Phila) 2013;6:455–65. 16. Vaid M, Singh T, Prasad R, Katiyar SK. Silymarin inhibits melanoma cell growth both in vitro and in vivo by targeting cell cycle regulators, angiogenic biomarkers and induction of apoptosis. Mol Carcinog 2015;54(11):1328–39. 17. Ng CY, Yen H, Hsiao HY, Su SC. Phytochemicals in skin cancer prevention and treatment: an updated review. Int J Mol Sci 2018;19(4):941. 18. Bang CI, Paik SY, Sun DI, Joo YH, Kim MS. Cell growth inhibition and down-regulation of survivin by silibinin in a laryngeal squamous cell carcinoma cell line. Ann Otol Rhinol Laryngol 2008;117:781–5. 19. Cheung CW, Gibbons N, Johnson DW, Nicol DL. Silibinin—a promising new treatment for cancer. Anticancer Agents Med Chem 2010;10(3):186–95. 20. Mateen S, Raina K, Agarwal C, Chan D, Agarwal R. Silibinin synergizes with histone deacetylase and DNA methyltransferase inhibitors in upregulating E-cadherin expression together with inhibition of migration and invasion of human non-small cell lung cancer cells. J Pharmacol Exp Ther 2013;345:206–14. 21. Kim S, Lee HS, Lee SK, Kim SH, Hur SM, Kim JS, et al. 12-O-Tetradecanoyl phorbol-13-acetate (TPA)-induced growth arrest is increased by silibinin by the down-regulation of cyclin B1 and cdc2 and the up-regulation of p21 expression in MDA-MB231 human breast cancer cells. Phytomedicine 2010;17:1127–32. 22. Wang HJ, Jiang YY, Wei XF, Huang H, Tashiro S, Onodera S, et al. Silibinin induces protective superoxide generation in human breast cancer MCF-7 cells. Free Radic Res 2010;44:90–100. 23. Tamaki H, Satoh H, Hori S, Ohtani H, Sawada Y. Inhibitory effects of herbal extracts on breast cancer resistance protein (BCRP) and structureinhibitory potency relationship of isoflavonoids. Drug Metab Pharmacokinet 2010;25:170–9. 24. Kim S, Han J, Kim JS, Kim JH, Choe JH, Yang JH, et al. Silibinin suppresses EGFR ligand-induced CD44 expression through inhibition of EGFR activity in breast cancer cells. Anticancer Res 2011;31:3767–73. 25. Noh EM, Yi MS, Youn HJ, Lee BK, Lee YR, Han JH, et al. Silibinin reduces ultraviolet B-induced apoptosis in MCF-7 human breast cancer cells. J Breast Cancer 2011;14:8–13. 26. Dunnick JK, Singh B, Nyska A, Peckham J, Kissling GE, Sanders JM. Investigating the potential for toxicity from the long-term use of the herbal products, goldenseal and milk thistle. Toxicol Pathol 2011;39:398–409. 27. Dastpeyman M, Motamed N, Azadmanesh K, Mostafavi E, Kia V, Jahanian-Najafabadi A, et al. Inhibition of silibinin on migration and adhesion capacity of human highly metastatic breast cancer cells line, MDA-MB-231, by evaluation of b1-integrin and down stream molecules, Cdc42, Raf-1 and D4GDI. Med Oncol 2011;29:2512–8. 28. Oh SJ, Jung SP, Han J, Kim S, Nam SJ, Lee JE, et al. Silibinin inhibits TPA-induced cell migration and MMP-9 expression in thyroid and breast cancer cells. Oncol Rep 2013;29:1343–8. 29. Ramakrishnan G, Jagan S, Kamaraj S, Anandakumar P, Devaki T. Silymarin attenuated mast cells recruitment thereby decreased the expressions of matrix metalloproteinases-2 and 9 in rat liver carcinogenesis. Invest New Drugs 2009;27:233–40. 30. Brandon-Warner E, Sugg JA, Schrum LW, McKillop IH. Silibinin inhibits ethanol metabolism and ethanol-dependent cell proliferation in an in vitro model of hepatocellular carcinoma. Cancer Lett 2010;291:120–9. 31. Nambiar D, Prajapati V, Agarwal R, Singh RP. In vitro and in vivo anticancer efficacy of silibinin against human pancreatic cancer BxPC-3 and PANC-1 cells. Cancer Lett 2012;334(1):109–17. 32. Bousserouel S, Bour G, Kauntz H, Gosse F, Marescaux J, Raul F. Silibinin inhibits tumor growth in a murine orthotopic hepatocarcinoma model and activates the TRAIL apoptotic signaling pathway. Anticancer Res 2012;32:2455–62. 33. Cho HJ, Suh DS, Moon SH, Song YJ, Yoon MS, Park DY, et al. Silibinin inhibits tumor growth through downregulation of extracellular signalregulated kinase and Akt in vitro and in vivo in human ovarian cancer cells. J Agric Food Chem 2013;61(17):4089–409. 34. Verschoyle RD, Greaves P, Patel K, Marsden DA, Brown K, Steward WP, et al. Evaluation of the cancer chemopreventive efficacy of silibinin in genetic mouse models of prostate and intestinal carcinogenesis: relationship with silibinin levels. Eur J Cancer 2008;44:898–906. 35. Raina K, Rajamanickam S, Singh RP, Deep G, Chittezhath M, Agarwal R. Stage-specific inhibitory effects and associated mechanisms of silibinin on tumor progression and metastasis in transgenic adenocarcinoma of the mouse prostate model. Cancer Res 2008;68:6822–30. 36. Mokhtari MJ, Motamed N, Shokrgozar MA. Evaluation of silibinin on the viability, migration and adhesion of the human prostate adenocarcinoma (PC-3) cell line. Cell Biol Int 2008;32:888–92. 37. Wu K, Zeng J, Li L, Fan J, Zhang D, Xue Y, et al. Silibinin reverses epithelial-to-mesenchymal transition in metastatic prostate cancer cells by targeting transcription factors. Oncol Rep 2010;23:1545–52. 38. Flaig TW, Glode M, Gustafson D, van Bokhoven A, Tao Y, Wilson S, et al. A study of high-dose oral silybin-phytosome followed by prostatectomy in patients with localized prostate cancer. Prostate 2010;70:848–55. 39. Vidlar A, Vostalova J, Ulrichova J, Student V, Krajicek M, Vrbkova J, et al. The safety and efficacy of a silymarin and selenium combination in men after radical prostatectomy—a six month placebo-controlled double-blind clinical trial. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2010;154:239–44. 40. Kavitha CV, Deep G, Gangar SC, Jain AK, Agarwal C, Agarwal R. Silibinin inhibits prostate cancer cells- and RANKL-induced osteoclastogenesis by targeting NFATc1, NF-kB, and AP-1 activation in RAW264.7 cells. Mol Carcinog 2012;53(3):169–80.

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41. Lu W, Lin C, King TD, Chen H, Reynolds RC, Li Y. Silibinin inhibits Wnt/b-catenin signaling y suppressing Wnt co-receptor LRP6 expression in human prostate and breast cancer cells. Cell Signal 2012;24:2291–6. 42. Ting H, Deep G, Agarwal R. Molecular mechanisms of silibinin-mediated cancer chemoprevention with major emphasis on prostate cancer. AAPS J 2013;15(3):707–16. 43. Colombo V, Lupi M, Falcetta F, Forestieri D, D’Incalci M, Ubezio P. Chemotherapeutic activity of silymarin combined with doxorubicin and paclitaxel in sensitive and multidrug resistant colon cancer cells. Cancer Chemother Pharmacol 2011;67:369–79. 44. Kauntz H, Bousserouel S, Gosse F, Raul F. Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells. Apoptosis 2011;16:1042–53. 45. Toyoda-Hokaiwado N, Yasui Y, Muramatsu M, Masumura K, Takamune M, Yamada M, et al. Chemopreventive effects of silymarin against 1,2dimethylhydrazine plus dextran sodium-sulfate-induced inflammation-associated carcinogenicity and genotoxicity in the colon of gpt delta rats. Carcinogenesis 2011;32:1512–7. 46. Yan X, Gardner TR, Grieco M, Herath SA, Jang JH, Kirchoff D, et al. Perioperative polyphenon E- and siliphos-inhibited colorectal tumor growth and metastases without impairment of gastric or abdominal wound healing in mouse models. Surg Endosc 2012;26:1856–64. 47. Kauntz H, Bousserouel S, Gosse F, Marescaux J, Raul F. Silibinin, a natural flavonoid, modulates the early expression of chemoprevention biomarkers in a preclinical model of colon carcinogenesis. Int J Oncol 2012;41:849–54. 48. Kauntz H, Bousserouel S, Gosse F, Raul F. The flavonolignan silibinin potentiates TRAIL induced apoptosis in human colon adenocarcinoma and in derived TRAIL-resistant metastatic cells. Apoptosis 2012;17:797–809. 49. Karim BO, Rhee KJ, Liu G, Zheng D, Huso DL. Chemoprevention utility of silibinin and Cdk4 pathway inhibition in Apc /+ mice. BMC Cancer 2013;13:157–66. 50. Raina K, Agarwal C, Wadhwa R, Serkova NJ, Agarwal R. Energy deprivation by silibinin in colorectal cancer cells: a double-edged sword targeting both apoptotic and autophagic machineries. Autophagy 2013;9:697–713. 51. Tyagi AK, Agarwal C, Singh RP, Shroyer KR, Glode LM, Agarwal R. Silibinin down-regulates survivin protein and mRNA expression and causes caspases activation and apoptosis in human bladder transitional-cell papilloma RT4 cells. Biochem Biophys Res Commun 2003;312(4):1178–84. 52. Tyagi A, Singh RP, Agarwal C, Agarwal R. Silibinin activates p53-caspase 2 pathway and causes caspase-mediated cleavage of Cip1/p21 in apoptosis induction in bladder transitional-cell papilloma RT4 cells: evidence for a regulatory loop between p53 and caspase 2. Carcinogenesis 2006;27 (11):2269–80. 53. Kaur G, Athar M, Alam MS. Dietary supplementation of silymarin protects against chemically induced nephrotoxicity, inflammation and renal tumor promotion response. Invest New Drugs 2010;28:703–13. 54. Garcia-Maceira P, Mateo J. Silibinin inhibits hypoxia-inducible factor-1alpha and mTOR/p70S6K/4E-BP1 signalling pathway in human cervical and hepatoma cancer cells: implications for anticancer therapy. Oncogene 2009;28:313–24. 55. Yu HC, Chen LJ, Cheng KC, Li YX, Yeh CH, Cheng JT. Silymarin inhibits cervical cancer cells through an increase of phosphatase and tensin homolog. Phytother Res 2012;26:709–15. 56. Ladas EJ, Kroll DJ, Oberlies NH, Cheng B, Ndao DH, Rheingold SR, et al. A randomized, controlled, double-blind, pilot study of milk thistle for the treatment of hepatotoxicity in childhood acute lymphoblastic leukemia (ALL). Cancer 2010;116:506–13. 57. Karbasforooshan H, Roohbakhsh A, Karimi G. SIRT1 and microRNAs: the role in breast, lung and prostate cancers. Exp Cell Res 2018;367:1–6. 58. Humphrey PA. Cancers of the male reproductive organs. In: Stewart BW, Wild CP, editors. World cancer report. Lyon: World Health Organization; 2014. 59. Kooshyar MM, Maruzi A, Fani Pakdel A, Elyasi S, Taghizadeh-Kermani A, Akbarzadeh M, et al. Adherence to a standardized chemotherapy order form for colorectal cancer in a referral teaching hospital, Mashhad, Iran. Iran J Pharm Res 2019;18(1):488–95. 60. Greenlee H, Abascal K, Yarnell E, Ladas E. Clinical applications of Silybum marianum in oncology. Integr Cancer Ther 2007;6(2):158–65. 61. Patel N, Joseph C, Corcoran GB, Ray SD. Silymarin modulates doxorubicin-induced oxidative stress, Bcl-xL and p53 expression while preventing apoptotic and necrotic cell death in the liver. Toxicol Appl Pharmacol 2010;245(2):143–52. 62. Shahbazi F, Sadighi S, Dashti-Khavidaki S, Shahi F, Mirzania M, Abdollahi A, et al. Effect of Silymarin administration on cisplatin nephrotoxicity: report from a pilot, randomized, double-blinded, placebo-controlled clinical trial. Phytother Res 2015;29(7):1046–53. 63. Zholobenko A, Modriansky M. Silymarin and its constituents in cardiac preconditioning. Fitoterapia 2014;97:122–32. 64. Elyasi S, Shojaee FSR, Allahyari A, Karimi G. Topical silymarin administration for prevention of capecitabine-induced hand-foot syndrome: a randomized, double-blinded, placebo-controlled clinical trial. Phytother Res 2017;31(9):1323–9. 65. Elyasi S, Hosseini S, Niazi Moghadam MR, Aledavood SA, Karimi G. Effect of oral silymarin administration on prevention of radiotherapy induced mucositis: a randomized, double-blinded, placebo- controlled clinical trial. Phytother Res 2016;30(11):1879–85. 66. Karbasforooshan H, Hosseini S, Elyasi S, Fani Pakdel A, Karimi G. Topical silymarin administration for prevention of acute radiodermatitis in breast cancer patients: a randomized, double-blind, placebo-controlled clinical trial. Phytother Res 2019;33(2):379–86.

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Chapter 44

Plants of the genus Terminalia: Phytochemical and antioxidant profiles, proliferation, and cancer Ian Edwin Cocka,b and Matthew Cheesmanc,d a

School of Environment and Science, Griffith University, Nathan, QLD, Australia, b Environmental Futures Research Institute, Griffith University, Nathan,

QLD, Australia, c School of Pharmacy and Pharmacology, Griffith University, Southport, QLD, Australia, d Menzies Health Institute Queensland, Quality Use of Medicines Network, Southport, QLD, Australia

List of abbreviations CAT DNA GSH GSH-Px IL MDA NF-kB RNA ROS SOD TEAC THx TNF

catalase deoxyribonucleic acid glutathione glutathione peroxidase interleukin malondialdehyde nuclear factor kappa-light-chain-enhancer of activated B cells ribonucleic acid reactive oxygen species superoxide dismutase trolox equivalent antioxidant content thioredoxin tumor necrosis factor

Introduction The genus Terminalia (Family Combretaceae) is of prominent ethnomedicinal importance due to its historical use in traditional medicine.1 It comprises approximately 250 species of medium-to-large flowering trees distributed through tropical and subtropical regions of Asia, Australia, and Africa. The Asian species account for the greatest number and diversity, and their therapeutic usage is most extensively documented. These species are found from the Middle East and Southern and Western Asia, across to the Malaysian peninsula and the Indonesian archipelago. T. arjuna and T. chebula are particularly well documented for their applications within Ayurveda. Approximately 30 African species are known, with most being found in the southern part of the continent. Numerous Terminalia species are also native to North, Central, and South America, and to Australia. High antioxidant levels are a common feature of the genus Terminalia. Indeed, the Australian species T. ferdinandiana contains the highest antioxidant capacity than any plant worldwide.2 The ascorbic acid levels in this plant are more than 900 times those of blueberry (g/g). The Asian species T. chebula and T. belerica are also rich in antioxidants,3 as are T. paniculata,4 T. arjuna, and T. catappa.3 The African species T. prunioides, T. brachystemma, T. gazensis, T. mollis, and T. sambesiaca also possess high antioxidant levels.5

Applications to cancers or other conditions The genus Terminalia consists of some of the most useful therapeutic plant species worldwide. Many species are used to treat cancer and also have antioxidant, anti-inflammatory, analgesic, antibacterial, antifungal, antiviral, and antiprotozoal Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00044-4 © 2021 Elsevier Inc. All rights reserved.

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properties.1 Indeed, T. chebula is commonly used in Indian Ayurveda for a wide variety of therapeutic purposes and is known as the “king of plants” due to its wide variety of medicinal uses. All species of this genus are known for their high antioxidant contents.1, 2 As the consumption of high antioxidant contents can modulate the cellular redox state, plants of this genus may protect the cell against oxidative stress and thus prevent cancer induction. Furthermore, high antioxidant contents may direct the cell to a prooxidant state and induce apoptosis. Thus, Terminalia spp. have the potential to block cancer, as well as treating it once it is established. Furthermore, all species of this genus are rich in phyto-constituents with potent anticancer properties, including tannins and stilbenes. Recent studies have reported potent anticancer properties of multiple members of this genus (as well as isolated compounds)1 and it is likely that future studies will highlight further anticancer properties.

Antioxidant content The ascorbic acid levels of T. ferdinandiana fruit2 are the highest of any plant globally, accounting for up to 6% of the wet weight. T. ferdinandiana fruit also contains many additional compounds including flavanoids that contribute to its high antioxidant activity. The fruit is also a good source of gallic and ellagic acids,1 and is rich in lutein, vitamin E, and vitamin E analogs.2 Other antioxidants found in T. ferdinandiana fruit include hesperidin, and the glycosides kaempferol, luteolin, and quercetin. Asian Terminalia species also contain high quantities of antioxidants, with the trolox equivalent antioxidant content (TEAC) being approximately 12.4, 1.6, 44.3, and 1.4 mg/mL extract for T. arjuna, T. belerica, T. catappa, and T. chebula, respectively.6 Tannins and flavonoids are abundant in T. catappa peels7 and T. arjuna bark.8 T. paniculata4 and T. alata3 also have high TEAC values. The African species T. brachystemma, T. gazensis, T. mollis, T. pruniodes, T. sambesiaca, and T. sericea also have high antioxidant contents.5 The antioxidant activities of Terminalia spp. correlate with their high tannin and flavonoid levels.9 Indeed, the tannin contents of T. chebula reach 32% and extracts from this plant exert a powerful cytoprotective effects in vivo.10

The relationship between oxidative stress and cancer Oxidative stress results from a disturbance in the antioxidant/prooxidant balance (Fig. 1). Cells use intracellular antioxidant systems including glutathione peroxidase (GSH-Px), catalase (CAT), thioredoxin (THx), superoxide dismutase (SOD), and extracellular antioxidants (vitamin E, vitamin C, etc.) to neutralize reactive oxygen species (ROS).11 These antioxidant systems also aid in the repair of oxidized cellular components. SOD catalyzes superoxide to H2O2, which is successively detoxified by CAT and GSH-Px. Failure in any of these enzyme systems results in damaged cells/tissue and the generation of free radicals, subsequently inducing the production of intracellular ROS, triggering protein, and DNA oxidation. Excessive oxygen free radicals may also induce peroxidation of polyunsaturated fatty acids in the cytosolic membranes of cells. The lipid peroxidate subsequently disintegrates and liberates carbonyl fragments including malondialdehyde (MDA). MDA is a reactive electrophilic aldehyde species and a marker of cellular injury. Oxidative stress and the subsequent cell damage also correlate with decreased Na+ K+-ATPase activity.12 Ultimately, the cell’s physiology and osmoregulatory ability is compromised and the ability of the plasma membrane to control the passage of ions and water is disrupted.

FIG. 1 Excessive oxidative stress causes disruption of the antioxidants/reactive oxygen species (ROS) balance, leading to the establishment of oxidative stress.

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Oxidative stress-induced damage may ultimately result in mortality via either apoptotic or necrotic pathways. Oxidative stress must reach a threshold level before apoptosis may occur. Beyond that level, necrosis occurs. The contents of the dying cells inflict further damage to neighboring cells.13 Changes in the cellular redox environment also affect signal transduction, DNA and RNA synthesis, protein synthesis, enzyme activation, regulation of the cell cycle, and nuclear translocation.14 Transcription factors are active in their reduced form and their translocation to the nucleus is redox dependent.15 Furthermore, high thiol concentrations (consistent with a reducing environment) stimulate cell proliferation. Conversely, an oxidizing environment initiates cell death. Prooxidants increase the levels of reactive oxygen species, thereby inducing apoptosis.16 Antioxidants such as GSH and thiol-containing proteins (e.g., thioredoxin) are reducing agents and prevent apoptosis.17 Therefore, the redox environment determines if a cell will proliferate, differentiate, or die. Cellular proliferation requires high GSH levels and low levels of the antioxidant proteins SOD, GPx, and CAT, favoring a reducing environment.18 GSH levels fluctuate in response to cell cycle progression.19 Low antioxidant levels induce cell proliferation, whilst high levels inhibit it.20 Therefore, proliferation favors a reducing environment whilst differentiation requires an oxidizing environment. Oxidizing environments may also predispose a cell to apoptosis or necrosis, depending on the degree and nature of the oxidative stimuli.

Phytochemistry of the genus Terminalia Terminalia spp. are characterized by their high antioxidant and high tannin contents. For the sake of brevity, this study concentrates on the tannin and stilbene components due to their notable anticancer properties.

Tannins Recent studies have identified a complex mixture of tannins, including ethyl gallic acid, 4-galloylpyrogallol, gallocatechin, ellagic acid, and corilagin in T. ferdinandiana extracts.21–23 Another Australian species, T. oblongifolia F.Muell., has similar tannin components and is particularly rich in gallic acid and tannic acid.24 The Indian species T. arjuna, T. catappa, and T. chebula also have similar tannin compositions, with gallic acid, chebulic acid, and ellagic acid predominating.25 Gallotannins, including gallic and chebulic acids, are particularly prevalent in Terminalia species. These tannins have antiproliferative and apoptotic effects against a variety of carcinoma cell lines (Fig. 2). They inhibit cellular growth by binding to cell surface lipoteichoic acid and cell surface proteins,26 and by inhibiting glucosyltransferase enzymes. They can modulate cellular p27 levels, resulting in G1 phase cell cycle arrest.27 Gallotannins also induce an upregulation in Bcl-2 FIG. 2 Anticancer mechanisms and pathways of gallo- and ellagitannins. Both classes of compound have cytotoxic activities and can also block the cell cycle in the G1 phase and inhibit cellular proliferation.

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levels and a subsequent increase in caspase 3 activity, ultimately triggering apoptosis.28 In addition, gallotannins may inhibit carcinoma proliferation by downregulating the synthesis of NFkB.29 Ellagitannins are also highly potent inhibitors of cell growth and also function via apoptotic, antiproliferative, and cell cycle arrest mechanisms (Fig. 2). The ellagitannin arjunin, extracted from T. arjuna, inhibits carcinoma proliferation and stimulates apoptosis via multiple mechanisms.30 Ellagitannins can modulate cellular levels of p21, p27, and p53 levels, resulting in G1 phase cell cycle arrest.31 Furthermore, ellagic acid downregulates the production of multiple cyclins including cyclins B1, D1, and E, which also arrests the cell cycle in G1 phase. The effects of ellagitannins on apoptosis-signaling proteins are variable and depend on the carcinoma cell type. For example, ellagic acid upregulates Bcl-2 protein levels in human kerationcyte (HaCaT) cells, whilst also upregulating Bax levels,31 but downregulates the Bcl-2 levels in PC3 human prostate cancer cells.32 In both cases, this results in a shift in the Bax/Bcl-2 ratio in favor of apoptosis via increased caspase 3 activity. Interestingly, ellagitannins can also induce apoptosis via Bcl-2-independent pathways. Ellagic acid supplementation in human HBP breast cancer cells induces IkB expression.33 This subsequently inhibits cellular proliferation by downregulating the synthesis of NFkB, thereby inhibiting cellular proliferation and inducing apoptosis.29

Stilbenes A number of stilbenes have also been identified in Terminalia spp. extracts. Stilbenes are phytoalexins produced by plants for protection against microbes, although they also have many other therapeutic properties. Resveratrol is present in T. ferdinandiana extracts in substantial levels.23 Its presence has also been documented in the African species T. sericea and T. pruinoides34 and the Indian species T. chebula.35 This stilbene is useful in the prevention and treatment of cancer.36 Furthermore, resveratrol is a potent specific inhibitor of NF-kB activation via its induction by TNF-a and IL-1b.37 Thus, resveratrol directly inhibits NF-kB activation, thereby potentiating apoptosis.38 The resveratrol glycoside piceid is also present in T. ferdinandiana22 and the African species T. sericea.39 Piceid (and other glycosylated stilbenes) blocks IL-17 production in stimulated human mononuclear cells.40 A variety of other stilbenes have also been reported in the southern and eastern African species T. brownii and T. laxiflora.41 Several combretastatins have been reported in T. ferdinandiana22, 23 and T. brownii (Fresen. Mus. Senckenb) Shaf extracts.41 Combretastatins are potent inhibitors of cancer cell progression. They induce apoptosis by binding intracellular tubulin, thereby disrupting microtubule formation.42 They act by a similar mechanism to that of colchicine by binding the colchicine binding site on the tubulin peptide.43 A generalized summary of the anticancer mechanisms of combretastatins is presented in Fig. 3. Interestingly, several other stilbenes also bind to tubulin monomers and have been reported to function via similar mechanisms as the combretastatins.44 Blocking tubulin polymerization has dramatic effects of several aspects of cancer development (Fig. 3). As microtubules are vital for formation of the mitotic spindle during cell division, inhibiting tubulin polymerization and destabilizing existing microtubules blocks cell cycle progression in the M phase of the cell cycle, accounting for the reported cytostatic effects of these compounds.45, 46 Furthermore, destabilization of endothelial cell microtubules induces apoptosis in A549 lung carcinoma46 and human epithelial45 cell lines. Combretastatins may also modulate the expression of Bcl-2, thereby altering the Bax/Bcl-2 ratio in favor of apoptosis.47 This subsequently increases caspase 3 activity and stimulates cell death. Other stilbenes may have similar effects although this is yet to be verified. Combretastatins also affect cell survival and the ability of a cancer to spread via other mechanisms. To develop beyond their threshold size, solid tumors must develop neovascularization. Therefore, inhibition of angiogenesis is an important cancer therapeutic target. Combretastatin a4 has pronounced angiogenic inhibitory properties. Within minutes of its infusion into solid tumors, a substantial reduction of blood flow occurs.48 The disruption of tumor circulation is relatively long lasting and can result in substantial necrosis. Furthermore, the inability of cancer cells to form microtubules in the presence of combretastatins directly affects metastasis. Indeed, several studies have reported that combretastatin a4 therapy inhibits metastasis in MDA-MB-231 breast adenocarcinoma cells and Lewis lung carcinoma cells.49

Other compounds with anticancer activities A number of other compounds with anticancer properties have also been reported in some Terminalia spp. The presence of the imidazo-alkaloid compound 1-(3H-imidazol-4-yl)-ethanone in several Terminalia spp. is especially noteworthy. The imidazole moiety has a wide variety of biological activities.50 Furthermore, imidazole derivatives are structural isomers of naturally occurring nucleotides, allowing them to be incorporated into new nucleic acids, resulting in inhibition of DNA and

FIG. 3 The anticancer properties of combretastatins are mediated via inhibition of tubulin polymerization. This inhibits cell cycle progression and induces apoptosis. Combretastatins also decrease angiogenesis and inhibit metastasis.

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RNA synthesis.51 For example, distamycin A is a potent inhibitor of transcription and an efficient inhibitor of cellular proliferation.52 A number of synthetic analogs of distamycin A have also been reported to have potent anticancer activity, with IC50 values as low as 1.53 mM.53 Furthermore, recent studies aimed at developing synthetic anticancer drugs have focused on using the imidazole nucleus as a precursor. A novel imidazole analog 2-b-D-ribofuranosylimidazole-4-carboxamide (imidazofurin) was synthesized and found to have potent anticancer activity.54 A number of other promising anticancer drugs containing imidazole moieties including diaryl-substituted imidazole analogs, pyrrole-imidazole polyamides, arylimidazonaphthalimides, and xantheno-imadazole derivatives have been developed.50 A recent study detected C17 sphingosine in T. ferdinandiana extracts.55 Interestingly, two sphingosine kinase isozymes (SphK1 and SphK2) are upregulated in many cancers, resulting in increased production of the potent bioactive compound sphingosine-1-phosphate (S1P).56 S1P induces a variety of responses including stimulation of TNF production, which in turn results in NF-kB activation and the subsequent downregulation of apoptosis.57 Thus, increased levels of S1P have tumor promoting effects and several recent studies have aimed at inhibiting SphK1 and SphK2 in order to inhibit cancer progression. Interestingly, whilst S1P is a potent mediator of cancer progression, its precursors and other related sphingolipids antagonize the activity of S1P and instead arrest carcinoma growth and induce apoptosis.58 Indeed, several sphingosine analogs, including C17 sphingosine, are potent specific inhibitors of SphK1.59 That study also reported that the sphingosine analogs were highly specific and did not inhibit other protein kinases. As a result, exposure to C17 sphingosine dramatically affected the growth and survival of human leukemia U937 and Jurkat cells. The effects were manifested in several ways: the sphingosine analogs dramatically decreased cellular growth and proliferation and enhanced apoptosis and cleavage of Bcl-2. A number of terepenoids with anticancer properties have also been reported in Terminalia spp. extracts. Indeed, several monoterpenoids (linalool, terpineol, and camphor) are particularly widespread and have been detected in extracts prepared from multiple Australian,25 Indian,60 and African61 species. Linalool has cytotoxic effects in SW620, T-47D, A549, and Hep G2 cells via a stimulation of TFN-a secretion.62 Similarly, terpineol inhibits RPMI 8226 myeloma, CCRF-CEM leukemia, U937-GTB lymphoma, NCI-H69 lung carcinoma, and ACHN renal adenocarcinoma cells.63 The same study also reported that the antiproliferative activity of terpineol was via NF-kB inhibition. Many other terpenoids have documented antiproliferative activity toward a variety of cancer cell lines. Lupeol inhibits the growth of human colorectal cancer cells.64 Limonene and carvone are useful in the prevention and treatment of cancer in a murine test model, reducing stomach tumor formation by approximately 60%.65 Another study reported potent antiproliferative activity for leaf extracts prepared from the Australian species T. carpentariae and T. grandiflora and highlighted the triterpenoid components, including the pentacyclic triterpenoid thurberogenone.66 Notably, several similar pentacyclic triterpenoids have good anticancer activities. Ursane triterpenoids are cytotoxic to NTUB1 bladder cancer cells.67 They induce apoptosis via p53-mediated p38 MAPK activation pathways. Furthermore, the same triterpenoids also inhibit cell cycle progression by downregulating the levels of cyclins D1 and E, as well as the cyclin-dependent kinases CDK2 and CDK4, thereby blocking cell cycle progression.67 Similar effects have been reported against other carcinoma cell lines. Ursolic acid is a potent inhibitor of MCF-7 mammary cancer cells via inhibition of cell cycle progression through the G1 phase.68 Other studies have also demonstrated cytotoxicity toward MCF-7 cells.69 Ursolic acid suppresses multiple kinases and activator proteins (including p38 MAPK) in 184B4/HER mammary carcinoma cells.70 Similarly, oleanane triterpenoids inhibit MCF-7/HER cell proliferation by decreasing HER2 phosphorylation and inhibiting cyclin D1 expression, as well as by inducing apoptosis.71 Lanostane-type triterpenoids are also present in T. grandiflora leaf extracts.66 A similar lanostane triterpenoid (inotodiol) inhibits proliferation in mouse P388 leukemia cells via the induction of DNA fragmentation and caspase 3/7 activation.72 Ganoderic acid inhibits MDA-MB-231 breast cancer growth and invasive behavior by inhibiting AP-1 and NF-kB transcription factors, resulting in a downregulation of Cdk4 expression and inhibition of uPA secretion.73 Several compounds identified in Terminalia spp. extracts contain lactone moieties (e.g., ascorbic acid and glucuronic acid). This is noteworthy as many lactone compounds are mediators of apoptosis. Perhaps, the most extensively studied of the natural lactone chemotherapeutics are sesquiterpene lactones such as eupatoriopicrin74 and parthenolide,75 which enhance apoptosis via multiple mechanisms. Furthermore, many clinically used cancer chemotherapeutics (e.g., camptothecin, testolactone, and topotecan) contain lactone moieties. Furthermore, diacylglycerol (DAG)-lactones are potent inducers of apoptosis in LNCaP cells.76 That study determined that the apoptotic induction of the DAG-lactones was mediated via protein kinase C activation. Enhancement of glutathione-S-transferase activity has also been reported for many lactones.77 Glutathione-S-transferase has a regulatory role in the mitogen-activated protein (MAP) kinase pathway that participates in cellular survival/apoptosis signals.78 As an increase in glutathione-S-transferase activity stimulates apoptosis, compounds which enhance this enzyme have emerged as promising new cancer therapy targets.79

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Summary points l

l l

l l l l

l l

Multiple Terminalia species, including T. chebula, T. bellerica, and T. ferdinandiana, have potent antiproliferative activity. High antioxidant and tannin contents are characteristic of the genus Terminalia. The high antioxidant contents of Terminalia spp. have been linked with protective effects against the establishment of cancers, and the induction of apoptosis once cancer is established. Terminalia spp. tannins have direct antiproliferative and apoptotic effects. They also block cell cycle progression. A number of stilbenes (including combretastatins) have been identified in multiple Terminalia spp. The anticancer properties of combretastatins are mediated via inhibition of tubulin polymerization. Combretastatins inhibit cell cycle progression and induce apoptosis. They also decrease angiogenesis and inhibit metastasis. Several other compounds with anticancer properties are also present in Terminalia spp. This chapter reviews the current knowledge regarding the phytochemistry and anticancer properties of the genus Terminalia.

References 1. Cock IE. The medicinal properties and phytochemistry of plants of the genus Terminalia (Combretaceae). Inflammopharmacology 2015;23 (5):203–29. 2. Kahkeshani N, Farzaei F, Fotouhi M, Alavi SS, Bahramsoltani R, Naseri R, et al. Pharmacological effects of gallic acid in health and disease: a mechanistic review. Iran J Basic Med Sci 2019;22(3):225–37. 3. Mety SS, Mathad P. Antioxidative and free radical scavenging activities of Terminalia species. Int Res J Biotechnol 2011;2(5):119–27. 4. Agrawal S, Kulkarni GT, Sharma VN. A comparative study on the antioxidant activity of methanolic extracts of Terminalia paniculata and Madhuca longifolia. Free Radicals Antioxid 2011;1(4):62–8. 5. Masoko P, Eloff JN. Screening of twenty-four South African Combretum and six Terminalia species (Combretaceae) for antioxidant activities. Afr J Tradit Complement Altern Med 2007;4(2):231–9. 6. Jaiwal BV, Shaikh FK, Waghire HB, Sarwade BP. Comparative anti-oxidative potential of barks phenolics of genus Terminalia. Int J Sci Pharm Educ Res 2012;1:6–10. 7. Kaneria MJ, Rakholiya KD, Marsonia LR, Dave RA, Golakiya BA. Nontargeted metabolomics approach to determine metabolites profile and antioxidant study of tropical almond (Terminalia catappa L.) fruit peels using GC-QTOF-MS and LC-QTOF-MS. J Pharm Biomed Anal 2018;160:415–27. 8. Kumar V, Sharma N, Sourirajan A, Khosla PK, Dev K. Comparative evaluation of antimicrobial and antioxidant potential of ethanolic extract and its fractions of bark and leaves of Terminalia arjuna from North-Western Himalayas. J Tradit Complement Med 2018;8(1):100–6. 9. Jain S, Yadav PP, Gill V, Vasudeva N, Singla N. Terminalia arjuna a sacred medicinal plant: phytochemical and pharmacological profile. Phytochem Rev 2009;8(2):491–502. 10. Punniyakotti P, Rengarajan RL, Velayuthaprabhu S, Vijayakumar K, Manikandan R, Anand AV. Protective effect of Terminalia catappa leaves and Terminalia chebula fruits on the enzymatic and non-enzymatic anti-oxidant levels in the doxorubicin induced toxicity rats. Pharmacogn J 2019;11 (2):346–9. 11. Shang YZ, Qin BW, Cheng JJ, Miao H. Prevention of oxidative injury by flavonoids from stems and leaves of Scutellaria baicalensis Georgi in PC12 cells. Phytother Res 2006;20(1):53–7. 12. Huang WH, Wang Y, Askari A. Na-K-ATPase: inactivation and degradation induced by oxygen radicals. Int J Biochem 1992;24:621–6. 13. Vaux DL, Korsmeyer SJ. Cell death in development. Cell 1999;96(2):245–54. 14. Makino Y, Yoshikawa N, Oamoto K, Hirota K, Yodoi J, Makino I, et al. Direct association with thioredoxin allows redox regulation of glucocorticoid receptor function. J Biol Chem 1999;274(5):3182–8. 15. Okamoto K, Tanaka H, Ogawa H, Makino Y, Eguchi H, Hayashi S, et al. Redox-dependent regulation of nuclear import of the glucocorticoid receptor. J Biol Chem 1999;274(15):10363–71. 16. Kim HS, Lee JH, Kim IK. Intracellular glutathione level modulates the induction of apoptosis by delta 12-prostaglandin J2. Prostaglandins 1996;51 (6):413–25. 17. Iwata S, Hori T, Sato N, Hirota K, Sasada T, Mitsui A, et al. Adult T cell leukemia (ATL)-derived factor/human thioredoxin prevents apoptosis of lymphoid cells induced by L-cystine and glutathione depletion: possible involvement of thiol-mediated redox regulation in apoptosis caused by prooxidant state. J Immunol 1997;158(7):3108–17. 18. Allen RG, Venkatraj VS. Oxidants and antioxidants in development and differentiation. J Nutr 1992;122:631–5. 19. Schafer FQ, Buettner GR. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 2001;30(11):1191–212. 20. Brown MR, Miller FJ, Li WG, Ellingson AN, Mozena JD, Chatterjee P, et al. Overexpression of human catalase inhibits proliferation and promotes apoptosis in vascular smooth muscle cells. Circ Res 1999;85(6):524–33.

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21. Wright MH, Shalom J, Matthews B, Greene AC, Cock IE. Terminalia ferdinandiana Exell. extracts inhibit Shewanella spp. growth and prevent fish spoilage. Food Microbiol 2019;78:114–22. 22. Shalom J, Cock IE. Terminalia ferdinandiana Exell. fruit and leaf extracts inhibit proliferation and induce apoptosis in selected human cancer cell lines. Nutr Cancer 2018;70(4):579–93. 23. Sirdaarta J, Matthews B, Cock IE. Kakadu plum fruit extracts inhibit growth of the bacterial triggers of rheumatoid arthritis: identification of stilbene and tannin components. J Funct Foods 2015;17:610–20. 24. Murdiati TB, McSweeney CS, Lowry JB. Metabolism in sheep of gallic acid, tannic acid and hydrolysable tannin from Terminalia oblongata. Aust J Agr Res 1992;43(6):1307–19. 25. Wright MH, Sirdaarta J, White A, Greene AC, Cock IE. Bacillus anthracis growth inhibitory properties of Australian Terminalia spp.: putative identification of low polarity volatile components by GC-MS headspace analysis. Pharmacogn J 2016;8(3):282–90. 26. Buzzini P, Arapitsas P, Goretti M, Branda E, Turchetti B, Pinelli P, et al. Antimicrobial and antiviral activity of hydrolysable tannins. Mini-Rev Med Chem 2008;8(12):1179–87. 27. Darvin P, Baeg SJ, Joung YH, Sp N, Kang DY, Byun HJ, et al. Tannic acid inhibits the Jak2/STAT3 pathway and induces G1/S arrest and mitochondrial apoptosis in YD-38 gingival cancer cells. Int J Oncol 2015;47(3):1111–20. 28. Zhang J, Chen D, Han DM, Cheng YH, Dai C, Wu XJ, et al. Tannic acid mediated induction of apoptosis in human glioma Hs 683 cells. Oncol Lett 2018;15(5):6845–50. 29. Lu Y, Jiang F, Jiang H, Wu K, Zheng X, Cai Y, et al. Gallic acid suppresses cell viability, proliferation, invasion and angiogenesis in human glioma cells. Eur J Pharmacol 2010;641(2–3):102–7. 30. Kandil FE, Nassar MI. A tannin anti-cancer promotor from Terminalia arjuna. Phytochemistry 1998;47(8):1567–8. 31. Hseu YC, Chou CW, Kumar KS, Fu KT, Wang HM, Hsu LS, et al. Ellagic acid protects human keratinocyte (HaCaT) cells against UVA-induced oxidative stress and apoptosis through the upregulation of the HO-1 and Nrf-2 antioxidant genes. Food Chem Toxicol 2012;50(5):1245–55. 32. Malik A, Afaq S, Shahid M, Akhtar K, Assiri A. Influence of ellagic acid on prostate cancer cell proliferation: a caspase–dependent pathway. Asian Pac J Trop Med 2011;4(7):550–5. 33. Anitha P, Priyadarsini RV, Kavitha K, Thiyagarajan P, Nagini S. Ellagic acid coordinately attenuates Wnt/b-catenin and NF-kB signaling pathways to induce intrinsic apoptosis in an animal model of oral oncogenesis. Eur J Nutr 2013;52(1):75–84. 34. Cock IE, van Vuuren SF. The potential of selected some South African plants with anti-Klebsiella activity for the treatment and prevention of ankylosing spondylitis. Inflammopharmacology 2015;23(1):21–35. 35. Naik GH, Priyadarsini KI, Naik DB, Gangabhagirathi R, Mohan H. Studies on the aqueous extract of Terminalia chebula as a potent antioxidant and a probable radioprotector. Phytomedicine 2004;11(6):530–8. 36. Aggarwal BB, Bhardwaj A, Aggarwal RS, Seeram NP, Shishodia S, Takada Y. Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res 2004;24(5A):2783–840. 37. Elmali N, Baysal O, Harma A, Esenkaya I, Mizrak B. Effects of resveratrol in inflammatory arthritis. Inflammation 2007;30(1–2):1–6. 38. Pikarsky E, Porat RM, Stein L, Abramovitch R, Amit S, Kasem S, et al. NF-kB functions as a tumour promoter in inflammation-associated cancer. Nature 2004;431(7007):461–6. 39. Joseph CC, Moshi MJ, Innocent E, Nkunya MH. Isolation of a stilbene glycoside and other constituents of Terminalia sericeae. Afr J Tradit Complement AlternMed 2007;4(4):383–6. 40. Lanzilli G, Cottarelli A, Nicotera G, Guida S, Ravagnan G, Fuggetta MP. Anti-inflammatory effect of resveratrol and polydatin by in vitro IL-17 modulation. Inflammation 2012;35(1):240–8. 41. Salih EY, Kanninen M, Sipi M, Luukkanen O, Hiltunen R, Vuorela H, et al. Tannins, flavonoids and stilbenes in extracts of African savanna woodland trees Terminalia brownii, Terminalia laxiflora and Anogeissus leiocarpus showing promising antibacterial potential. S Afr J Bot 2017;108:370–86. 42. Dark GG, Hill SA, Prise VE, Tozer GM, Pettit GR, Chaplin DJ. Combretastatin A-4, an agent that displays potent and selective toxicity toward tumor vasculature. Cancer Res 1997;57(10):1829–34. 43. Bhardwaj S, Bakshi S, Chopra B, Dhingra A, Dhar KL. Synthesis of combretastatin analogues with their potent anticancer activity. Int J Res Pharm Sci 2010;1:414–6. 44. Mikstacka R, Zieli
nska-Przyjemska M, Dutkiewicz Z, Cichocki M, Stefa
nski T, Kaczmarek M, et al. Cytotoxic, tubulin-interfering and proapoptotic activities of 40 -methylthio-trans-stilbene derivatives, analogues of trans-resveratrol. Cytotechnology 2018;70(5):1349–62. 45. Zhu H, Zhang J, Xue N, Hu Y, Yang B, He Q. Novel combretastatin A-4 derivative XN0502 induces cell cycle arrest and apoptosis in A549 cells. Invest New Drugs 2010;28(4):493–501. 46. Kanthou C, Greco O, Stratford A, Cook I, Knight R, Benzakour O, et al. The tubulin-binding agent combretastatin A-4-phosphate arrests endothelial cells in mitosis and induces mitotic cell death. Am J Pathol 2004;165(4):1401–11. 47. Biersack B, Effenberger K, Schobert R, Ocker M. Oxazole-bridged combretastatin A analogues with improved anticancer properties. ChemMedChem 2010;5(3):420–7. 48. Delmonte A, Sessa C. AVE8062: a new combretastatin derivative vascular disrupting agent. Expert Opin Investig Drugs 2009;18(10):1541–8. 49. Nathwani SM, Hughes L, Greene LM, Carr M, O’Boyle NM, McDonnell S, et al. Novel cis-restricted b-lactam combretastatin A-4 analogues display anti-vascular and anti-metastatic properties in vitro. Oncol Rep 2013;29(2):585–94. 50. Narasimhan B, Sharma D, Kumar P. Biological importance of imidazole nucleus in the new millennium. Med Chem Res 2011;20(8):1119–40. 51. Starcevi
c K, Kralj M, Ester K, Sabol I, Grce M, Paveli
c K, et al. Synthesis, antiviral and antitumor activity of 2-substituted-5-amidino-benzimidazoles. Bioorg Med Chem 2007;15(13):4419–26.

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52. Arcamone FM, Animati F, Barbieri B, Configliacchi E, D’Alessio R, Geroni C, et al. Synthesis, DNA-binding properties, and antitumor activity of novel distamycin derivatives. J Med Chem 1989;32(4):774–8. 53. Baraldi PG, Beria I, Cozzi P, Bianchi N, Gambari R, Romagnoli R. Synthesis and growth inhibition activity of a-Bromoacrylic heterocyclic and benzoheterocyclic derivatives of distamycin A modified on the amidino moiety. Bioorg Med Chem 2003;11(6):965–75. 54. Franchetti P, Marchetti S, Cappellacci L, Yalowitz JA, Jayaram HN, Goldstein BM, et al. A new C-nucleoside analogue of tiazofurin: synthesis and biological evaluation of 2-b-d-ribofuranosylimidazole-4-carboxamide (imidazofurin). Bioorg Med Chem Lett 2001;11(1):67–9. 55. Sirdaarta JP. Phytochemical study and anticancer potential of high antioxidant Australian native plants [Ph.D. dissertation]. Griffith University; 2016. 56. Spiegel S, Milstien S. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol 2003;4(5):397–407. 57. Xia P, Wang L, Moretti PA, Albanese N, Chai F, Pitson SM, et al. Sphingosine kinase interacts with TRAF2 and dissects tumor necrosis factor-a signaling. J Biol Chem 2002;277(10):7996–8003. 58. Ogretmen B, Hannun YA. Biologically active sphingolipids in cancer pathogenesis and treatment. Nat Rev Cancer 2004;4(8):604–16. 59. Paugh BS, Paugh SW, Bryan L, Kapitonov D, Wilczynska KM, Gopalan SM, et al. EGF regulates plasminogen activator inhibitor-1 (PAI-1) by a pathway involving c-Src, PKCd, and sphingosine kinase 1 in glioblastoma cells. FASEB J 2008;22(2):455–65. 60. Wright MH, Greene AC, Cock IE. Investigating the pharmacognostic potential of Indian Terminalia spp. in the treatment and prevention of yersiniosis. Pharmacogn Commun 2017;7(3):108–13. 61. Gu B, Shalom J, Cock IE. Anti-proliferative properties of Terminalia sericea Burch. Ex DC leaf extracts against Caco2 and Hela cancer cell lines. Pharmacogn J 2018;10(3):73–80. 62. Chang MY, Shen L. Linalool exhibits cytotoxic effects by activating antitumor immunity. Molecules 2014;19(5):6694–706. 63. Hassan SB, Gali-Muhtasib H, G€oransson H, Larsson R. Alpha terpineol: a potential anticancer agent which acts through suppressing NF-kB signalling. Anticancer Res 2010;30(6):1911–9. 64. Tarapore RS, Siddiqui IA, Adhami VM, Spiegelman VS, Mukhtar H. The dietary terpene lupeol targets colorectal cancer cells with constitutively active Wnt/b-catenin signaling. Mol Nutr Food Res 2013;57(11):1950–8. 65. Wattenberg LW, Sparnins VL, Barany G. Inhibition of N-nitrosodiethylamine carcinogenesis in mice by naturally occurring organosulfur compounds and monoterpenes. Cancer Res 1989;49(10):2689–92. 66. Courtney R, Sirdaarta J, White A, Cock IE. Inhibition of Caco-2 and HeLa proliferation by Terminalia carpentariae CT White and Terminalia grandiflora Benth. extracts: identification of triterpenoid components. Pharmacogn J 2017;9(4):441–51. 67. Lin KW, Huang AM, Lin CC, Chang CC, Hsu WC, Hour TC, et al. Anti-cancer effects of ursane triterpenoid as a single agent and in combination with cisplatin in bladder cancer. Mol Cell Pharmacol 2014;740:742–51. 68. Es-Saady D, Simon A, Jayat-Vignoles C, Chulia AJ, Delage C. MCF-7 cell cycle arrested at G1 through ursolic acid, and increased reduction of tetrazolium salts. Anticancer Res 1996;16:481–6. 69. Neto CC, Vaisberg AJ, Zhou B-N, Kingston DGI, Hammond GB. Cytotoxic triterpene acids from the Peruvian medicinal plant Polylepis racemosa. Planta Med 2000;66:483–4. 70. Subbaramaiah K, Michaluart P, Sporn MB, Dannenberg AJ. Ursolic acid inhibits cyclooxygenase-2 transcription in human mammary epithelial cells. Cancer Res 2000;60:2399–404. 71. Konopleva M, Zhang W, Shi Y, McQueen T, Tsao T, Abdelrahim M, et al. Synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid induces growth arrest in HER2-over expressing breast cancer cells. Mol Cancer Ther 2006;5:317–28. 72. Nomura M, Takahashi T, Uesugi A, Tanaka R, Kobayashi S. Inotodiol, a lanostane triterpenoid, from Inonotus obliquus inhibits cell proliferation through caspase-3-dependent apoptosis. Anticancer Res 2008;28(5A):2691–6. 73. Jiang J, Grieb B, Thyagarajan A, Sliva D. Ganoderic acids suppress growth and invasive behavior of breast cancer cells by modulating Ap-1 and NfKappa B signaling. Int J Mol Med 2008;21(5):577–84. 74. Woerdenbag HJ, Lemstra W, Malingr
e T, Konings AW. Enhanced cytostatic activity of the sesquiterpene lactone eupatoriopicrin by glutathione depletion. Br J Cancer 1989;59(1):68. 75. Lesiak K, Koprowska K, Zalesna I, Nejc D, D€uchler M, Czyz M. Parthenolide, a sesquiterpene lactone from the medical herb feverfew, shows anticancer activity against human melanoma cells in vitro. Melanoma Res 2010;20(1):21–34. 76. Garcia-Bermejo ML, Leskow FC, Fujii T, Wang Q, Blumberg PM, Ohba M, et al. Diacylglycerol (DAG)-lactones, a new class of protein kinase C (PKC) agonists, induce apoptosis in LNCaP prostate cancer cells by selective activation of PKCa. J Biol Chem 2002;277(1):645–55. 77. Sparnins VL, Chuan J, Wattenberg LW. Enhancement of glutathione S-transferase activity of the esophagus by phenols, lactones, and benzyl isothiocyanate. Cancer Res 1982;42(4):1205–7. 78. Yin Z, Ivanov VN, Habelhah H, Tew K, Ronai Z. Glutathione S-transferase p elicits protection against H2O2-induced cell death via coordinated regulation of stress kinases. Cancer Res 2000;60(15):4053–7. 79. Townsend DM, Tew KD. The role of glutathione-S-transferase in anti-cancer drug resistance. Oncogene 2003;22(47):7369–75.

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Chapter 45

Uncaria tomentosa: A promising source of therapeutic agents for prevention and treatment of oxidative stress and cancer Francesca Ciania, Natascia Cocchiaa, Viola Calabro`b, Alessandra Polliceb, Lucianna Maruccioa, Domenico Carotenutoc, Luigi Espositoa, Luigi Avallonea, and Simona Tafuria a

Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy, b Department of Biology, Complesso

Universitario Monte S. Angelo, University of Naples Federico II, Naples, Italy, c UNMSM, Universidad Nacional Mayor San Marcos, Lima, Peru

Introduction The reactive species, in particular, those of the oxygen (ROS) and nitrogen species (RNS), play roles in the activation of signaling pathways in animal and plant cells in response to changes in environmental conditions of intra- and extracellular compartments. During the last two decades, extensive research has revealed some of the mechanisms by which continued oxidative stress (OS) can lead to chronic inflammation and subsequently to diseases including cancer, cardiovascular failure, diabetes, neurological, and pulmonary diseases. Reactive species and mediators of inflammation can cause genetic lesions such as mutations in tumor suppressor genes leading to genome instability and change in gene expression patterns. These modifications have been described in cancer diseases before the development of cancer itself. However, ROS and mediators of inflammation can perform their action of either pro- or antitumorigenic activities increasing the complexity of cancer biology. Cancer is a multistage process in which three steps are recognized: initiation, promotion, and progression.1 OS, ROS, and RNS affect all the stages of carcinogenesis: they are able to induce DNA damage, during the initiation stage, provoking structural modifications of the DNA and gene mutations. During the promotion stage, ROS can induce aberrant gene expression, alteration in cell-cell interaction or signal transduction pathways, which lead to uncontrolled cell proliferation and apoptosis failure. At the progression stage, OS takes part in neoplastic development inducing further DNA damage to the initiated cell population. To complicate matters, OS also intervene in the transition from the inflammatory state to the initiation of the cancer process. In fact ROS are produced in high amounts by inflammatory cells. Further, the tumor cells produce factors able to attract inflammatory cells. Therefore OS, chronic inflammation, and cancer are closely linked and their activity creates a closed circle destined to progressively aggravate the pathological state.2, 3 Several recent studies both in cellular systems and animals have been conducted to find out novel substances with antioxidant activities, with the aim to reduce chronic inflammation injuries and hinder the neoplastic promoters. Thorough attention by means of chemopreventive must be given to patients with oxyradical overload diseases to stop, or even reverse, carcinogenesis before cancer becomes clinically observable. Several agents are able to interfere with redox cell signaling pathways, including nutraceuticals from vegetables, fruits, spices, and plants. South America, particularly Brazil, Colombia, and Peru, is the source and reservoir of many medicinal plants. Ashaninka priests Sancoshi educated by their mentors in complete seclusion in the forest are traditionally meant to be able to recognize good spirits in individual plants that can be used to eliminate disturbances between body and spirit, and thereby restore health. According to folklore, one of the plants containing good spirits is Uncaria tomentosa. It grows as a woody vine and is found at the base of tall trees in the rainforest, winding its way up around the tree with curved thorns that resemble cat’s claws at the base of its leaves. The root and bark extracts have traditionally been used by priests to treat various diseases, including asthma, arthritis, rheumatism, abscesses, gastric ulcers, inflammation, menstrual irregularity, viral infections, and cancer.

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00045-6 © 2021 Elsevier Inc. All rights reserved.

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Antitumor and antioxidant effects of U. tomentosa have been shown in in vitro systems and some animal models. However, the adoption of different extraction protocols (e.g., aqueous vs organic solvents) has produced different mixtures of bioactive compounds making the systematic evaluation of cat’s claw biological activities quite problematic. However, U. tomentosa has been largely investigated for its antineoplastic properties, which is not surprising considering that cancer is regarded as a chronic inflammatory disease. To date, compounds isolated from cat’s claw have shown growth inhibitory activity in vitro against a wide range of human cancer cell lines, by increasing the cell death and DNA damage. The present chapter is an overview of the biological effects ascribed to U. tomentosa extracts and derived metabolites. They have been shown to suppress tumorigenesis in preclinical models.4 Therefore, U. tomentosa-derived compounds offer great promise for its use in cancer prevention and therapy by targeting redox-sensitive pathways.

Applications to other cancers or conditions In this chapter, we reviewed how U. tomentosa extracts can have effects as anticancer and antioxidant factors. Several human cancer cell lines were studied to evaluate the anticancer properties of U. tomentosa extracts. U. tomentosainduced apoptosis is the main mechanism involved in antitumor efficacy of this plant. Some adenocarcinoma from colon, cervix, breast, and lung and melanoma cell lines were found to be sensitive to the apoptotic effects of U. tomentosa extracts. Furthermore, SAOS, MCF7, and HeLa human cancer cell lines were also sensitive to the U. tomentosa apoptotic action, activating it via caspase3. Interestingly, U. tomentosa extracts have been reported to inhibit the proliferation of HL-60 and K562 human leukemia and Raji EBV-transformed B lymphoma cell lines without inducing apoptosis. U. tomentosa has been demonstrated to enhance chemotherapy-induced apoptosis, establishing a role for cancer patients as a complementary therapy. Moreover, human epidermoid cancer cells (A431) were found to be sensitive to aqueous extract of U. tomentosa bark, antagonizing the oxidative stress-induced DNA repair, supporting the use of the extract for the treatment of precancerous and early forms of squamous cell carcinomas. Reports have shown that U. tomentosa extracts and preparations are able to inhibit tumor growth and metastasis in mouse and rat models. Authors have identified in the interference on the metabolism of ROS and in some redox processes the mechanisms responsible for the beneficial effects. Chemotherapeutics are used for the treatment of many cancers, whose side effects are in many cases related to alteration in the cellular part of the blood, leading to erythropenia and leukopenia. U. tomentosa has been used as an adjuvant in the treatment of breast cancer to reduce toxic effects and restore cellular DNA damage. Even in patients with colorectal cancer, U. tomentosa has minimized the deleterious effects of chemotherapy treatment. The beneficial effect is assumed to be due to the ROS’s scavenging ability.

Uncaria tomentosa As reported by the World Health Organization (WHO) about 80% of the population use medicinal plants as alternative or complementary procedures for the treatment of their diseases. Among the plants the U. tomentosa [Willdenow ex Roemer and Schultes (Willd) de Caundolle (DC)], known as “cat’s claw,” has been used in South American countries, over the centuries, as a traditional medicine for its several supposed health benefits.5 Already since 1994 the WHO recognized U. tomentosa as an important medical plant authorizing its marketing. In fact in Europe the use of U. tomentosa in different forms, such as capsules, extracts, and tea has been introduced, for the complementary treatment of patients affected by cancer or viral diseases. The U. tomentosa is a tropical vine belonging to the family of Rubiaceae. It is a scrambling liana, up to 20–30 m long, and with a stem over 25 cm in diameter.6 This plant is a thick woody vine that grows at an altitude of 500–600 m above sea level, in high forests with abundant insolation, from the Amazon rainforest and other tropical areas of South and Central America7 (Figs. 1 and 2). The presence of hook-like thorns, growing along the vine in a leafy pattern, resembles the claws of a cat and for this reason, it is known as “cat’s claw” (Fig. 3). U. tomentosa was first described in 1830 and first studied in Peru` by the German biologist Brell in 1950.8 In Peru, many native tribes such as the Aguaruna, Ashaninka, Cashibo, and Shipibo use U. tomentosa for its multiple properties in the medical field. Keplinger, studied the medicinal system of the Ashaninka, one of the most numerous indigenous people of South America, to learn more about the healing properties found in cat’s claw extracts9; he spent several years with this population and discovered that Sanchosha priests attributed miraculous powers to some plants. In 1980, Kindberg10 reported in the Ashaninka dictionary a prickly plant, probably referable to Uncariae. So according to this folklore, one of

FIG. 1 Traditional method of drying barks of Uncaria tomentosa. Traditional method of air drying of barks. The arrow indicates a particular bark of U. tomentosa. (Photograph courtesy of Prof. Domenico Carotenuto.)

FIG. 2 Stem with and without bark of Uncaria tomentosa. The stem of U. tomentosa is observed in the presence and absence of bark. (Photograph courtesy of Prof. Domenico Carotenuto.)

FIG. 3 Leaves of Uncaria tomentosa with characteristic hooked thorns. At the base of U. tomentosa leaves are the hooked thorns (arrow), reason for the name “un˜a de gato.”. The arrow indicates a particular hooked thorn of U. tomentosa. (Photograph courtesy of Prof. Domenico Carotenuto.)

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these plants was U. tomentosa, whose root and bark extracts were traditionally used by Sanchosha to treat various diseases including asthma, arthritis, rheumatism, inflammation, and viral infections.11

Botanical classification of Uncaria genus In 1978, Ridsdale12 reported that the Uncaria genus is constituted by a total of 39 species, 34 of them are distributed in Southeast Asia, 3 in Africa, and 2 in tropical America. Andersson and Taylor in 19946 reported about 60 species of Uncaria distributed worldwide. Actually, about 40 currently known species and most of the native species are distributed in tropical areas such as Asia, Africa, the Mediterranean, and the neotropics.13 Regardless of the number of species known so far, all of them have the same general characteristics, in fact, all are lianas with monopodial main shoots and more or less horizontal patent.6 The two American species of Uncaria are U. tomentosa and U. guianensis [(Aublet), Gmell], and according to Peruvian scientists, they have often been confused.5 U. tomentosa is characterized by densely tomentose buds, and the meshing and tightening of the longer hairs, which help stipules to remain connected to each other along the margins. Moreover, the thorns are straight, very sharp, and insensitive sickles.14

Chemical composition of U. tomentosa U. tomentosa is used in Peru` and Europe and since 1994 the WHO recognized this plant as an adjunctive treatment for cancer and other diseases.15 The edible part of U. tomentosa is the bark that in the pharmaceutical industry is processed into capsules, tablets, ointments, and even tea. Heitzman et al.7 isolated about 50 different components from all plant parts of this species and 35 of which have been identified in only a couple of other species. In particular, three classes seem to play an important role in cat’s claw activity. These compounds are the alkaloids, quinovic acid glycosides, and polyhydroxylated triterpenes (Table 1).16 The alkaloid content can vary 10–40-fold depending on cultivation techniques and the season when the plant is harvested17 (Fig. 4). The oxindole alkaloids are considered the main active compounds responsible for the medicinal activities and are classified into two chemotypes: tetracyclic and pentacyclic indole alkaloids.18 The pentacyclic indole alkaloids stimulate the cellular immune system, the tetracyclic indole alkaloids the central nervous system, and have an antagonistic effect when combined.18 The chemical composition of U. tomentosa, and in particular the concentration of certain analytes present, can vary from the collection area of the plant, seasonality, maturity, and extraction method. The presence of the chemical components in U. tomentosa, in particular of oxindole alkaloids, is affected during seasons by changes in environmental factors such as water supply, temperature, and light.19

Oxidative stress Free radicals and, in particular, ROS play an important role in the regulation of metabolic activity and the functioning of certain organs. They are produced by the mitochondria during normal aerobic metabolism.20 ROS, which include the hydroxyl radicals (OH), superoxide anion (O2 ), hydrogen peroxide (H2O2), are highly reactive molecules due to the presence of an unpaired electron in their outer shell. They have a very short half-life in the range of nanoseconds to TABLE 1 Chemical composition of Uncaria tomentosa. Oxindole alkaloids Indole alkaloids Quinovic acid glycosides Pyroquinovic acid glycosides Organic acids Proanthocyanidines Sterols Triterpenes

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FIG. 4 Cut stems of Uncaria tomentosa. Stems of U. tomentosa traditionally cut from a native inhabitant. (Photograph courtesy of Prof. Domenico Carotenuto.)

milliseconds. ROS are produced as a consequence of natural cell machinery and participate in the normal function of a cell. The normal production of ROS is necessary for the functions of the body, while excessive ROS production is harmful. For instance, physiological and low levels of ROS in the spermatozoa play an important role in processes such as capacitation, hyperactivation, acrosome reaction, and sperm-oocyte fusion, thus ensuring appropriate fertilization, whereas high levels of ROS may determine sperm pathologies, such as ATP depletion and loss of sperm motility and viability.21 However, when ROS production overcomes cellular antioxidant defenses overcoming the physiological range, they cause deleterious effects due to oxidative stress which results in the oxidation of lipids, proteins, carbohydrates, and nucleotides.22 OS is a particular kind of chemical stress, which is induced, locally and/or systemically, by an excess of potentially oxidant reactive species; it appears to be a health risk factor for aging disorders and several diseases in humans and in animals.23 The production of free radicals by cells can increase considerably due to external stimulation; physical agents (ionizing radiations and UV rays), chemicals (ozone, polycyclic aromatic hydrocarbons or drugs), biological agents (bacteria), pollution can induce an increase in the production of free radicals through a specific metabolic stimulation.24 A large amount of ROS is able to attack any substrate with which they come into contact, tearing from them the electron or electrons needed to reach their own stability. This triggers radical chain processes which, if not blocked in a timely manner, can cause serious consequences on the plan, first functional, then also structural. The cell is the first target of oxidative damage. The damage, initially cellular, if prolonged through time, spreads to the tissues, organs, and then becomes systemic. Indeed, evidence shows that OS can influence the onset of diseases such as diabetes, cardiovascular diseases, depression, anxiety, neurodegenerative diseases, early senescence, inflammation, and cancers.25 Many studies have been carried out to counteract the oxidative conditions with the use of specific antioxidants.26–29 The cellular antioxidant defense systems are able to control the deleterious effects of ROS.24, 30, 31 Antioxidants are groups of substances of different chemical nature and present at low concentrations with respect to the substrate, and act with different mechanisms by inactivating or eliminating the free radicals. They can be classified into enzymatic and nonenzymatic. The enzymatic antioxidants include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). SOD catalyzes the dismutation reaction of the superoxide anion into hydrogen peroxide and is found in the mitochondria, cytosol, and extracellular space.32 CAT catalyzes the conversion of H2O2 to O2 and H2O, acting mainly in the endoplasmic reticulum, peroxisomes, mitochondria, and the cytosol of several cell types.33 Glutathione peroxidase (GPX), which is located mainly in the mitochondria,32 catalyzes the reduction of H2O2 and organic peroxides. Among the nonenzymatic antioxidants are vitamins (C and E), carotenoids, carnitine, cysteine, some metals, taurine, and albumin.34 Exogenous antioxidants can also be classified according to their mechanism of action into three groups: vitamin C, vitamin E, vitamin A, carotenoids, and phenolic compounds that react directly with free radicals transforming them into less reactive molecules; those that perform a chelating action against metal ions such as iron and copper avoiding Fenton’s reactions (albumin, transferrin, and ceruloplasmin); and minerals that are structural components of antioxidant enzymes as copper, zinc, and selenium.24 Fig. 5 shows the power hierarchy of antioxidant pyramid. Endogenous enzyme antioxidants have a higher antioxidant capacity and are located at the top of the pyramid.

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FIG. 5 The antioxidant pyramid. The power hierarchy of antioxidant pyramid is shown in this figure. Endogenous enzyme antioxidants have a higher antioxidant capacity and are located at the top of the pyramid. (From Tafuri S, Cocchia N, Landolfi F, Iorio EL, Ciani F. Redoxomics and oxidative stress: from the basic research to the clinical practice. In: Wu B, editor. Free radicals and diseases, vol. 8. Intechopen; 2016. p. 149–169. https://doi.org/10.5772/ 64577.)

Epidemiological evidence shows that the diet and an appropriate lifestyle can play a very important role in the modulation of OS and, in particular, on the deleterious effects that OS can have on organisms. Indeed, through the diet the organism introduces both oxidizable substrates and antioxidant substances which can prevent the formation and progression of degenerative diseases thus underlying the strict relationships among diet, OS, and pathologies. Some plants from South America are increasingly used for their therapeutic properties because they produce metabolites that modulate the effects of the OS.28, 29, 35 Such metabolites are polyphenols, sterols, and alkaloids, some of which are considered important nonenzymatic antioxidants able to scavenge free radicals and protect cells from OS.36

Oxidative stress and U. tomentosa U. tomentosa, the tropical rainforest vine plant, distributed naturally from Peru to Guatemala,37 is used in raw form or as extracts of root, stem bark, or leaves. Depending on different extraction protocols adopted, e.g., aqueous vs ethanol at various temperatures, Uncaria extracts may have different compositions, making systematic research on the biological activities of cat’s claw problematic. To overcome the variability in Uncaria extract composition due to the application of different extraction protocols, a method has been suggested to standardize the preparations based on U. tomentosa, by using a defined ratio of polar and nonpolar solvents. To standardize the protocol for Uncaria extract preparation, Pilarski et al.38 suggested an easy method for their extraction that produced reproducible antioxidant activity mainly ascribable to polyphenol content. The analysis of alkaloids was carried out on the bark of U. tomentosa, while ethanol and aqueous bark extracts were prepared for the quantitative determination of tannins, the total phenolic compounds, and the antioxidant capacity. The evaluation of the results has evidenced that the ethanol bark extract has greater antioxidant activity and is more effective than aqueous extract. Phyto-complex is a set of active and inactive molecules present in the plants. Each plant species requires, for its growth, a certain soil and climate, in terms of altitude, latitude, mean temperature, average rainfall, light availability, and physicochemical soil properties. Climatic conditions and soil characteristics can certainly affect the availability of metabolites necessary for the compound biosynthesis. For instance, a plant can lose the ability to synthesize specific bioactive molecules if it grows outside its specific habitat. Thus, the knowledge of the agricultural techniques, the territory, and of the phyto-complex pattern of a specific plant in relation to environment can help to obtain a standardized extract indispensable to improve the herbal product final quality and ensure the clinical effectiveness. Vera-Reyes et al.39 have highlighted how the OS elicitation can influence the gene expression of some enzymes in U. tomentosa and direct the metabolism toward the synthesis of biomolecules, in this case alkaloids with antioxidant activity, which defend the plant against adverse environmental conditions. Similarly, OS status in U. tomentosa cell cultured in a

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bioreactor, due to generation of ROS, induced pentacyclic monoterpenoid oxindole alkaloid (MOA) accumulation. MOA is known to act as immunomodulatory, cytotoxic, antileukemic, and anti-AIDS. Interestingly, the aim of the study was to use biotechnological tools to produce MOA as an alternative to chemical synthesis, that results too much elaborate and to avoid deforestation.40 Cancer cells are sensitive to OS, whereby the mechanism of action for many cancer chemotherapeutic drugs involves ROS-mediated apoptosis. Many studies have evaluated the effects of substances, herbal plants, or drugs on neoplastic cells with the aim of tracing back to the apoptosis induction via OS. In fact, hydroalcoholic extract of U. tomentosa is able to enhance chemotherapy-induced apoptosis, establishing a role for cancer patients as complementary therapy.41 Also, Ciani et al. reported the ability of an aqueous bark extract of U. tomentosa to induce oxidative DNA damage and antagonize the mechanism of DNA repair in A431 cancer cells (human epidermoid cancer cells) with a defective G1/S checkpoint with a consequent accumulation of G2/M arrested cells followed by massive apoptosis.42 By now it is everyone’s opinion that the environment must be safeguarded and that pollution, in general, can cause problems for the health of humans, animals, and other living organisms. One of the causes of pollution is the presence of xenobiotics, including pesticides, which are the reason for many harmful effects on health. Some studies assessed if U. tomentosa extracts are able to counteract the deleterious effects of pesticides responsible for OS. Catechol and 2,4DCP, derived from 2,4-dichlorophenoxyacetic (2,4-D) transformation, used for the cultivation of rice, cereals, and so on, are hormonal herbicides characterized by high toxicity, persistence of residues in the environment and capacity to bioaccumulate in living organisms. U. tomentosa leaf and bark extracts were tested to verify if they possess antioxidants properties against the OS provoked by 2,4-DCP and catechol in red blood cells.43 Although no striking effect was observed on most of the parameters studied, U. tomentosa was found to be effective in preventing oxidation of hemoglobin by decreasing the amount of ROS in parallel with the onset of hemolysis caused by 2,4-DCP. Furthermore, no direct action of the extracts was observed on catechol, which is a precursor of semiquinones. On the other hand, semiquinones, radicals usually formed as a result of catechol interaction with red blood cells, are not affected by the action of U. tomentosa. However, the ethanolic extracts were more effective than the aqueous ones. The antioxidant effect of leaf and bark ethanolic extracts from U. tomentosa was also detected in mononuclear blood human cells.44 Moreover, Dal Santo et al.45 evaluated the protective effect of U. tomentosa, in the form of hydroalcoholic extract, on acute exposure to glyphosate-Roundup using zebrafish as a model system. The exposure to the Roundup, a glyphosate-based herbicide, induces a whole series of effects on plants and plant-eating animals, including the onset of OS and genotoxicity. U. tomentosa extract was able to avoid oxidative damage induced by Roundup in zebrafish probably because of its ROS scavenger ability. The extract was found to contain essentially phenols and flavonoids which are really effective antioxidant substances.

Cancer and U. tomentosa U. tomentosa has been traditionally used in the treatment of various diseases, including cancer. U. tomentosa is one of the best-selling plants in the world and is used as immunomodulatory, anti-inflammatory, and anticancer. For the last couple of decades, researchers have experimented several methods of extraction to study pharmacological properties of the plant with antitumor activities. Antitumor effects of U. tomentosa have been shown in vitro and in some animal models. Compounds isolated from cat’s claw have shown inhibitory activity against different human cancer cell lines, causing increased DNA damage and cell death. Several reports have shown that U. tomentosa extracts and preparations inhibit tumor growth and metastasis in mouse and rat models.35, 37, 46–50 In a previous study, Coussens and Werb51 suggested, in light of the well-established role of chronic inflammation in cancer progression, that the anti-inflammatory activity of U. tomentosa may be at least partially responsible for its antitumor activity.37 The U. tomentosa component called mitraphylline demonstrated in vitro antitumoral activity against human neuroblastoma and glioma cell lines.52 Despite the ineffectiveness of U. tomentosa extract in regulating glutathione (GSH) and lipidic peroxidation (LPO) level, it may be suggested that U. tomentosa extract is interesting as an adjuvant in the treatment of solid tumors. The fundament of this statement occurs, partially at least, because of its action on enzymes that regulate OS. It is well known that the interference in some redox processes and ROS metabolism are a possible way of achieving apoptosis in neoplastic cells.53, 54 Dreifuss et al.35 showed in vivo the importance of the modulation of OS as part of the antineoplastic activity of U. tomentosa, this effect is possibly due to a synergic combination of substances, most of them with antioxidant properties.

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Also, Ciani et al.42 showed that U. tomentosa cytotoxicity can be ascribed, at least in part, to its ability both to induce oxidative DNA damage and antagonize the mechanism of DNA repair relying on activity of YB-1, a protein involved in the recognition and repair of DNA lesions. They also showed that squamous carcinoma cells are more susceptible to U. tomentosa treatment than untransformed keratinocytes supporting the use of U. tomentosa extract for the treatment of precancerous and early forms of squamous cell carcinomas. Another study investigated the possible proapoptotic mechanism of root bark extracts of U. tomentosa in three different tumoral cell lines: SAOS (human osteosarcoma cell line), MCF7 (human breast carcinoma cell line), and HeLa (human cervical carcinoma cell line). Data obtained clearly showed induction of apoptosis, by the n-BuOH soluble fraction of U. tomentosa, via caspase3 activation.53 U. tomentosa methanolic and aqueous extracts were tested against cancer cell lines, in particular against Caco2 (human epithelial colorectal adenocarcinoma cell line) and Hela (human epithelioid cervix carcinoma cell line) cells. The methanolic extract was particularly effective against both cell lines suggesting their potential use in the treatment and prevention of some cancers.55 In contrast, organic solvent extracts (ethyl acetate, chloroform, and hexane) were less potent in the control of cancer cell proliferation. Moreover, aqueous U. tomentosa extract has also been reported to inhibit the proliferation of HL-60 and K562 human leukemia and Raji EBV-transformed B lymphoma cell lines without inducing apoptosis.56 U. tomentosa aqueous extract was also demonstrated to be effective against lymphoblastic and breast cancer cell lines by inducing apoptosis.57, 58 Similarly, apoptotic activity was reported for several U. tomentosa hydro-alcohol extracts against human HT-29 and SW707 (colon adenocarcinoma), KB (cervical carcinoma), MCF7 (breast carcinoma), A549 (nonsmall cell lung carcinoma), HL-60 (promyelocytic leukemia) cells, as well as mouse LL/2 (lung carcinoma) and B16 (melanoma) cells.50 Chemotherapy is often the recommended treatment for cancer, alone or in combination with other drugs and/or radiotherapy. A frequent consequence of chemotherapy is the appearance of undesirable side effects, including OS. Complementary and alternative medicine is a tool to eventually ameliorate and reduce chemotherapy discomfort. Breast cancer is the most frequent neoplasm affecting women worldwide. Some of the recommended treatments involve chemotherapy whose toxic effects include leukopenia and neutropenia. U. tomentosa has been used to counteract the adverse effects of chemotherapy in a randomized clinical trial. U. tomentosa reduced the neutropenia caused by chemotherapy and was also able to restore cellular DNA damage.59 A similar study demonstrated the effectiveness of U. tomentosa in minimizing the side effects of chemotherapy in patients affected by colorectal cancer.48 Farias et al.47 demonstrated that U. tomentosa, in mice, had a positive effect on the number of myeloid progenitors and this result is promising for the utilization of the plant extract associated with chemotherapy, with the aim to minimize the side effects of this treatment. Remarkably, U. tomentosa has shown a positive effect on myeloid progenitor cells and was suggested as a promising therapy to minimize the adverse effects of chemotherapy.47 Antioxidant and antitumoral activities of U. tomentosa were demonstrated by Dreifuss et al.46 in a study performed on a solid tumor. In this experimental study, Walker 256 cells, deriving from rat breast carcinoma cell line syngeneic to Wistar rats, were subcutaneously inoculated in male Wistar rats. The authors revealed a dose-dependent role of U. tomentosa hydroalcoholic extract in the reduction of tumor mass and volume and an adjuvant of redox state in the treatment of solid tumors. Both effects are supposed to be due to a noteworthy correlation between the antitumoral properties of U. tomentosa and its potential as a free radical scavenger and as a guarantor of ROS homeostasis. A series of clinical trials have been performed on people affected by various forms of cancer to assess whether U. tomentosa, in various forms, could mitigate side effects of chemotherapy. On the basis of these findings, colorectal cancer (CRC) patients were administered U. tomentosa in herbal preparation form, corresponding to aqueous extracts, during the chemotherapy treatment. The results showed no significant reduction in the adverse effects of chemotherapy. The authors ascribed this negative result to colectomy, which patients underwent, as the reason why U. tomentosa was not adequately absorbed. However, further studies are needed to understand the real reasons for these unsatisfactory results.48

Conclusions The studies on U. tomentosa have been conducted using extracts obtained from bark, root or leaves of the plant with aqueous or alcoholic extraction methods. Scientific studies attribute to the extracts of this plant favorable antioxidant and anticancer activities. The chemical analyses of the extracts show the presence of numerous substances, including tannins and phenolic compounds, which act as antioxidants, and alkaloids, in particular the pentacyclic oxindoles, which are considered responsible for numerous anticancer effects in experiments performed on cell cultures and in clinical trials. The combined antineoplastic and antioxidant actions of U. tomentosa are indeed promising for its use as therapeutic alone or in combination with other drugs, however, further studies are necessary to improve and standardize the pharmaceutical preparations.

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Summary points l

l l

l

l

This chapter focuses on U. tomentosa, which is a thorny liana that grows wild in the upper Amazon region of Peru and neighboring countries. The root and bark extracts have traditionally been used by indigenous people of South America to treat various diseases. Many active compounds have been isolated from U. tomentosa and extensively studied for their potential use as antioxidant and anticancer agents. The chemical analyses of the extracts show the presence of tannins and phenolic compounds, which act as antioxidants, and alkaloids, which are considered responsible for numerous anticancer effects. The combined antineoplastic and antioxidant actions of U. tomentosa are indeed promising for its use as therapeutic alone or in combination with other drugs.

References 1. Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med 2010; 49(11):1603–16. 2. Frenkel K. Carcinogen-mediated oxidant formation and oxidative DNA damage. Pharmacol Ther 1992;53:127–66. 3. Tafuri S, Cocchia N, Landolfi F, Iorio EL, Ciani F. Redoxomics and oxidative stress: from the basic research to the clinical practice. In: Wu B, editor. Free radicals and diseases. vol. 8. Intechopen; 2016. p. 149–69. https://doi.org/10.5772/64577. 4. Fang J, Seki T, Maeda H. Therapeutic strategies by modulating oxygen stress in cancer and inflammation. Adv Drug Deliv Rev 2009;61:290–302. 5. Obregon-Vilches LE. Cat’s claw, genus Uncaria. Botanical, chemical and pharmacological studies of Uncaria tomentosa (Willd.) D.C.(Rubiaceae) and Uncaria guianensis (Aubl.) Gmel. Lima, Peru: Institute of American Phytotherapy; 1994. 6. Andersson L, Taylor CM. Rubiaceae-Cinchoneae-Coptosapeltaea. In: Harling G, Andersson L, editors. Flora of Ecuador. vol. 50. Copenhagen: Council for Nordic Publications in botany; 1994. p. 1–17. 7. Heitzman M, Neto C, Winiarz E, Vaisbergb A, Hammondc G. Ethnobotany, phytochemistry and pharmacology of Uncaria (Rubiaceae). Phytochemistry 2005;66:5–29. 8. Cabieses F. The saga of the cat’s claw. Lima, Peru`: Lactea Editores; 1994. 9. Keplinger K. Das Shevatari. Eine vergessene Schrift aus dem peruanischen Urwald. Innsbruck, Austria: Studien-Verlag; 1993. 10. Kindberg L. Dictionario Ashaninka. Pucallpa, Peru`: Instituto linguistico de verano; 1980. 11. Keplinger K, Laus G, Wurm M, Dierich M, Teppner H. Uncaria tomentosa (Willd.) DC. Ethnomedicinal use and new pharmacological, toxicological and botanical results. J Ethnopharmacol 1999;64:23–34. 12. Ridsdale CE. A revision of Mitragyna and Uncaria (Rubiaceae). Blumea 1978;24:43–100. 13. Mabberley DJ. Mabberley’s plant book. 3rd ed. Cambridge: Cambridge University Press; 2008. 14. Lindorf H. Bark and wood anatomy of Uncaria guianensis and Uncaria tomentosa (cat’s claw). IAWA J 2005;26(2):239–51. 15. Sandoval M, Okuhama NN, Zhang XS, Condezo LA, Lao J, Angeles’ FM, et al. Anti-inflammatory and antioxidant activities of cat’s claw (Uncaria tomentosa and guianensis) are independent of their alkaloid content. Phytomedicine 2002;9(4):325–37. 16. Valerio Jr. LG, Gonzales GF. Toxicological aspects of the south American herbs cat’s claw (Uncaria tomentosa) and Maca (Lepidium meyenii): a critical symposium. Rev Toxicol 2005;24(1):11–35. 17. Laus G, Keplinger K. Separation of stereoisomeric oxindole alkaloids from Uncaria tomentosa by high performance chromatography. J Chromatogr 1994;662:243–9. 18. Reinhard KH. Uncaria tomentosa (Willd.) D.C.: cat’s claw, un˜a de gato, or saventaro. J Altern Complement Med 1999;5(2):143–51. 19. Alvarenga-Venutolo S, Rosales-Lo´pez C, Sa´nchez-Chinchilla L, Mun˜oz-Arrieta R, Aguilar-Cascante F. Seasonality effect on the composition of oxindole alkaloids from distinct organs of Uncaria tomentosa from the Caribbean region of Costa Rica. Phytochemistry 2018;151:26–31. 20. Adam-Vizi V. Production of reactive oxygen species in brain mitochondria: contribution by electron transport chain and non-electron transport chain sources. Antioxid Redox Signal 2005;7:1140–9. 21. Tafuri S, Ciani F, Iorio EL, Esposito L, Cocchia N. Reactive oxygen species (ROS) and male fertility. In: Wu B, editor. New discoveries in embryology. London: Intechopen; 2015. p. 19–40. 22. Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. Oxidative stress and antioxidant defense. World Allergy Organ J 2012;5:9–19. 23. Ray PD, Huang BW, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal 2012;24(5):981–90. 24. Sies H. Strategies of antioxidant defense. Eur J Biochem 1993;21:213–9. 25. Cheng L, Miao X, Li F, Wang S, Liu Q, Wang Y, et al. Oxidative stress-related mechanisms and antioxidant therapy in diabetic retinopathy. Oxid Med Cell Longev 2017;2017:1–15. 26. Cocchia N, Corteggio A, Altamura G, Tafuri S, Rea S, Rosapane I, et al. The effects of superoxide dismutase addition to the transport medium on cumulus-oocyte complex apoptosis and IVF outcome in cats (Felis catus). Reprod Biol 2015;15(1):56–64. 27. Lu JM, Lin PH, Yao Q, Chen C. Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems. J Cell Mol Med 2010;14(4):840–60. 28. Del Prete C, Tafuri S, Ciani F, Pasolini MP, Ciotola F, Albarella S, et al. Influences of dietary supplementation with Lepidium meyenii (Maca) on stallion sperm production and on preservation of sperm quality during storage at 5°C. Andrology 2018;6:351–61.

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29. Tafuri S, Cocchia N, Carotenuto D, Vassetti A, Staropoli A, Mastellone V, et al. Chemical analysis of Lepidium meyenii (Maca) and its effects on redox status and on reproductive biology in stallions. Molecules 2019;24(10):1981. 30. Costantino M, Giuberti G, Caraglia A, Caraglia M, Lombardi A, Misso G, et al. Possible antioxidant role of SPA therapy with chlorine-sulphurbicarbonate mineral water. Amino Acids 2009;36:161–5. 31. Del Prete C, Ciani F, Tafuri S, Pasolini MP, Valle GD, Palumbo V, et al. Effect of superoxide dismutase, catalase, and glutathione peroxidase supplementation in the extender on chilled semen of fertile and hypofertile dogs. J Vet Sci 2018;19:667–75. 32. Galecka E, Jacewicz R, Mrowicka M, Florkowski A, Galecki P. Antioxidative enzymes-structure, properties, functions. Pol Merkur Lekarski 2008;25:266–8. 33. Scibior D, Czeczot H. Catalase: structure, properties, functions. Postepy Hig Med Dosw 2006;60:170–80. 34. Halliwell B. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet 1997;344:721–4. 35. Dreifuss AA, Bastos-Pereira AL, Fabossi IA, Lı´vero FAR, Stolf AM, Alves de Souza CE, et al. Uncaria tomentosa exerts extensive anti-neoplastic effects against the Walker-256 tumour by modulating oxidative stress and not by alkaloid activity. PLoS One 2013;8(2)e54618. 36. Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 1996; 20(7):933–56. 37. Fazio AL, Ballen D, Cesari IM, Abad MJ, Arsenak M, Taylor P. An ethanolic extract of Uncaria tomentosa reduces inflammation and B16-BL6 melanoma growth in C57BL/6 mice. Bol Latinoam Caribe Plant Med Aromat 2008;7:217–24. 38. Pilarski R, Zielinski H, Ciesiołka D, Gulewicz K. Antioxidant activity of ethanolic and aqueous extracts of Uncaria tomentosa (Willd.) DC. J Ethnopharmacol 2006;104:18–23. 39. Vera-Reyes I, Huerta-Heredia AA, Ponce-Noyola T, Flores-Sanchez IJ, EsparzaGarcıa F, Cerda-Garcıa-Rojas CM, et al. Strictosidine-related enzymes involved in the alkaloid biosynthesis of Uncaria tomentosa root cultures grown under oxidative stress. Biotechnol Prog 2013;29(3):621–30. 40. Trejo-Tapia G, Sepulveda-Jimenez G, Trejo-Espino JL, Cerda-Garcıa-Rojas AM, de la Torre M, Rodrıguez-Monroy M, et al. Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst. Biotechnol Bioeng 2007;98:230–8. 41. de Oliveira LZ, Farias ILG, Rigo ML, Glanzner WG, Gonc¸alves PBD, Cadona´ FC, et al. Effect of Uncaria tomentosa extract on apoptosis triggered by oxaliplatin exposure on HT29 cells. Evid Based Complement Alternat Med 2014;2014:274786. 42. Ciani F, Tafuri S, Troiano A, Cimmino A, Fioretto BS, Guarino AM, et al. Anti-proliferative and proapoptotic effects of Uncaria tomentosa aqueous extract in squamous carcinoma cells. J Ethnopharmacol 2018;211:285–94. 43. Bors M, Bukowska B, Pilarski R, Gulewicz K, Oszmianski J, Michałowicz J, et al. Protective activity of the Uncaria tomentosa extracts on human erythrocytes in oxidative stress induced by 2,4-dichlorophenol (2,4-DCP) and catechol. Food Chem Toxicol 2011;49:2202–11. 44. Bors M, Michalowicz J, Pilarski R, Sicinska P, Gulewicz K, Bukowska B. Studies of biological properties of Uncaria tomentosa extracts on human blood mononuclear cells. J Ethnopharmacol 2012;142:669–78. 45. Dal Santo G, Grotto A, Boligon AA, Da Costa B, Rambo CL, Fantini EA, et al. Protective effect of Uncaria tomentosa extract against oxidative stress and genotoxicity induced by glyphosate-roundup® using zebrafish (Danio rerio) as a model. Environ Sci Pollut Res 2018;25:11703–15. 46. Dreifuss AA, Bastos-Pereira AL, Avila TV, Soley Bda S, Rivero AJ, Aguilar JL, et al. Antitumoral and antioxidant effects of a hydroalcoholic extract of cat’s claw (Uncaria tomentosa) (Willd. Ex Roem. & Schult) in an in vivo carcinosarcoma model. J Ethnopharmacol 2010;130:127–33. 47. Farias I, do Carmo Arau´jo M, Zimmermann ES, Dalmora SL, Benedetti A, AlvarezSilva M, et al. Uncaria tomentosa stimulates the proliferation of myeloid progenitor cells. J Ethnopharmacol 2011;137(1):856–63. 48. Farias I, do Carmo Arau´jo M, Farias JG, Rossato LV, Elsenbach LI, Dalmora SL, et al. Uncaria tomentosa for reducing side effects caused by chemotherapy in CRC patients: clinical trial. Evid Based Complement Alternat Med 2012;8:892182. 49. Caballero M, Arsenak M, Abad MJ, Cesari IM, Taylor PG. Effect of 3 plant extracts on B16-BL6 melanoma cell growth and metastasis in C57Bl/6 mice. Acta Cient Venez 2005;55:21–7. 50. Pilarski R, Filip B, Wietrzyk J, Kuras M, Gulewicz K. Anticancer activity of the Uncaria tomentosa (Willd.) DC. Preparations with different oxindole alkaloid composition. Phytomedicine 2010;17(14):1133–9. 51. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420(6917):860–7. 52. Garcı´a Prado E, Garcı´a Gimenez MD, De la Puerta Va´zquez R, Espartero Sa´nchez JL, Sa´enz Rodrı´guez MT. Antiproliferative effects of mitraphylline, a pentacyclic oxindole alkaloid of Uncaria tomentosa on human glioma and neuroblastoma cell lines. Phytomedicine 2007;14(4):280–4. 53. De Martino L, Martinot JL, Franceschelli S, Leone A, Pizza C, De Feo V. Proapoptotic effect of Uncaria tomentosa extracts. J Ethnopharmacol 2006;107(1):91–4. 54. Cheng AC, Jian CB, Huang YT, Lai CS, Hsu PC, Pan MH. Induction of apoptosis by Uncaria tomentosa through reactive oxygen species production, cytochrome c release, and caspases activation in human leukemia cells. Food Chem Toxicol 2007;45(11):2206–18. 55. Shen J, Shalom J, Cock IE. The Antiproliferative properties of Uncaria tomentosa Willd. DC. Extracts against Caco2 and HeLa cancer cell lines. Pharmacol Commun 2018;8(1):8–14. 56. Sheng Y, Akesson C, Holmgren K, Bryngelsson C, Giamapa V, Pero RW. An active ingredient of cat’s claw water extracts: identification and efficacy of quinic acid. J Ethnopharmacol 2005;96(3):577–84. 57. Bacher N, Tiefenthaler M, Sturm S, Stuppner H, Ausserlechner MJ, Kofler R, et al. Oxindole alkaloids from Uncaria tomentosa induce apoptosis in proliferating, G0/G1-arrested and bcl-2- expressing acute lymphoblastic leukaemia cells. Br J Haematol 2006;132(5):615–22. 58. Riva L, Coradini D, Di Fronzo G, De Feo V, De Tommasi N, De Simone F, et al. The antiproliferative effects of Uncaria tomentosa extracts and fractions on the growth of breast cancer cell line. Anticancer Res 2001;21(4A):2457–61. 59. do Carmo Santos Arau´jo M, Farias IL, Gutierres J, Dalmora SL, Flores N, Farias J, et al. Uncaria tomentosa-adjuvant treatment for breast cancer: clinical trial. Evid Based Complement Alternat Med 2012;2012:676984.

Chapter 46

Pharmacological ascorbate and use in pancreatic cancer Rory S. Carroll, Garry R. Buettner, and Joseph J. Cullen Free Radical and Radiation Biology Program, Departments of Surgery and Radiation Oncology, Holden Comprehensive Cancer Center, The University of Iowa Hospitals and Clinics, The University of Iowa College of Medicine, and the Iowa City Veterans Affairs, Iowa City, IA, United States

List of abbreviations DHA MnPs P-AscH2 PDAC SVCT

dehydroascorbic acid manganoporphyrins pharmacological ascorbate pancreatic ductal adenocarcinoma sodium-dependent vitamin C transporter

Introduction Pancreatic cancer is the fourth leading cause of cancer death in the United States, with an estimated 56,770 new cases and 45,750 deaths in the United States for 2019.1 Surgical resection of the primary tumor provides the only potential for curative intent, but less than 20% of these patients survive past 5 years. Unfortunately, the majority of patients present with locally advanced disease, with median survival less than 6 months. Chemotherapy and radiation therapy regimens have evolved over the years, however, very little progress has been made in improving survival.2 There is obviously a great need for increasing effectiveness of these modalities. In this chapter, we review the promising data on vitamin C (ascorbate or ascorbic acid), as a safe and potentially effective adjuvant treatment to this deadly disease. Since it was first fully described in 1933, vitamin C has been found to play many roles in biochemical processes based on its ability to act as an enzyme cofactor and as a donor antioxidant.3 One vitamin C function is the maintenance of dioxygenases which are involved in collagen synthesis among other physiologic processes, with deficiencies resulting in the well-known condition scurvy. In the 1970s, several hypotheses also promoted cancer as a disease associated with collagen and vitamin C deficiency.4–6 Thus, vitamin C became an early unorthodox therapy for the treatment of cancer; clinical studies by Cameron et al. showed increased overall survival in terminal cancer patients treated with high-dose ascorbate.7–9 Subsequent randomized controlled trials by Moertel and colleagues found no benefit in administering ascorbate to cancer patients.10, 11 These randomized trials administered oral ascorbate, while the studies by Cameron included both oral and high-dose intravenous ascorbate. The differences between oral and IV ascorbate bioavailability were unknown at that time. Although there are epidemiologic data suggesting that dietary vitamin C is protective against development of cancer, randomized control trials with oral vitamin C as an intervention have not shown protective effects.12–14 However, recent discoveries in the pharmacokinetics of IV ascorbate demonstrate a significant increase in achievable plasma levels using pharmacological ascorbate (P-AscH
) when compared to orally administered ascorbate.15, 16 In this chapter, we review the role of P-AscH
as a promising adjuvant therapy for pancreatic cancer.

Ascorbate biochemistry Ascorbate is a water-soluble ketolactone which, at physiologic pH, exists predominantly as the monoanion, AscH
. It is an effective donor antioxidant, able to undergo two consecutive oxidations to form ascorbate radical and dehydroascorbic acid (DHA) (Fig. 1).17 Ascorbate is also an enzyme cofactor for many physiologic processes.18 Importantly, but not limited to, Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00046-8 © 2021 Elsevier Inc. All rights reserved.

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FIG. 1 The oxidation of ascorbate generates H2O2. At pharmacological concentrations ascorbate can act as a prooxidant by donating an electron to oxygen, ultimately generating H2O2 and dehydroascorbic acid (DHA).

maintaining 2-oxogluturate-dependent dioxygenases that are involved in collagen synthesis, epigenetics regulation, and cellular responses to hypoxia.19 Humans depend on dietary ascorbate, which is absorbed by small intestine enterocytes via sodium-dependent vitamin C transporters (SVCTs). These transporters are located on the apical surface of small bowel enterocytes, and also on renal tubular cells where reabsorption is controlled. DHA on the other hand is absorbed via a sodium-independent glucose transporters (GLUTs).20 Ascorbate absorption from the intestinal tract is tightly controlled.16 Bioavailability of oral ascorbate is limited as doses reach 200 mg. At this point absorption decreases and urine excretion increases. Plasma concentrations are therefore limited to less than 200 mM when ascorbate is administered orally.15, 16 When ascorbate is infused intravenously, on the other hand, with doses of up to 50–100 g, plasma levels of 15–25 mM can be achieved.21 With pharmacological doses, ascorbate undergoes oxidation to produce hydrogen peroxide (H2O2) (Fig. 1),22 while intracellular antioxidant enzymes like catalase, glutathione peroxidase, and peroxiredoxins efficiently remove H2O2.23

Selective toxicity to cancer cells P-AscH
is selectively cytotoxic to various cancer cell lines.23–27 The mechanism of cytotoxicity is mediated by H2O2 formation and dependence on redox active metals.28–30 Ascorbate has been shown to be cytotoxic, in a dose-dependent manner, to multiple pancreatic cancer cell lines, as demonstrated by clonogenic survival assays. These results are seen with ascorbate doses that can be achieved safely in humans via IV administration. As mentioned, the mechanism by which PAscH
selectively kills cancer cells appears to be dependent on H2O2. Du et al. have shown that when catalase is overexpressed, by either adding it to the extracellular environment, or intracellularly through viral vector overexpression, ascorbate-induced cytotoxicity to pancreatic cancer cells can be completely reversed, strongly suggesting the role of PAscH
generated H2O2.23 As it oxidizes to form H2O2, P-AscH
acts as a prodrug to deliver increasing fluxes of H2O2 to cancer cells. In normal cells, intracellular antioxidant enzymes like catalase, glutathione peroxidases, and peroxiredoxins efficiently remove H2O2. Most cancer cell lines have been found to have low levels of catalase, rendering them inefficient at removing H2O2. In fact, in a study by Doskey et al. which tested 10 different normal tissue cell types and 15 different cancer cell lines, the normal cells on average removed H2O2 with a rate constant that was twofold higher than the cancer cell lines.31 The catalase activity varied among the different tumor cell lines. Notably, there was a differential sensitivity to ascorbate across pancreatic cancer cell lines which correlated with each cell line’s ability to remove H2O2. Furthermore, in the cell line that was most resistant to P-AscH
(PANC-1 cells), the addition of a catalase inhibitor significantly increased sensitivity to ascorbate.31 In addition to increased influx of H2O2, ascorbate-induced cytotoxicity is thought to be dependent on catalytic metals. Redox active transition metals like iron, copper, and manganese can increase the oxidation rate of ascorbate, which in turn increases the flux of H2O2.32 Redox active iron, or labile iron, activates H2O2 in the cell, catalyzing a Fenton reaction which produces hydroxyl radicals.30 This can induce oxidative DNA damage and cell death.33 To test this, Schoenfeld and colleagues inhibited intracellular iron redox activity with the iron chelator desferrioxamine (DFO), which diminished ascorbate toxicity in sarcoma cells.26 They also showed that increasing the steady-state levels of superoxide, by deleting superoxide dismutase (SOD) gene, increased labile iron pools in cancer cells. This subsequently sensitized the cells to ascorbate-induced cytotoxicity.26 Manganoporphyrins (MnPs), redox-active metal chelates, have also proven to be catalysts for AscH
oxidation. Together, MnPs and ascorbate synergize to enhance cytotoxicity.34 This finding was expanded upon by Cieslak et al., who showed that a similar synergistic effect was achieved when gemcitabine was added as a third therapeutic agent to MnPs and P-AscH
.35 In summary, our model for the mechanism of how P-AscH
contributes to cancer therapy has three nodes (Fig. 2). Node 1 incorporates the fundamental aspects of the production of a high flux of H2O2; Node 2 is how cells handle this high flux of

Pharmacological ascorbate and use in pancreatic cancer Chapter

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FIG. 2 Overview of our model for the mechanism of P-AscH
.31 (Node 1) P-AscH
readily oxidizes to produce a high flux of H2O2 in both the extracellular and intracellular space. The ordinate reflects in part this flux of H2O2 and the resulting biological response. This H2O2 is the essential mediator of the cytotoxicity of AscH
toward cancer cells. The greater the flux of H2O2, the greater the cellular effects; this flux is a function of [AscH
], pH, and catalytic metals. (Node 2) Represents how cells handle H2O2. An important concept is that the effect on cells is a function of cell density, i.e., the critical parameter is flux per cell, i.e., moles of H2O2 cell
1 s
131, 36; the response will vary with this flux and the ability of the intracellular peroxide-removal enzyme system to remove this H2O2. Cells with high capacity for the removal of H2O2 are less affected; on the other hand, inhibiting the removal of H2O2 will result in greater effects on the cell. (Node 3) These modulators represent the effects of small molecules as well as pathways and networks on the response to the intracellular H2O2 resulting from 1 to 2, e.g., the influence of intracellular labile iron (Fe3+/Fe2+), ATP levels, GAPDH, GSH, SVCT2 activity, NOX activation, HIF, autophagy, apoptotic signaling, DNA damage and repair, etc.

FIG. 3 Liver metastases eradicated with P-AscH
. Left: PDAC liver metastases prior to treatment (red arrow). Right: Following 2 months of treatment with P-AscH
and gemcitabine, the liver metastases disappeared on CT scan.

H2O2; and Node 3 represents the many downstream factors that alter its effectiveness. Naturally, there is an overlap in these nodes.31

P-AscH2 as chemosensitizer In addition to being selectively cytotoxic, P-AscH
has been shown to sensitize pancreatic cancer cells to the effects of standard of care chemotherapeutic agents. Espey et al. demonstrated that P-AscH
synergizes with gemcitabine to enhance therapeutic efficacy in multiple pancreatic cancer cell lines. These findings were consistent with the growth inhibition response of pancreatic tumor xenograft models in mice treated with P-AscH
and gemcitabine.37 The following clinical trials have expanded on these preclinical findings. In one of the first phase I studies done in patients with metastatic, unresectable, or recurrent pancreatic cancer, patients at The University of Iowa underwent twice weekly infusions of ascorbate, achieving plasma levels of 20 mM, combined with the gemcitabine regimen established by Burris and colleagues. There were no adverse events or change in toxicity of the chemotherapy regimen. Although not powered for efficacy, the mean progression-free survival was 26 weeks and overall survival was 12 months which compared favorably to the historical data demonstrating overall survival of only 6 months in this patient population.21, 38 With the completion of our phase I trial (NCT 01049880), initial data from this small sample size suggest some therapeutic efficacy. Most importantly, for our current proposal, metastatic disease did not occur in patients undergoing treatment with P-AscH
. In two patients who did have hepatic metastases, treatment resulted in the disappearance of the lesions (Fig. 3). Average treatment duration was 6 months (177 days, range 69–556 days) whereupon no patients were removed from the trial due to new metastatic disease. Thus, we hypothesize that P-AscH
can reduce metastatic disease in PDAC.

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In another phase I trial by Monti et al. (NCT 00954525), all nine patients had metastatic disease when enrolled. Although treatment duration was shorter in this study (8 weeks), primary tumor size decreased in 8 out of the 9 patients by the end of treatment period.39 Similar dose and treatment durations compared to our phase I trial were used in the phase I/IIa trial (NCT 01364805) by Polireddy et al. This group recorded an overall survival of 15.1 months in a study of 12 patients who had a combination of metastatic and locally advanced PDAC.40 In all three aforementioned clinical trials, all significant adverse events were attributed to either disease progression or expected consequences of chemotherapy, again demonstrating the low-toxicity profile of ascorbate as a cancer therapeutic.

P-AscH2 as radiosensitizer Radiation therapy plays an important role in the treatment of locally advanced pancreatic cancer. Previous studies have demonstrated that radiation therapy in combination with gemcitabine increased overall survival from 9.2 to 11.1 months compared to patients who underwent gemcitabine alone.41 Radiation induces DNA damage directly and via the generation of reactive oxygen species, which damage DNA in addition to proteins and lipids. The formation of H2O2 from P-AscH
also causes DNA damage by reacting with Fe2+ to create hydroxyl radical. It was therefore hypothesized that combining ascorbate with radiation would increase the effectiveness of radiation therapy. Indeed, Du et al. demonstrated that ascorbate acts as a radiosensitizer to cancer cells, including increased DNA damage. This was measured by increased levels of gamma-H2AX, a sensor of DNA damage and promoter of repair, when ascorbate was added to radiation treatment on pancreatic cancer cell lines. In the same study, radiosensitization was also dependent on H2O2, as the addition of catalase in these experiments decreased cytotoxicity in clonogenic cell survival assays. In an in vivo model tumor growth decreased and overall survival increased in mice. These preclinical data led to the first-in-human phase I trial (NCT 01852890), where patients with locally advanced pancreatic cancer were treated with P-AscH
in conjunction with standard of care chemoradiation therapy. Patients received P-AscH
intravenously daily during radiation therapy in addition to standard of care gemcitabine. In another group of patients used as comparators, the same standard of care chemoradiation therapy was given. As of August 2019, Fig. 4 presents data demonstrating that patients receiving P-AscH
had an increased median overall survival (12.7 vs 22.8 months) and progression-free survival (4.6 vs 13.7 months, Fig. 5) when compared to the comparator group of patients at The University of Iowa and compared to patients from the E4201 trial.42

P-AscH2 as a protector of normal tissue during chemoradiation As discussed previously, ascorbate seems to act as a prooxidant locally for tumor cells while maintaining its role as an antioxidant in normal tissue. The reducing capacity of P-AscH
is therefore hypothesized to decrease oxidative distress caused by chemoradiation on noncancer tissues. The dosing of radiation targeted to intra-abdominal tumors like pancreatic cancer is limited by the damage induced in surrounding organs. The small bowel is particularly sensitive to radiation injury during the treatment of pancreatic cancer. In mouse models, radiation injury has been measured by the degree of villous blunting, crypt cell loss, and collagen deposition of the jejunum. These changes are partially ameliorated when ascorbate is

FIG. 4 Overall survival from phase I trial (NCT 01049880). Kaplan-Meier curve estimating median overall survival in subjects treated with P-AscH
plus gemcitabine and radiation therapy as of August 2019 (n ¼ 14) as 22.8 vs 12.7 months in institutional controls treated with gemcitabine and radiation therapy (n ¼ 19). (Log-Rank test p ¼ 0.04). Institutional controls from The University of Iowa are parallel to historical controls as published by Loehrer et al.41

Pharmacological ascorbate and use in pancreatic cancer Chapter

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FIG. 5 Progression free survival from phase I trial (NCT 01049880). Kaplan-Meier curve demonstrating median progression-free survival as of August 2019 in subjects treated with P-AscH
plus gemcitabine and radiation therapy (n ¼ 14) as 4.6 in institutional controls treated with gemcitabine and radiation therapy vs 13.7 months in patients receiving the same chemoradiation therapy and also P-AscH
(n ¼ 19, p ¼ 0.01). Institutional controls from University of Iowa are equivalent to historical controls as published by Loehrer et al.41

administered.42, 43 To characterize ascorbate’s antioxidant mechanism of radioprotection, glutathione assays on irradiated mice jejunum revealed lower levels of glutathione disulfide, the primary oxidation product of glutathione, on samples taken from mice that received ascorbate in addition to radiation compared to those treated with radiation alone.42 Radiation injury to other organs has also been blunted by ascorbate in mouse models. A delayed onset of alopecia and achromotrichia was noted in mice that underwent radiation with ascorbate vs radiation alone.19 Plasma F2-isoprostanes are also a known marker of oxidative injury.44 In a phase I trial (NCT 01852890) of pancreatic cancer patients treated with ascorbate and gemcitabine/radiation therapy vs chemoradiation alone, there were decreased levels of plasma F2-isoprostanes in the patients who received P-AscH
.42 These findings suggest that P-AscH
may function as a systemic antioxidant when administered in conjunction with chemotherapy and radiation therapy.

Conclusions In this chapter, we have reviewed the significance of understanding the difference in pharmacokinetics between orally administered and intravenously administered ascorbate. Pharmacological levels of ascorbate, which can only be achieved parenterally, create an environment of oxidative stress in pancreatic cancer cells while appear to maintain its antioxidant properties systemically with normal tissue. This dual effect of prooxidant and antioxidant functions make P-AscH
a promising adjuvant to cancer therapy, where cancer cells must be targeted while limiting damage to normal cells. Furthermore, P-AscH
has been shown to be safe and tolerable in phase I clinical trials with suggestions of efficacy. Phase II trials are currently underway to further advance P-AscH
as a potential standard of care adjuvant treatment.

Applications in other cancers In this chapter, we have reviewed the role of P-AscH
as a promising therapy for the treatment of pancreatic cancer. Its role as a chemoradiation sensitizer is promising as a generalizable strategy for enhancing other cancer treatment regimens that include chemoradiation. Indeed, preclinical and clinical trials have shown similar results in other cancer cell lines. O’Leary et al. have shown that P-AscH
-induced clonogenic cell killing is also mediated by H2O2 in gastric cancer cell lines. In the same study, clonogenic survival was also dependent on availability of redox active iron, as previously demonstrated in pancreatic cancer cell lines.23, 25 Similar to the pancreatic cancer murine models previously discussed, cancer cell xenografts from sarcoma and gastric cancer cell lines have shown increased overall survival when P-AscH
is added to standard of care chemotherapy or chemoradiation therapy.25, 26 Additionally, increased effectiveness (as measured by decreased tumor weight and volume of ascites) of chemotherapy when combined with P-AscH
has also been shown in ovarian cancer xenograft models.24 In terms of clinical trials, P-AscH
has also been tested in patients with ovarian cancer, glioblastoma, and nonsmall cell lung carcinoma (NSCLC). In a phase I clinical trial (NCT01752491), Schoenfeld et al. showed an increased progressionfree survival and overall survival in patients with glioblastoma multiforme who underwent P-AscH
infusions in

520 SECTION

B Antioxidants and cancer

conjunction with standard of care chemoradiation.24, 27 In a phase I/IIa clinical trial of patients with stage three or four ovarian cancer (NCT 00228319), patients treated with P-AscH
plus standard carboplatin/paclitaxel experienced an increased 8.75 months progression-free survival, with a noted trend toward increasing overall survival compared to patients who received chemotherapy only.24 Finally, in a phase II trial (NCT 02420314), patients with advanced stage NSCLC showed improved disease control states and objective response rates when P-AscH
was added to platinum doublet chemotherapy.27 Importantly, these clinical studies have shown P-AscH
to be safe and well tolerated. In fact, P-AscH
has been shown to decrease many of the side effects that make chemoradiation intolerable. In a study by Vollbracht et al., a significant decrease in side effects and improved performance status were noted in postsurgical breast cancer patients receiving weekly IV ascorbate infusions during adjuvant chemoradiation compared to patients undergoing chemoradiation alone.45 Similarly, the phase I/IIa clinical trial by Ma et al. showed decreased toxicities in the ovarian cancer patients who were treated with adjuvant P-AscH
.24

Summary points l

l l

l

l

Intravenously administered vitamin C allows plasma concentrations to reach 20 mM, levels that are unattainable via oral intake. P-AscH
is selectively cytotoxic to cancers cells, including multiple pancreatic cancer cell lines. P-AscH
acts as a prooxidant, creating high influx of H2O2 into cancer cells, while maintaining its antioxidant properties among normal tissues. P-AscH
synergizes with both chemotherapeutic agents and radiation to enhance cytotoxic effects toward pancreatic cells in vitro and in vivo. Multiple clinical trials have shown P-AscH
to be safe and tolerable as an adjuvant treatment for pancreatic cancer, with suggestions of efficacy.

Acknowledgment Supported by NIH grants CA184051, P01 CA217797, and CA148062.

References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69(1):7–34. 2. Balaban EP, Mangu PB, Khorana AA, Shah MA, Mukherjee S, Crane CH, et al. Locally advanced, unresectable pancreatic cancer: American society of clinical oncology clinical practice guideline. J Clin Oncol 2016;34(22):2654–68. 3. Svirbely JL, Szent-Gyorgyi A. The chemical nature of vitamin C. Biochem J 1933;27(1):279–85. 4. McCormick WJ. Cancer: the preconditioning factor in pathogenesis; a new etiologic approach. Arch Pediatr 1954;71(10):313–22. 5. McCormick WJ. Cancer: a collagen disease, secondary to a nutritional deficiency. Arch Pediatr 1959;76(4):166–71. 6. Cameron E, Rotman D. Ascorbic acid, cell proliferation, and cancer. Lancet 1972;1(7749):542. 7. Cameron E, Campbell A. Innovation vs. quality control: an ’unpublishable’ clinical trial of supplemental ascorbate in incurable cancer. Med Hypotheses 1991;36(3):185–9. 8. Cameron E, Pauling L. Supplemental ascorbate in the supportive treatment of cancer: prolongation of survival times in terminal human cancer. Proc Natl Acad Sci U S A 1976;73(10):3685–9. 9. Cameron E, Pauling L. Supplemental ascorbate in the supportive treatment of cancer: reevaluation of prolongation of survival times in terminal human cancer. Proc Natl Acad Sci U S A 1978;75(9):4538–42. 10. Creagan ET, Moertel CG, O’Fallon JR, Schutt AJ, O’Connell MJ, Rubin J, et al. Failure of high-dose vitamin C (ascorbic acid) therapy to benefit patients with advanced cancer. A controlled trial. N Engl J Med 1979;301(13):687–90. 11. Moertel CG, Fleming TR, Creagan ET, Rubin J, O’Connell MJ, Ames MM. High-dose vitamin C versus placebo in the treatment of patients with advanced cancer who have had no prior chemotherapy. A randomized double-blind comparison. N Engl J Med 1985;312(3):137–41. 12. Block G. Vitamin C and cancer prevention: the epidemiologic evidence. Am J Clin Nutr 1991;53(1 Suppl):270S–82S. 13. Gaziano JM, Glynn RJ, Christen WG, Kurth T, Belanger C, MacFadyen J, et al. Vitamins E and C in the prevention of prostate and total cancer in men: the physicians’ health study II randomized controlled trial. JAMA 2009;301(1):52–62. 14. Lin J, Cook NR, Albert C, Zaharris E, Gaziano JM, Van Denburgh M, et al. Vitamins C and E and beta carotene supplementation and cancer risk: a randomized controlled trial. J Natl Cancer Inst 2009;101(1):14–23. 15. Levine M, Conry-Cantilena C, Wang Y, Welch RW, Washko PW, Dhariwal KR, et al. Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance. Proc Natl Acad Sci U S A 1996;93(8):3704–9. 16. Padayatty SJ, Sun H, Wang Y, Riordan HD, Hewitt SM, Katz A, et al. Vitamin C pharmacokinetics: implications for oral and intravenous use. Ann Intern Med 2004;140(7):533–7.

Pharmacological ascorbate and use in pancreatic cancer Chapter

46

521

17. Du J, Cullen JJ, Buettner GR. Ascorbic acid: chemistry, biology and the treatment of cancer. Biochim Biophys Acta 2012;1826(2):443–57. 18. Buettner GR. The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate. Arch Biochem Biophys 1993;300(2):535–43. 19. Schoenfeld JD, Alexander MS, Waldron TJ, Sibenaller ZA, Spitz DR, Buettner GR, et al. Pharmacological ascorbate as a means of sensitizing cancer cells to radio-chemotherapy while protecting normal tissue. Semin Radiat Oncol 2019;29(1):25–32. 20. Savini I, Rossi A, Pierro C, Avigliano L, Catani MV. SVCT1 and SVCT2: key proteins for vitamin C uptake. Amino Acids 2008;34(3):347–55. 21. Welsh JL, Wagner BA, van’t Erve TJ, Zehr PS, Berg DJ, Halfdanarson TR, et al. Pharmacological ascorbate with gemcitabine for the control of metastatic and node-positive pancreatic cancer (PACMAN): results from a phase I clinical trial. Cancer Chemother Pharmacol 2013;71(3):765–75. 22. Calcutt G. The formation of hydrogen peroxide during the autoxidation of ascorbic acid. Experientia 1951;7(1):26. 23. Du J, Martin SM, Levine M, Wagner BA, Buettner GR, Wang SH, et al. Mechanisms of ascorbate-induced cytotoxicity in pancreatic cancer. Clin Cancer Res 2010;16(2):509–20. 24. Ma Y, Chapman J, Levine M, Polireddy K, Drisko J, Chen Q. High-dose parenteral ascorbate enhanced chemosensitivity of ovarian cancer and reduced toxicity of chemotherapy. Sci Transl Med 2014;6(222):222ra18. 25. O’Leary BR, Houwen FK, Johnson CL, Allen BG, Mezhir JJ, Berg DJ, et al. Pharmacological ascorbate as an adjuvant for enhancing radiationchemotherapy responses in gastric adenocarcinoma. Radiat Res 2018;189(5):456–65. 26. Schoenfeld JD, Sibenaller ZA, Mapuskar KA, Bradley MD, Wagner BA, Buettner GR, et al. Redox active metals and H2O2 mediate the increased efficacy of pharmacological ascorbate in combination with gemcitabine or radiation in pre-clinical sarcoma models. Redox Biol 2018;14:417–22. 27. Schoenfeld JD, Sibenaller ZA, Mapuskar KA, Wagner BA, Cramer-Morales KL, Furqan M, et al. O2(
) and H2O2-mediated disruption of Fe metabolism causes the differential susceptibility of NSCLC and GBM cancer cells to pharmacological ascorbate. Cancer Cell 2017;32(2):268. 28. Chen Q, Espey MG, Krishna MC, Mitchell JB, Corpe CP, Buettner GR, et al. Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues. Proc Natl Acad Sci U S A 2005;102(38):13604–9. 29. Chen Q, Espey MG, Sun AY, Lee JH, Krishna MC, Shacter E, et al. Ascorbate in pharmacologic concentrations selectively generates ascorbate radical and hydrogen peroxide in extracellular fluid in vivo. Proc Natl Acad Sci U S A 2007;104(21):8749–54. 30. Du J, Wagner BA, Buettner GR, Cullen JJ. Role of labile iron in the toxicity of pharmacological ascorbate. Free Radic Biol Med 2015;84:289–95. 31. Doskey CM, Buranasudja V, Wagner BA, Wilkes JG, Du J, Cullen JJ, et al. Tumor cells have decreased ability to metabolize H2O2: implications for pharmacological ascorbate in cancer therapy. Redox Biol 2016;10:274–84. 32. Buettner GR. In the absence of catalytic metals ascorbate does not autoxidize at pH 7: ascorbate as a test for catalytic metals. J Biochem Biophys Methods 1988;16(1):27–40. 33. Buranasudja V, Doskey CM, Gibson AR, Wagner BA, Du J, Gordon DJ, et al. Pharmacological ascorbate primes pancreatic cancer cells for death by rewiring cellular energetics and inducing DNA damage. Mol Cancer Res 2019;17:2102–14. 34. Rawal M, Schroeder SR, Wagner BA, Cushing CM, Welsh JL, Button AM, et al. Manganoporphyrins increase ascorbate-induced cytotoxicity by enhancing H2O2 generation. Cancer Res 2013;73(16):5232–41. 35. Cieslak JA, Strother RK, Rawal M, Du J, Doskey CM, Schroeder SR, et al. Manganoporphyrins and ascorbate enhance gemcitabine cytotoxicity in pancreatic cancer. Free Radic Biol Med 2015;83:227–37. 36. Doskey CM, van ‘t Erve TJ, Wagner BA, Buettner GR. Moles of a substance per cell is a highly informative dosing metric in cell culture. PLoS One 2015;10(7):e0132572. 37. Espey MG, Chen P, Chalmers B, Drisko J, Sun AY, Levine M, et al. Pharmacologic ascorbate synergizes with gemcitabine in preclinical models of pancreatic cancer. Free Radic Biol Med 2011;50(11):1610–9. 38. Burris III HA, Moore MJ, Andersen J, Green MR, Rothenberg ML, Modiano MR, et al. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol 1997;15(6):2403–13. 39. Monti DA, Mitchell E, Bazzan AJ, Littman S, Zabrecky G, Yeo CJ, et al. Phase I evaluation of intravenous ascorbic acid in combination with gemcitabine and erlotinib in patients with metastatic pancreatic cancer. PLoS One 2012;7(1):e29794. 40. Polireddy K, Dong R, Reed G, Yu J, Chen P, Williamson S, et al. High dose parenteral ascorbate inhibited pancreatic cancer growth and metastasis: mechanisms and a phase I/IIa study. Sci Rep 2017;7(1):17188. 41. Loehrer Sr PJ, Feng Y, Cardenes H, Wagner L, Brell JM, Cella D, et al. Gemcitabine alone versus gemcitabine plus radiotherapy in patients with locally advanced pancreatic cancer: an Eastern Cooperative Oncology Group trial. J Clin Oncol 2011;29(31):4105–12. 42. Alexander MS, Wilkes JG, Schroeder SR, Buettner GR, Wagner BA, Du J, et al. Pharmacologic ascorbate reduces radiation-induced normal tissue toxicity and enhances tumor radiosensitization in pancreatic cancer. Cancer Res 2018;78(24):6838–51. 43. Du J, Cieslak III JA, Welsh JL, Sibenaller ZA, Allen BG, Wagner BA, et al. Pharmacological ascorbate radiosensitizes pancreatic cancer. Cancer Res 2015;75(16):3314–26. 44. Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts II LJ. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc Natl Acad Sci U S A 1990;87(23):9383–7. 45. Vollbracht C, Schneider B, Leendert V, Weiss G, Auerbach L, Beuth J. Intravenous vitamin C administration improves quality of life in breast cancer patients during chemo
/radiotherapy and aftercare: results of a retrospective, multicentre, epidemiological cohort study in Germany. In Vivo 2011;25 (6):983–90.

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Chapter 47

Antioxidant vitamins and genetic polymorphisms in breast cancer Daehee Kanga, Sang-Ah Leeb, and Woo-Kyoung Shina a

Department of Preventive Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea, b Department of Preventive Medicine,

Kangwon National University School of Medicine, Chuncheon-si, Gangwon-do, Republic of Korea

List of abbreviation ADP CAT CBS CpG CPS-II DCH dTMP E3N eNOS EPIC ER/PR FTHFD GPX IWHS MDC MnSOD MPO MTHF MTHFD1 MTR MTRR NAD NADPH NHS PARPs PLCO ROS RR SHMT1 SWHS TYMS V WHS

adenosine diphosphate catalase cystathionine-beta-synthase C-phosphate-G Cancer Prevention Study II Nutrition cohort diet, cancer and health cohort deoxythymidylate Etude Epide’miologique aupre’ s de femmes de la Mutuelle Ge0 ne’rale de l’Education Nationale endothelial nitric oxide synthase European Prospective Investigation into Cancer and nutrition estrogen receptor/progesterone receptor formyltetrahydrofolate dehydrogenase glutathione peroxidase Iowa Women’s Health Study Malm€ o Diet and Cancer manganese superoxide dismutase myeloperoxidase methylenetetrahydrofolate methylenetetrahydrofolate dehydrogenase1 methionine synthase methionine synthase reductase nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate Nurses’ Health Study poly-ADP-ribose polymerase prostate, lung, colorectal, and ovarian cancer screening trial reactive oxygen species risk ratio serine hydroxymethyltransferase1 Shanghai Women’s Health Study thymidylate synthase valine Women’s Health Initiative Observational Study

Introduction As current knowledge, the effect of diet on carcinogenesis is still limited because of the difficult evaluation of actual nutrient intake, such as recall bias, measurement error, the lack of evaluation tool for the dietary antioxidant intake, the

Cancer. https://doi.org/10.1016/B978-0-12-819547-5.00047-X © 2021 Elsevier Inc. All rights reserved.

523

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short half-time of blood antioxidant vitamins, etc. In recent, many epidemiological studies have tried to identify the role of dietary factors in cancer development using well-designed studies and to overcome the mentioned limitation in nutritional epidemiological research. Nevertheless, considering genetic factors in the impact of dietary factors on cancer development is very valuable to understand the role of the dietary factor for cancer development. Therefore, an approach using the genediet interaction in breast cancer is valuable to understand individual variability in the potential relationship between dietary constituents and breast cancer risk; in particular, for investigating relevant mechanisms, identifying susceptible populations/individuals, and applying their results in preventive strategies of breast cancer. The role of diet for the risk of breast cancer is of great interest as a potentially modifiable risk factor. Dietary factors are of particular interest in the context of breast cancer including fruits and vegetables, whole grain, red meat and processed meat, salted or fermented foods, soybean (isoflavone), fat intake (saturated fat), antioxidant vitamins, carbohydrate (glycemic index/load), green tea, heterocyclic amines, etc. An association of diet/nutrients in breast cancer etiology may be ascribed to the antioxidant properties of selected nutrients, influence on DNA repair, DNA mutations, DNA adducts, metabolic detoxification, stimulation of growth factors, and potential antiestrogenic influence of some nutrients. Recently, DNA methylation has been proposed as a biological mechanism by which alterations of diet can modify predisposition to breast cancer. Future studies on gene-diet interactions are also warranted and perhaps particularly important in the field of diet and breast cancer because most of the existing evidence has not revealed strong associations with risk. The beneficial or harmful effects of dietary exposures may be restricted to a subgroup of women defined by specific genetic characteristics with direct relevance to the biological pathway underlying the associations. Studies of gene-diet interactions should be grounded solidly in biological plausibility (biological relationship of gene and dietary exposure of interest and demonstration of functional significance) and must have enough statistical power to detect suspected modifying effects of a candidate genetic polymorphism on the association between dietary exposure and breast cancer risk.

Effect of antioxidant vitamin on breast cancer incidence Although many prospective epidemiologic studies conducted on diet and breast cancer to date, the association was not consistent. Deficiency of folate and methyl-related B vitamins could induce defective DNA repair and chromosomal fragile site expression and thus cause chromosomal breaks and micronucleus formation. These functions could play an important role in the development of breast cancers. Most prospective studies do not provide evidence of an association between folate intake and breast cancer risk, however, there was inverse association between high consumption of folate intake and methyl-related B vitamins and risk of breast cancer in three different populations (Table 1). Also, the results from TABLE 1 Dietary antioxidants intake and breast cancer risk in prospective studies. No. of subject

Antioxidant vitamins

Cohort

Country

Cases

Total

Results

Folate

SWHS

Chinese

718

73,237

No association, even stratified by hormone-receptor status or menopausal status

DCH

Denmarka

1072

27,296

No association

CPS II

a

3898

70,656

No association

USA

MDC

Sweden

392

11,699

Decreased risk (Q1–Q5, HR ¼ 0.56, 95% CI ¼ 0.35–0.95)

E3N

Francea

1812

62,739

Decreased risk (Q1–Q5, RR ¼ 0.78, 95% CI ¼ 0.67–0.90)

PLCO

USAa

691

15,400

No association

NHS

USA

3797

88,744

No association, even stratified by hormone-receptor status

IWHS

USA

1875

34,393

No association, even stratified by hormone-receptor status

Mexico

Mexico

475

2341

Decreased risk (Q1-Q5, HR ¼ 0.64, 95% CI ¼ 0.45–0.90, p for trend ¼ 0.009)

a

Antioxidant vitamins and genetic polymorphisms Chapter

47 525

TABLE 1 Dietary antioxidants intake and breast cancer risk in prospective studies—cont’d Antioxidant vitamins Beta-carotene

No. of subject Cohort DCH EPIC

Country a

Denmark b

EU

a

Cases

Total

Results

1072

27,296

No association

1480

118,437

No association

WHS

USA

2879

84,805

Decreased risk among women with ER+/PR+ (Q1vs.5, RR ¼ 0.78, 95% CI ¼ 0.66–0.94)

Netherlands

Netherland

650

62,573

No association

Sweden

Sweden

1721

59,036

No association

SWHS

Chinese

718

73,237

No association, even stratified by hormone-receptor status or menopausal status

IWHS

USAa

1586

35,973

No association

Vitamin B3

SWHS

Chinese

718

73,237

Increased risk among women with ER+/PR+ (Q1vs.4, HR ¼ 1.62, 95% CI ¼ 1.07–2.46)

Vitamin B6

SWHS

Chinese

718

73,237

No association, even stratified by hormone-receptor status or menopausal status

CPS II

USAa

3898

70,656

No association

IWHS

a

USA

1586

35,973

No association

SWHS

Chinese

718

73,237

No association, even stratified by hormone-receptor status or menopausal status

CPS II

USAa

3898

70,656

No association

IWHS

a

USA

1586

35,973

No association

Mexico

Mexico

475

2341

Decreased risk (Q1-Q5, HR ¼ 0.32, 95% CI ¼ 0.22–0.49, p for trend