Molecular Biochemical Aspects of Cancer [1st ed.] 9781071607398, 9781071607411

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Undurti N. Das

Molecular Biochemical Aspects of Cancer

Molecular Biochemical Aspects of Cancer

Undurti N. Das

Molecular Biochemical Aspects of Cancer

Undurti N. Das BioScience Research Centre and Department of Medicine GVP Medical College and Hospital Visakhapatnam, India UND Life Sciences, Battle ground WA, USA

ISBN 978-1-0716-0739-8    ISBN 978-1-0716-0741-1 (eBook) https://doi.org/10.1007/978-1-0716-0741-1 © Springer Science+Business Media, LLC, part of Springer Nature 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

To My wife Lakshmi Friend, philosopher, critic, supporter, and soul and for her understanding of my long hours at work.

Preface

Cancer is one of the leading causes of morbidity and mortality in both developing and developed countries. Despite many advances both in our understanding of its molecular biology and development of many newer therapeutics, it continues to be a major health issue. Tumor cells differ from normal cells in many aspects (including gene expressions, metabolism, behavior in terms of proliferation, and ability to metastasize, etc.,) but the role of lipids in cancer has largely been ignored. The cell membrane (including nuclear membrane, mitochondrial membrane, etc.,) lipid composition of tumor cells is different from normal, a fundamental property that has not been paid much attention. Lipids, especially polyunsaturated fatty acids (PUFAs) and their metabolites, both pro- and anti-inflammatory products, seem to play a critical role in the pathobiology of cancer. Tumor cells are deficient in the activity of desaturases that are essential for the formation of long-chain metabolites from dietary essential fatty acids: cis-linoleic acid (LA) and α-linolenic acid (ALA). As a result, tumor cells contain low quantities of γ-linolenic acid (GLA, 18: n-6), dihomo-GLA (DGLA, 20:3 n-6), arachidonic acid (AA, 20:4 n-6) formed from LA and eicosapentaenoic acid (EPA, 20:5 n-3), and docosahexaenoic acid (DHA, 22:6 n-3) from ALA that can be referred to as bioactive lipids (which also include their metabolites) . As a result, the tumor cell membrane is uniquely different from the normal especially in terms of expression of several receptors on its surface, the way messages are conveyed from the membrane to the DNA and vice versa, and its antigenicity including the expression of immune checkpoint proteins/receptors such as PD-1, PD-L1, and CTLA4. These altered properties of tumor cell may render it to escape the immune surveillance system and acquire unique properties in terms of its proliferation ability, metastasis, triggering angiogenesis to survive, and drug-­ resistance. Studies performed by us and several others showed that tumor cells have low rates of peroxidation and are exquisitely sensitive to the cytotoxic action of lipid peroxides compared to their normal counter parts. Furthermore, some of these bioactive lipids protect normal cells from the cytotoxic action of chemicals, chemotherapeutic drugs, cytokines, and radiation. This differential action of bioactive lipids on normal and tumor cells (protecting normal cells but produce apoptosis of tumor cells) could be exploited to develop them as potential drugs for cancer. These vii

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Preface

bioactive lipids seem to regulate the proliferation and action of Treg and Teff cells; cytokines production and action; expression of PD-1, PD-L1, and CTLA4; macrophage function; immune response; and finally as potential mediators of the tumoricidal action of T cells, macrophages, and immune checkpoint inhibitors. They possess antimicrobial properties. These pleiotropic actions of bioactive lipids suggest that they are likely to be useful in several diseases. Our recent observation that drug-resistant Hodgkin’s lymphoma could be successfully treated by bioactive lipids when supplemented with chemotherapeutic drugs with substantial attenuation of side-effects of conventional anti-cancer drugs implies that they have the potential to be exploited as unconventional yet specific and selective anti-cancer molecules. The therapeutic potential of bioactive lipids is just being realized and it is only the beginning, and I foresee a bright future for lipid-based drugs for several diseases. Visakhapatnam, India Battle Ground, WA, USA

Undurti N. Das

Contents

1 Introduction to Genes, Oncogenes, and Anti-oncogenes������������������������    1 Introduction������������������������������������������������������������������������������������������������     1 Retinoblastoma Gene ��������������������������������������������������������������������������������     3 Function and Properties of RB1����������������������������������������������������������������     3 Neurons������������������������������������������������������������������������������������������������������     4 Interaction of Retinoblastoma Protein with Several Other Receptors, Proteins, and Genes������������������������������������������������������������������������������������     5 p53�������������������������������������������������������������������������������������������������������������     6 Functions of p53����������������������������������������������������������������������������������������     6 Factors Regulating p53������������������������������������������������������������������������������     8 p53 in Some Diseases��������������������������������������������������������������������������������     9 Interaction(s) of p53 with Other Genes, Proteins, and Molecules������������    10 p53 and Inflammation��������������������������������������������������������������������������������    10 Phosphatase and Tensin Homolog (PTEN) in Cancer ������������������������������    12 Clinical Significance of PTEN������������������������������������������������������������������    12 PTEN, Brain Function, and Autism ����������������������������������������������������������    14 Immune Checkpoint Inhibitors in Cancer��������������������������������������������������    20 PD-L1 Inhibitors Include ��������������������������������������������������������������������������    21 Bioactive Lipids and Immune Response in Cancer ����������������������������������    22 Prostaglandin E2 (PGE2) and Immune Checkpoint Inhibition������������������    24 LXA4 Versus PGE2 in Cancer������������������������������������������������������������������    24 References��������������������������������������������������������������������������������������������������    29 2 Introduction to Free Radicals, Antioxidants, Lipid Peroxidation, and Their Effects on Cell Proliferation����������������������������������������������������   41 Introduction������������������������������������������������������������������������������������������������    41 Free Radicals����������������������������������������������������������������������������������������������    43 Formation of Free Radicals������������������������������������������������������������������������    44 Persistence and Stability of Free Radicals ������������������������������������������������    44 Diradicals ��������������������������������������������������������������������������������������������������    45 Free Radical Reaction��������������������������������������������������������������������������������    45 ix

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Combustion������������������������������������������������������������������������������������������������    45 Polymerization ������������������������������������������������������������������������������������������    46 Radicals Present in the Atmosphere����������������������������������������������������������    46 Biological Significance of Free Radicals��������������������������������������������������    46 Identification and Diagnostic Techniques to Detect Free Radicals������������    48 Nitric Oxide (NO)��������������������������������������������������������������������������������������    48 Methods of Preparation of NO������������������������������������������������������������������    49 Methods of Measurement of NO ��������������������������������������������������������������    50 Biological Functions/Significance of NO��������������������������������������������������    50 NO in Inflammation, Immune Response, and Cancer ������������������������������    53 NO Induces Apoptosis of Cancer Cells ����������������������������������������������������    54 Oxidative Stress in Cancer Initiation and Progression Including Apoptosis and Necrosis ������������������������������������������������������������    55 References��������������������������������������������������������������������������������������������������    57 3 Immune System, Inflammation, and Essential Fatty Acids and Their Metabolites in Cancer��������������������������������������������������������������   67 Introduction to the Immune System����������������������������������������������������������    67 Innate Immune System������������������������������������������������������������������������������    68 Inflammation����������������������������������������������������������������������������������������������    69 NK Cells����������������������������������������������������������������������������������������������������    70 Adaptive Immune System or Acquired Immune System��������������������������    71 Lymphocytes����������������������������������������������������������������������������������������������    71 Killer T Cells������������������������������������������������������������������������������������������    72 Helper T Cells����������������������������������������������������������������������������������������    73 γδ T Cells ����������������������������������������������������������������������������������������������    74 TH1/TH 2 Model for Helper T Cells ������������������������������������������������������    75 Suppressor T Cells ��������������������������������������������������������������������������������    75 Treg Cells and Autoimmune Diseases ����������������������������������������������������    78 Methods to Analyze and Monitor Treg Cells ������������������������������������������    79 B Lymphocytes��������������������������������������������������������������������������������������    80 Toxic Shock Syndrome��������������������������������������������������������������������������    81 Superantigens and the Immune System ����������������������������������������������������    81 Immunological Aspects of Cancer ������������������������������������������������������������    82 Immunosurveillance and Immunoediting����������������������������������������������    83 PD-1 (Programmed Cell Death Protein 1) ��������������������������������������������    88 TH17–Treg Cell Balance������������������������������������������������������������������������    89 IL-17, Synaptic Plasticity, and Memory������������������������������������������������    91 Cancer and Autoimmune Diseases Are Two Sides of the Same Coin������������������������������������������������������������������������������������    94 IL-17 and Fibrosis����������������������������������������������������������������������������������    97 Macrophages in Tissue Repair (Regeneration) and Fibrosis ����������������    98 PUFAs, Ion Channels, Cell Proliferation, or Apoptosis������������������������   101

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Prostanoids in Inflammation������������������������������������������������������������������   106 PGE2 and PD-1 and CTL (Cytotoxic T Lymphocyte) Function ����������   107 Tumor Cells and Lipid Peroxidation����������������������������������������������������������   109 Tumor Cells Are Relatively Rich in Vitamin E��������������������������������������   109 SOD and Tumor Cells����������������������������������������������������������������������������   113 Tumor Necrosis Factor (TNF) and PUFAs��������������������������������������������   115 Anti-cancer Drugs, ROS, and SOD ������������������������������������������������������   116 Free Radicals, Lipid Peroxides, p53, Caspases, and Apoptosis������������   117 Oxidant Stress, Bcl-2, and Apoptosis����������������������������������������������������   118 Connecting the Plasma Membrane to the Nucleus������������������������������������   120 PUFAs Enhance Lipid Peroxidation and Inhibit Cell Proliferation������   121 PUFA-Activated Macrophages Possess Tumoricidal Action ����������������   122 PUFAs Are Involved in Various Mitochondrial Processes��������������������   123 PUFAs Augment Cytotoxic Action of Anti-cancer Drugs and Reverse Drug Resistance����������������������������������������������������������������   124 Differential Metabolism of PUFAs by Normal and Tumor Cells����������   125 PUFAs and Telomerase Activity������������������������������������������������������������   129 PUFAs Can Modulate G-Protein-Mediated Signals����������������������������������   130 Differential Action Due to Differences in the Metabolism of PUFAs Makes All the Difference������������������������������������������������������   130 References��������������������������������������������������������������������������������������������������   131 4 PUFAs and Their Metabolites in Carcinogenesis������������������������������������  159 Introduction������������������������������������������������������������������������������������������������   159 Genomic Instability and Cancer����������������������������������������������������������������   161 Bioactive Lipids and Mutagenesis/Carcinogenesis������������������������������������   163 PGE2, LXA4, and Treg and Teff Cells and Inflammation ������������������������   163 Bioactive Lipids in Mutagenesis and Carcinogenesis��������������������������������   169 References��������������������������������������������������������������������������������������������������   175 5 Molecular Mechanism of Anti-cancer Action of PUFAs with Particular Reference to GLA in Glioma ����������������������������������������  181 Introduction������������������������������������������������������������������������������������������������   181 Metabolism of Essential Fatty Acids (EFAs)��������������������������������������������   182 Anti-cancer Action of PUFAs��������������������������������������������������������������������   184 ROS and Lipid Peroxides as Mediators of the Anti-cancer Action of PUFAs (Bioactive Lipids: BAL)������������������������������������������������������������   187 PUFAs and Immune System in Cancer������������������������������������������������������   188 PUFAs (Especially GLA) Have Anti-angiogenic Action��������������������������   193 Mechanism of Action of PUFAs/GLA on Tumor Cells����������������������������   194 GLA for Glioma Management������������������������������������������������������������������   197 Examples����������������������������������������������������������������������������������������������������   198 Methods of Administration������������������������������������������������������������������������   198 References��������������������������������������������������������������������������������������������������   200

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6 Bioactive Lipid (BAL)-Based Therapeutic Approach to Cancer That Enhances Antitumor Action and Ameliorates Cytokine Release Syndrome of Immune Checkpoint Inhibitors���������������������������  207 Introduction������������������������������������������������������������������������������������������������   208 Immune Checkpoint Inhibitors������������������������������������������������������������������   208 CTLA-4������������������������������������������������������������������������������������������������������   210 Indoleamine-Pyrrole 2,3-Dioxygenase (IDO or INDO)����������������������������   212 Immune Checkpoint Inhibitors in Cancer��������������������������������������������������   214 Bioactive Lipids Have Tumor Growth Inhibitory and Tumoricidal Action������������������������������������������������������������������������������   216 PUFAs Enhance the Tumoricidal Action of TILs and/or CAR T-Cell Therapy����������������������������������������������������������   217 PD-1, PD-L1, CTLA-4, IDO, and PUFAs ������������������������������������������������   221 Conclusions and Therapeutic Implications������������������������������������������������   223 References��������������������������������������������������������������������������������������������������   225 7 A New Therapeutic Strategy to Treat Cancer Based on Bioactive Lipids (BAL) ������������������������������������������������������������������������  237 Introduction������������������������������������������������������������������������������������������������   237 Bioactive Lipids for Cancer ����������������������������������������������������������������������   238 Intratumoral Delivery by Injection/Infusion����������������������������������������������   238 Lithium-GLA-Iodized Solution Complex (LGIOC) for Intra-­arterial Injection/Infusion������������������������������������������������������������   238 BAL Conjugated to Growth Factors (EGF/VEGF)/Antigrowth Factor(s) Antibodies����������������������������������������������������������������������������������   239 Conjugation of BAL with Angiostatin and Endostatin������������������������������   239 TNF-α–/IL-6–BAL Complex��������������������������������������������������������������������   239 PUFAs Anti-cancer Drug Complexes��������������������������������������������������������   240 PUFA-Based Nanoparticles in Combination with Growth Factors/Monoclonal Antibodies to Growth Factors/TNF-α/Angiostatin/ Endostatin/Anti-cancer Drugs��������������������������������������������������������������������   240 PUFAs Delivered Using Biodegradable Membranes��������������������������������   240 BAL–CAR T-Cell Immune Checkpoint Inhibitors������������������������������������   240 Conclusion and Future Perspective������������������������������������������������������������   241 References��������������������������������������������������������������������������������������������������   243 Index������������������������������������������������������������������������������������������������������������������  245

About the Author

Undurti  N.  Das  is an MD in Internal Medicine from Osmania Medical College, Hyderabad, India; a Fellow of the National Academy of Medical Sciences, India; and Shanti Swaroop Bhatnagar prize awardee. Apart from clinical work, he is researching the role of polyunsaturated fatty acids, cytokines, nitric oxide, free radicals, and anti-oxidants in cancer, inflammation, metabolic syndrome, schizophrenia, autism, and tropical diseases. His current interests include cancer, molecular biological aspects of diabetes mellitus, hypertension, cardiovascular diseases, and metabolic syndrome. Dr Das was formerly scientist at Efamol Research Institute, Kentville, Canada; Professor of Medicine at Nizam’s Institute of Medical Sciences, Hyderabad, India; and Research Professor of Surgery and Nutrition at SUNY (State University of New  York) Upstate Medical University, Syracuse, USA.  At present, he is the Chairman and Research Director of UND Life Sciences LLC, Battle Ground, WA, USA, and Chairman and Professor of Department of Medicine, GVP Hospital and Medical College, Visakhapatnam, India, and serves as a consultant to both Indian and US-based biotech and pharmaceutical companies. Dr Das is the Founding Editor of the international journal: Lipids in Health and Disease and serves on the editorial board of another 10 international journals. Dr Das has authored more than 500 international publications, written 4 books, and has been awarded 6 US patents. He is now developing lipid-based drugs for cancer, diabetes mellitus, and sepsis.

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Chapter 1

Introduction to Genes, Oncogenes, and Anti-oncogenes

Abstract  Cancer is a major cause of significant morbidity and mortality in many countries across the globe though the type of cancers is different in different countries. The exact cause of majority of the cancers is not clear. Environmental agents (that include many mutagens and carcinogens) are considered to cause more than 50% of cancers. DNA damage leading to activation of oncogenes seems to be the underlying cause of cancer. In addition, suppression of the tumor suppressor genes is also at the center of the onset of cancer. Under normal physiological conditions, the immune system of the body recognizes tumor cells as foreign and mounts an attack to eliminate them. Cancer-specific antigens being weak antigens’ stimulation to the immune system is not adequate to mount a successful attack and eliminate them. As a result of DNA damage, there will be alterations in the activity and/or expression of p53, PTEN, ghrelin, leptin, Ras/Raf/ERK1/2, and PI3K/Akt and PIP3 that cause mitochondrial dysfunction, which results in changes in cell survival and function, growth, proliferation, migration, and cell size that ultimately leads to the development of cancer. Keywords  Cancer. Oncogenes · Anti-oncogenes · p53 · PTEN

Introduction Studies have identified cellular genes that play a significant role in the initiation and progression of cancer. Thus, proto-oncogenes, which participate in the normal cellular process of proliferation and differentiation, may turn to become oncogenes due to significant alterations in their expression. This change in their expression is due to structural alterations in their coding sequences. In contrast to this, the cellular DNA also contains anti-oncogenes or tumor suppressor genes or recessive oncogenes that have a negative regulatory role in the cellular proliferation process. It is believed that a loss or alteration in the function of anti-oncogenes may lead to cancer. Alteration in the delicate balance that is maintained between oncogenes and anti-oncogenes, when tilted more toward oncogenes, can lead to the development of cancer. © Springer Science+Business Media, LLC, part of Springer Nature 2020 U. N. Das, Molecular Biochemical Aspects of Cancer, https://doi.org/10.1007/978-1-0716-0741-1_1

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1  Introduction to Genes, Oncogenes, and Anti-oncogenes

Several chemicals, drugs, viruses, radiation, and environmental agents have the ability to produce mutations in the expression and function of suppressor gene, anti-­ oncogene, or both that can result in the abnormal proliferation of cells leading to the onset of cancer. In oncogenesis, there could occur loss of function of anti-oncogenes or enhancement of the activity and function of oncogenes or both events may occur (simultaneous inactivation of anti-oncogenes and activation of oncogenes) leading to the development of cancer. Since, at times, inactivation of anti-oncogenes and activation of oncogenes need to occur for the development of cancer, it is reasonable to suggest that it is not easy for cancer to develop despite the fact that the incidence of cancer continues to rise. For cancer to occur, in general, both the alleles that code for a specific protein have to be affected and are called as the “two-hit hypothesis.” But it needs to be emphasized that there are exceptions to this rule. This concept of two-hit hypothesis was first described for retinoblastoma [1]. In an occasional instance, oncogene mutations can also involve only a single allele leading to gain-­ of-­function mutation that leads to the onset of cancer. Such mutations are seen in the p53 and PTCH in medulloblastoma, NF1 in neurofibroma, and p27Kip1 mutations that is a cell cycle inhibitor, all of which can enhance the risk of cancer [2, 3]. Tumor suppressor genes and their proteins generally dampen or suppress cell cycle regulatory events and/or promote apoptosis. Some of the functions of tumor suppressor proteins are [4]: 1 . Inhibit the action of genes that are needed for the cell cycle. 2. Since cell cycle and DNA damage are closely related, whenever DNA damage occurs, the cell cycle arrest happens. Only when the damaged DNA is repaired, the cell cycle can resume. 3. In the event DNA damage cannot be repaired, the cell is initiated to undergo apoptosis so that cells harboring DNA damage do not progress to the stage of evolving into cancer. This is a protective mechanism developed by the cells to prevent the development of cancer. 4. Cell adhesion molecules generally expressed on the cell surface are meant to prevent tumor cells from detaching and metastasizing. By blocking loss of contact inhibition, the abnormal cells (tumor cells) do not form metastasis. These proteins are known as metastasis suppressors. 5. In general, proteins involved in DNA repair are regarded as tumor suppressors since alterations or mutations in their genes enhance the risk of cancer (e.g., mutations in HNPCC, MEN1, and BRCA). It is noteworthy that increased mutation rate due to decreased DNA repair can result in an increase in the inactivation of other tumor suppressors and activation of oncogenes, events that can result in higher incidence of cancer. Retinoblastoma protein (pRb) is the first tumor suppressor protein identified. It may be noted that recent studies suggested that pRb may also function as a tumor-­ survival factor.

Function and Properties of RB1

3

Retinoblastoma Gene Retinoblastoma usually develops in early childhood and generally affects one eye though bilateral involvement is not uncommon. Retinoblastoma is curable if diagnosed early. Retinoblastoma is due to mutations in the RB1 gene, a tumor suppressor gene. Most mutations in the RB1 gene interfere with making any functional protein. Retinoblastomas due to deletions in the region of chromosome 13 that contains the RB1 gene may also result in other features such as intellectual disability, slow growth, and distinctive facial features (such as prominent eyebrows, a short nose with a broad nasal bridge, and ear abnormalities). Some 40% of all retinoblastomas are germinal with a family history of the disease. The other 60 percent are non-germinal. Germinal retinoblastoma is inherited in an autosomal dominant pattern. The non-germinal form of retinoblastoma generally occurs in one eye with no family history of the disease. These individuals develop, in early childhood, mutations in both copies of the RB1 gene.

Function and Properties of RB1 The retinoblastoma protein (abbreviated as Rb, while the gene name is abbreviated as RB or RB1) is a tumor suppressor protein which is dysfunctional in several cancers as discussed above. Its main function is to suppress inappropriate cell division by inhibiting cell cycle progression. In other words, Rb protein is able to inhibit cell division till the cell is in a position to divide. When it is appropriate for the cell to divide, Rb is phosphorylated to pRb that renders it inactive, and so cell cycle progresses. Rb has the ability to recruit a number of chromatin remodeling enzymes including methylases and acetylases [5–7]. Rb belongs to the pocket protein family since other proteins can bind to it and induce the development of cancer. For instance, oncogene proteins of human papillomaviruses can bind to inactivate Rb and, thus, lead to the development of cancer [8]. In humans, the RB1 gene is located in 13q14.1-q14.2 (cytogenetic location, 13q14.2; molecular location on chromosome 13, base pairs 48,303,746 to 48,481,889) as shown below (Fig. 1.1): Mutation(s) of both alleles of Rb gene in early life leads to inactivation of its protein, and this results in the development of retinoblastoma. Rb restricts DNA replication and, thus, prevents the progression of G1 to S phase of the cell cycle. It is known that Rb can bind to and inhibit E2F family of transcription factors. These E2F families of transcription factors consist of dimers of an E2F protein and a dimerization partner (DP) protein [9]. E2F-DP can render a cell to progress to S phase of the cell cycle [9–11]. Inactivation of E2F-DP inhibits the cell cycle in G1 phase. Binding of Rb to E2F leads to growth suppression and prevention

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1  Introduction to Genes, Oncogenes, and Anti-oncogenes

13q33.1 13p33.3

13q31.3 13p32.2

13q22.2 13p31.1

13q21.33

13q21.1

13q21.31

13q14.2 13q14.12

13q13.3

13p122 13q13.1

13q12.12

13p12

13p112

13p12

Fig. 1.1  The location of the RB1 gene is depicted in the figure

of progression of the cell cycle. Rb-E2F/DP complex can attract a histone deacetylase (HDAC) protein to the chromatin to reduce transcription of S-phase-promoting factors and, thus, suppresses DNA synthesis. Cyclin-dependent kinases (CDKs) phosphorylate Rb to pRb that, in turn, prevents pRb to complex with E2F so that progression of cell cycle from G1 phase to the S phase will not occur [12]. It is known that the phosphorylation occurs over a series of steps that include initial phosphorylation by cyclin D/CDK4/CDK6 and subsequently by cyclin E/CDK2. Throughout the cell cycle, phases S, G2, and M pRb remain phosphorylated. Phosphorylation of Rb allows E2F-DP to dissociate from pRb to become active, and the free E2F activates cyclins (e.g., cyclin E and A) such that cell cycle proceeds. This is so since activation of cyclin-dependent kinases and proliferating cell nuclear antigen (PCNA) enhances DNA replication and repair due to the attachment of polymerase to DNA. Rb family proteins are components of the DREAM complex (also named LINC complex), which is composed of LIN9, LIN54, LIN37, MYBL2, RBL1, RBL2, RBBP4, TFDP1, TFDP2, E2F4, and E2F5. It is now possible to detect RB1 gene mutations including large deletions that correlate with advanced stage retinoblastoma [13]. RB1 is not only involved in the pathobiology of retinoblastoma but also seems to have a role in mammalian hair cell growth and development of the cochlea and neurons [14–16].

Neurons Disruption of Rb expression induces dendrites to branch out farther. Schwann cells, which are needed for neuronal survival, travel along with the neurites extending farther than normal when Rb is disrupted. This suggests that that inhibition of Rb is essential for the continued growth of nerve cells [17].

Interaction of Retinoblastoma Protein with Several Other Receptors, Proteins, and Genes

5

I nteraction of Retinoblastoma Protein with Several Other Receptors, Proteins, and Genes Retinoblastoma protein is known to interact with a variety of proteins, receptors, and genes. Some of these include but not limited to Abl gene; apoptosis-­antagonizing transcription factor; aryl hydrocarbon receptor; BRCA1; BRF1; C-jun; C-Raf; CDK9; cyclin A1; cyclin D1; cyclin T2; transcription factors E2F1, E2F2, E4F1, etc.; several histone deacetylases; insulin; peroxisome proliferator-activated receptor gamma; etc. Despite the fact that retinoblastoma protein interacts with many genes, proteins, and receptors, the actual significance of this interaction is not clear. Rb regulates MMP-1 (matrix metalloproteinaise-1) and interleukin-6 and, thus, has a role in inflammation and inflammatory conditions such as rheumatoid arthritis (RA). Proliferating synovial fibroblasts during the acute phase of RA show enhanced expression of the MAP kinase p38 that is known to augment the production of pro-­ inflammatory cytokines: interleukin-6 (IL-6) and MMPs. Both IL-6 and MMPs promote inflammation, osteoporosis, and joint destruction. These results imply a close association among Rb-regulated MMP-1 expression IL-6 levels and inflammation. Increased expression of inactive hyperphosphorylated Rb (inactive Rb/total Rb) is seen during the quiescent phase of RA. Increased expression or levels of the active Rb isoform suppress fibroblast cell cycle progression and inhibit IL-6 and MMP-1 secretion. In contrast, Rb overexpression does not have any effect on spontaneous or IL-1β-induced production of IL-6 or MMP-1 in normal or non-RA synovial fibroblasts. These and other results imply that Rb a role in the pathogenesis and joint destruction seen in RA by negatively regulating p38 activation and suppressing MMP-1 secretion by RA synovial fibroblasts [18, 19]. In a similar fashion, cell cycle proteins seem to have a role in renal hypertrophy seen in diabetes mellitus. Whole kidney and glomerular hypertrophy seen in type 1 diabetes mellitus due to hyperglycemia has been shown to be associated with early and sustained increase in the expression of cyclin D1 and activation of cyclin D1-cdk4 complexes with no change in the expression of cyclin E or cdk2 activity. It is noteworthy that overexpression of RB alone can cause hypertrophy that has been shown to be associated with an increase in cyclin D1-cdk4 activity only. It is interesting that no further increase in hypertrophy was noted even if hyperglycemia was further enhanced. Mitogenic signals can transiently and sequentially increase both cyclin D1-cdk4 and cyclin E-cdk2 expression especially in  vitro cell cultures. Hyperglycemia produces partial phosphorylation of Rb both in vivo and in cultured mesangial cells, an event that is catalyzed by cyclin D1-cdk4. This suggests that hyperglycemia-induced mesangial hypertrophy (especially in diabetes) is due to sustained cyclin D1-cdk4-dependent phosphorylation of RB that leads to maintenance of mesangial cells in the early-to-middle G1 phase of the cell cycle [20]. Based on these results, it is reasonable to suggest that RB plays a significant role in renal abnormalities seen in diabetes mellitus such as diabetic nephropathy. These evidences suggest that it is prudent to evaluate the role of RB in several inflammatory and noninflammatory conditions.

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1  Introduction to Genes, Oncogenes, and Anti-oncogenes

p53 p53 is a tumor suppressor protein encoded by the TP53 gene. Several mutations of p53 have been described in majority of the cancers, especially in colon cancer, breast cancer, and lung cancer. p53 mutations are also seen in leukemias, lymphomas, sarcomas, and neurogenic tumors. It needs to be noted that p53 gene abnormalities can be inherited that may explain occasional occurrence of cancer seen in several generations of the same family. p53 is essential to regulate cell cycle and, thus, is a tumor suppressor. In view of its regulatory role in cell cycle, it is considered as the “the guardian of the genome.” p53 is needed to maintain stability and prevent genome mutations [21–24]. TP53 gene is located on the short arm of chromosome 17 (17p13.1) with the gene spanning 20 kb, with a noncoding exon 1 and a very long first intron of 10 kb with high degree of conservation in several species, especially in exons 2, 5, 6, 7, and 8. In invertebrates, there is only a distant resemblance to mammalian TP53 [24]. In most mammals, TP53 orthologs are present that have been identified and their complete genome data are available. Some of the important structural aspects of p53 have been widely described [25–28]. Mutations in p53 that result in deactivation of p53 are seen in the DBD region leading to the inability of the protein to bind to its target DNA sequences. Mutations in p53 can result in recessive loss of function or a dominant negative effect on the function of p53.

Functions of p53 Functions of p53 include its ability to regulate apoptosis, enhance genomic stability, and modulate angiogenesis. In view of its anti-angiogenic action, p5 is able to restrict generation of new blood vessels that are needed for the growing tumor cells. As a result, tumor cells cannot proliferate and grow. In addition, p53 (see Fig. 1.2): • Activates DNA repair proteins as and when DNA damage occurs. Since aging is associated with accumulation of DNA damage, it can be said that p53 may have a role in aging process. • Arrests cell cycle at the G1/S phase such that the cell has adequate time to repair the DNA damage that has occurred. Once the DNA damage that has occurred is repaired, the cell cycle resumes. • In some instances, the DNA damage that has occurred cannot be repaired. In such instance, p53 induces apoptosis so that the cells harboring DNA damage are eliminated. This results in reducing the chances of occurrence of cancer. One of the negative regulators of p53, in a normal cell, is mdm2 complex. The activation of p53 is performed by mdm2 complex that, in turn, activates p53 to result in cell cycle arrest. Such an arrest of cell cycle induced by p53 is expected to

Functions of p53

7

DNA damage Cell cycle abnormalities Hypoxia

mdm2

p53

Cell cycle arrest

p53

Apoptosis

DNA repair Death and elimination of damaged cells Cell cycle restart

CELLULAR AND GENETIC STABILITY Fig. 1.2  Scheme showing the function of p53 in cell cycle regulation, apoptosis, and prevention of malignant transformation

give enough time for the cell to repair the DNA damage that has occurred. When such a repair process is not possible, the cell will die by apoptosis. The exact manner in which cell determines whether DNA repair has to occur or the cell has to undergo apoptosis is not fully understood. One thing that is certain is the observation that activated p53 can bind to DNA leading to the activation and expression of several genes and microRNA miR-34a [29] and WAF1/CIP1 encoding for p21 that binds to the G1-S/CDK(CDK4/CDK6, CDK2, and CDK1) complexes, which regulate G1/S transition in the cell cycle. When p21 (WAF1) is complexed with CDK2, cell division is halted. In contrast to this, when a mutant p53 is present, it will not be able to bind to DNA appropriately, and, hence, the p21 protein fails to bring about its action to stop the cell division, and this can lead to inappropriate cell division and ultimately the development of cancer. In human embryonic stem cells (hESCs), it was observed that p53-p21 is an effective G1/S checkpoint, and thus, it plays an important role in cell cycle regulation and DNA damage response (DDR). It was reported that p21 mRNA is upregulated after DNA damage in hESCs. p53 seems to have the ability to activate several microRNAs (like miR-302a, miR-302b, miR-­302c, and miR-302d) that can have a direct inhibitory action on p21 expression [30]. It is noteworthy that the p53 and RB1 pathways are linked via p14ARF, suggesting that these pathways interact and regulate each other [31–35].

8

1  Introduction to Genes, Oncogenes, and Anti-oncogenes

It is also interesting to note that p53 by regulating leukemia inhibitory factor (LIF) may enhance implantation in the mouse and humans [36]. In addition, p53 expression is enhanced by UV light, a known DNA damage inducer. But, in this instance, p53 initiates events leading to skin tanning [37, 38].

Factors Regulating p53 p53 can be activated by many factors including but not limited to DNA damage-­ inducing agents such as UV, IR, or chemical agents (such as hydrogen peroxide), both endogenous and exogenous oxidative stress, ribonucleotide depletion, and impaired or enhanced oncogene expression. The activation of p53 enhances half-life of the p53 protein, and as a result, it accumulates in the stressed cells leading to a conformational change in p53 that ultimately results in a change in its function as a transcription regulator in these cells. The activation of p53 occurs as a result of the phosphorylation of its N-terminal domain that contains a large number of phosphorylation sites that is considered as the primary target for protein kinases transducing stress signals. There are two groups of protein kinases that induce transcriptional activation domain of p53: the first one is the protein kinases of the MAPK family (JNK1–3, ERK1–2, p38 MAPK) that respond to cell membrane damage, oxidative stress, and other stresses; and the second set of protein kinases are ATR, ATM, CHK1 and CHK2, DNA-PK, CAK, and TP53RK, which are needed to maintain the genome integrity by detecting and responding to DNA damage. Several oncogenes are also capable of stimulating p53 activation. In normal cells, p53 levels remain low due to continuous degradation of p53. Mdm2 (also called HDM2 in humans) protein is a product of p53 and binds to p53 to prevent its action and transports it from the nucleus to the cytosol. Mdm2 is an ubiquitin ligase and covalently attaches ubiquitin to p53 to mark it for degradation by the proteasome. It is noteworthy that ubiquitylation of p53 is a reversible process. USP7 (or HAUSP), a known ubiquitin-specific protease, can cleave ubiquitin off p53 and, thus, prevent p53 degradation by proteasome, a mechanism by which p53 is stabilized in response to oncogenic insults. USP42 can deubiquitinate p53 and is needed to enable p53 to respond to stress [39]. It is noteworthy that protein ubiquitination is a reversible process. Several enzymes have deubiquitinating activity. Some of these include the ovarian tumor proteases (OTU), ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), Machado-Joseph disease proteins (MJD), and the Jab1/MPN/Mov34 metalloenzymes (JAMM) [40]. Deubiquitination of MDM2 stabilizes p53 to induce apoptosis [41, 42]. MDM2-specific DUB USP2a, USP42, and USP5 also take part in the p53 pathway that can degrade K48-linked polyubiquitin chains and, thus, so indirectly regulate levels of p53 [43, 44]. OTUD5 that belongs to the subfamily of 14 DUBs which suppresses type I interferon-dependent innate immune response seems to have a regulatory role in p53 stability by deubiquitinating p53 [45].

p53 in Some Diseases

9

It is interesting that overexpression of HAUSP that is generally localized to the nucleus stabilizes p53 and HAUSP depletion results in an increase of p53 levels. This is so since HAUSP binds and deubiquitinates Mdm2. HAUSP binds to Mdm2 more avidly than to p53  in unstressed cells. USP10, located in the cytoplasm in unstressed cells, deubiquitinates cytoplasmic p53 and reverses Mdm2 ubiquitination. In the event there is DNA damage, USP10 is translocated to the nucleus to stabilize p53 but (USP10) does not interact with Mdm2 [41]. Several protein kinases described above phosphorylate the N-terminal end of p53. This results in the disruption of Mdm2-binding. This leads to recruitment of protein Pin1 by p53. As a result of this, a conformational change occurs in preventing p53 from binding to Mdm2. In addition, phosphorylation exposes the DNA binding domain of p53 that can activate or repress specific genes. Sirt1 and Sirt7, which are deacetylase enzymes, deacetylate p53 leading to inhibition of apoptosis [46]. Some oncogenes are capable of stimulating transcription of proteins that have the ability to bind to MDM2 and, thus, inhibit its activity.

p53 in Some Diseases Mutations of TP53 gene compromise tumor suppression capacity and, thus, may lead to the development of several types of cancers. Inheritance of only one functional copy of the TP53 gene seems to be capable of inducing tumors in early adulthood. Several mutagens are capable of altering the TP53 gene that can lead to inappropriate cell division leading to the development of various cancers. It has been documented that ~50% of human tumors show a mutation or deletion of the TP53gene [47–51] that results in loss of p53 resulting in genomic instability that causes an aneuploidy phenotype. Based on these observations, it has been postulated that increasing the amount of p53 may suppress generation of tumors though such efforts have been unsuccessful. This is in part due to the development of premature aging [52–55]. Since restoration of p53 function to near normal can induce apoptosis of tumor cells, it has been proposed that pharmacological reactivation of p53 may offer a viable option for cancer treatment [56]. Human papillomavirus (HPV)-encoded protein, E6, binds to the p53 protein. This leads to inactivation of p53 resulting in development of warts. HPV types, in particular types 16 and 18, can make the benign warts to progress to low- or highgrade cervical dysplasia that are reversible precancerous lesions. It is known that persistent HPV infection of the cervix for many years can cause cervical cancer. Since this discovery, now a potent HPV vaccine is available for the prevention of genital warts and cervical cancer [57]. To maintain cell DNA integrity and prevent a normal cell from transforming into a cancer cell, p53 protein is continually produced and degraded in cells. The degradation of the p53 protein is due to the binding of MDM2 to p53. Paradoxically, MDM2 is induced by the p53 protein that serves as a negative feedback control on

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1  Introduction to Genes, Oncogenes, and Anti-oncogenes

the activity of p53. On the other hand, mutant p53 proteins are incapable of inducing MDM2 that results in the accumulation of p53 at very high levels. In general, it is expected that presence of excess of p53 is beneficial since it enhances the integrity of DNA as it serves as a DNA defender. But, the mutant p53 protein inhibits normal p53 protein levels and leads to disruption of p53 stability and function. DNA sequencing can be used to detect p53 mutations.

I nteraction(s) of p53 with Other Genes, Proteins, and Molecules Similar to retinoblastoma gene/protein, p53 is also known to interact with many other genes/proteins/molecules highlighting the possible role of p53  in several physiological and disease processes. The role of p53 in inflammation is particularly interesting and suggests its role in cancer and inflammatory conditions.

p53 and Inflammation There seems to be a close interaction among p53, HMGB1, a pro-inflammatory molecule, and IκBα. IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha) inhibits the NF-κB (nuclear factor kappa-light-chain-­ enhancer of activated B cells) transcription factor and, thus, suppresses inflammation, whereas NF-κB is a pro-inflammatory signal. The function of IκBα is to keep the NF-kB proteins sequestered in an inactive state in the cytoplasm [58]. IκBα inhibits NF-κB transcription factors ability to bind to DNA [59]. Thus, whenever there are mutations to the gene encoding the IκBα protein, it would lead to inactivation of the IκBα protein, and so NF-κB becomes active that contributes to the development of cancer [60]. NF-κB is needed for the for production pro-inflammatory cytokine TNF-α (tumor necrosis factor-α). TNF-α and IL-6 (interleukin-6) are needed to fight infections. But, paradoxically, certain tumor cells are able to thrive better in the presence of IL-6 and TNF-α. In fact, some recent studies showed that IL-6 (and possibly, TNF-α) may actually promote metastasis of tumor cells. NF-κB is activated by various stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, oxidized LDL, and various bacterial and viral antigens [61–63]. NF-κB is essential for the regulation of immune response against various infections. Hence, inappropriate NF-κB function can be harmful as is seen in cancer, autoimmune diseases, and sepsis. But NF-κB is also needed for synaptic plasticity and memory [64–70]. Similar to NF-kB, HMGB1 is also a pro-inflammatory molecule that has a role in several inflammatory conditions including sepsis [71–76].

p53 and Inflammation

11

Cancer cells with mutant p53 have a more aggressive behavior and show enhanced invasive capacity especially when exposed to TNF. It was also found that mutant p53 activated NF-κB suggesting that p53 under normal physiological conditions suppresses inflammation. Interfering with such interaction reduced aggressiveness of cancer cells. This interaction between p53 and NF-kB suggests that p53 has a role in the regulation of inflammation and inflammation associated with cancer [50, 77]. Thus, it can be argued that under normal physiological conditions, p53 may suppress inflammation, partly, by inducing apoptosis of not only cells harboring DNA damage (including mutations and oncogene(s) expression) but also immune cells that produce excess of pro-inflammatory cytokines. If this proposal is correct, it suggests that p53 is a suppressor of inappropriate pro-inflammatory cytokine secretion (such as IL-6, TNF-α, and HMGB1). By virtue of this role, p53 may be expected to have a role in rheumatoid arthritis, lupus, sepsis, and other inflammatory conditions. p53 expression is abnormal in rheumatoid arthritis and lupus [78–92]. CD4+ T cells from p53−/− mice show decreased activity of STAT-5, lower phosphorylated STAT-5 status, and inappropriate Treg cell differentiation. In mice with rheumatoid arthritis when the inflammatory activity was present, Th17 cell differentiation was suppressed by p53 and enhanced Treg cell differentiation by activating STAT-5 signaling cascades. Injection of a p53 overexpression vector or an antagonist of Mdm2 suppresses collagen-induced arthritis in experimental animals. These results are supported by the observation that decreased p53 levels corresponded with the severity of rheumatoid arthritis (RA) indicating a close interaction between p53- and STAT-mediated regulation of Th17 cells/Treg cells in RA [86]. Subsequent studies revealed that Cyr61 (cysteine-rich angiogenic inducer 61), a matricellular protein that is capable of regulating cell adhesion, migration, proliferation, differentiation, apoptosis, and senescence by interacting with cell surface integrin receptors and heparan sulfate proteoglycans, is overexpressed in rheumatoid arthritis. CYR61 is critical for cardiac septal morphogenesis, angiogenesis in placenta, and vascular integrity during embryogenesis. CYR61 is also involved in inflammation and tissue repair. Thus, CYR61 may have a role in chronic inflammation, rheumatoid arthritis, atherosclerosis, diabetes-related nephropathy and retinopathy, and some cancers. CYR61 promotes fibroblast-like synoviocyte (FLS) proliferation and Th17 cell differentiation. MicroRNA-22 (miR22) inhibits Cyr61 expression. In rheumatoid arthritis synovial tissue, miR-22 expression was found to be downregulated and negatively correlated with Cyr61 expression. Wild-type p53 can activate miR-22 transcription by binding to the promoter region of the miR-22 gene. In rheumatoid arthritis synovial tissue, mutant forms of p53 are frequent with downregulation of miR-22 and failure of activation of miR-22 by p53. These events lead to overexpression of Cyr61 in rheumatoid arthritis synovium. Based on these results, it can be suggested that p53 regulation of Cyr61 expression via miRNA-22 is abnormal [85] in rheumatoid arthritis and, possibly, in lupus [78–92] that may explain the abnormalities of p53 seen in these conditions.

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1  Introduction to Genes, Oncogenes, and Anti-oncogenes

Phosphatase and Tensin Homolog (PTEN) in Cancer PTEN is a protein encoded by the PTEN gene, whose mutations can cause cancer (see Fig. 1.3 for the crystallographic structure of PTEN). PTEN is a tumor suppressor, and its mutations are seen in many cancers. PTEN encodes a phosphatidylinositol-­ 3,4,5-trisphosphate 3-phosphatase that has a tensin-like domain as well as a catalytic domain that is similar to a dual-specificity protein tyrosine phosphatases, which negatively regulates intracellular levels of phosphatidylinositol-­3,4,5-trisphosphate and functions as a tumor suppressor by negatively regulating Akt/PKB signaling pathway. PTEN opposes the action of PI3K that is needed for the activation of Akt, an anti-apoptotic and tumor-promoting molecule. The PTEN protein is present in several tissues of the body. PTEN protein dephosphorylates phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5) P3 or PIP3) and catalyzes the dephosphorylation of the 3` phosphate of PIP3 that results in the formation of PIP2 (PtdIns(4,5)P2), events that eventually result in the inhibition of the AKT signaling pathway. The phosphatase protein product of PTEN regulates cell cycle [93].

Clinical Significance of PTEN Mutations in PTEN in several cancers are common, especially in prostate cancer [94]. In addition, mutations I PTEN are also seen in glioblastoma, endometrial cancer, lung cancer, and breast cancer. PTEN mutations can lead to increased inherited predispositions to cancer [95–99]. PTEN gene mutations lead to the formation of a

Fig. 1.3 Crystallographic structure of human PTEN. The N-terminal phosphatase domain is colored blue while the C-terminalC2 domain is colored red

13

Clinical Significance of PTEN

a

PBM

PD

C2D

C-Tail

b

Transcription Me

Me Me

c

Me

PTEN gene

Promoter hyper-methylation

TGF MKK4, IGF1, 17-βE2 Transcription SMAD

ID1 BMI1

EVI1

NFkB SNAIL

SALL4

PTEN gene

c-Jun

Transcriptional repression Indole 3-carbinol Quercetin Transcription

Phytoestrogens IGF2 p53 EGR1

Transcriptional activation

SPRY2 Resistin

d

PTEN gene

PPAR-γ

Resveratrol

PTEN gene

PTENP1 gene Transcription

Transcription 3’-UTR

PTEN mRNA

3’-UTR

PTENP1 mRNA

miRNA PTEN mRNA

3’-UTR

3’-UTR

PTENP1 mRNA

PTENP1 as a decoy 3’-UTR

PTEN mRNA Translation PTEN protein

Translation PTEN protein

Fig. 1.4  Genomic mechanisms of PTEN regulation. (Data from Uversky et al. [236])

dysfunctional protein (see Fig.  1.4) that is incapable of stopping cell division or induce apoptosis resulting in tumor formation [95, 100–110]. Types of mutations in PTEN gene are shown in Fig. 1.4. Mutations in the PTEN gene cause uncontrolled cell division and formation of tumors.

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1  Introduction to Genes, Oncogenes, and Anti-oncogenes

PTEN, Brain Function, and Autism PTEN gene defects are present in autism [111]. Defective PTEN protein interaction with the protein of Tp53 gene produces an abnormal increase in the levels of energy production in the cerebellum and hippocampus. Since PTEN and PI3K pathways regulate cell proliferation, migration, cellular size, synaptic transmission, and plasticity, it is no wonder that PTEN gene defects could result in neurological abnormalities seen in autism, seizures, and ataxia [112, 113] (Fig. 1.5). These two studies [112, 113] are interesting and suggest a critical role for PTEN in autism and related neurological conditions. At this juncture it is important to note that phosphoinositide 3-kinase (PI 3-kinase) is an ubiquitous enzyme that catalyzes the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-trisphosphate (PIP3). PI 3-kinase-driven generation of PIP3 activates various downstream signaling cascades which in turn regulate numerous biological processes including growth, metabolism, proliferation, migration, and cell size. These actions of PIP3 on various cellular events may explain its role in cancer (in which cellular morphology is changed and so also their ability to

Fig. 1.5  (continued)  that can result in the activation of the PI3K/Akt pathway and increase in oxidative stress and features of autism. This indicates that PTEN is a negative regulator of PI3K/ Akt pathway. Mitochondrial dysfunction occurs as a result of downregulation of p53 signaling and lower protein expression of p21 [141]. Thus, there is a close interaction between PTEN and p53 pathways. PTEN has a regulatory role in B-cell function. PTEN expression is inversely correlated with lupus disease activity. Interleukin-21 (IL-21) induces PTEN expression and suppresses Akt phosphorylation. IL-21-induced STAT3 (signal transducer and activator of transcription 3) phosphorylation can upregulate PTEN mRNA in lupus B cells that express enhanced levels of miR-7, miR-21, and miR-22, which downregulate the expression of PTEN. IL-21 enhances the expression of miR-7 and miR-22. A miR-7 antagonist is capable of correcting PTEN-related abnormalities seen in lupus B cells that are dependent on PTEN [209]. Thus, a defective miR-7 regulation of PTEN seems to be the underlying cause of B-cell hyperresponsiveness in lupus. miR-21 is upregulated and correlates with lupus disease activity. Suppressing miR-21 activity decreases interleukin-10 production seen in lupus [210]. Thus, miR-21 seems to have a role in lupus. High-mobility group box 1 (HMGB1, a pro-inflammatory molecule) also has a role in lupus, partly, through the cyclin D1/CDK4/p16 system. HMGB1 can downregulate PTEN expression, and overexpression of PTEN suppresses HMGB1 levels and its downstream events including proliferation of mesangial cells of kidney by blocking Akt activity [211]. These results indicate that HMGB1 interacts with PTEN/phosphoinositide 3-kinase (PI3K)/Akt/NF-κB signaling pathway. These results [209–211] imply that suppressing HMGB1, miR-21, and PTEN overexpression/activation is of benefit in lupus and other inflammatory conditions. Previously, we and others [212–229] showed that certain polyunsaturated fatty acids (PUFAs) and their anti-inflammatory metabolites such as lipoxins, resolvins, protectins, maresins, and nitrolipids have the ability to suppress HMGB1, miR-21, and PTEN expression/activation. Downregulation of miR-21 enhances susceptibility of colon cancer cells to therapeutic regimens and induces their differentiation [230]. PUFAs inhibit miR-21 expression [226], implying that PUFAs are of benefit in drug-resistant cancer. PUFAs have a role in stem cell survival, proliferation, and differentiation [231–235], suggesting that these bioactive lipids are likely to be of benefit in cancer, diabetes mellitus, Alzheimer’s disease, lupus, etc., wherein stem cell abnormalities are present

PTEN, Brain Function, and Autism

15

Mutagens and Carcinogens, other environmental agents

Retinoblastoma Gene

P53

PTEN

PUFAs PI3K/Akt pathway

MAPK family

PtdIns (3,4,5) P3 or PIP3

PUFAs Inflammation

Immune response

Apoptosis

PUFAs Neuronal and other cell proliferation, migration, cellular size, cell-cell communication and synaptic transmission PUFAs

PUFAs

Cancer, inflammatory, and autoimmune diseases, septic shock, viral infection, autism, Alzheimer’s disease, lupus, rheumatoid arthritis, multiple sclerosis, sepsis, etc.

Fig. 1.5  Scheme showing the role of p53, Rb, and PTEN (and other anti-oncogenes) in the regulation of inflammation, immune response, and apoptosis and consequently their role in various diseases. Mutagens, carcinogens, and other environmental agents (such as viruses) may produce changes in the expression and action of p53, Rb, and PTEN and other anti-oncogenes that leads to alterations in their downstream events. This will eventually alter events related to inflammation, changes in immune response (both cellular and humoral), and apoptosis. As a result of these events, cell and neuronal cell proliferation, their migration and cell-to-cell communication, and in the case of cerebral neurons synaptic transmission will be altered. These changes in cell/neuronal behavior will lead to the development of cancer, sepsis, susceptibility to various infections, lupus, rheumatoid arthritis, and other diseases. Alterations in neuronal cell proliferation, migration, cell size, and synaptic transmission could lead to Alzheimer’s disease, autism, multiple sclerosis, and other neuronal disorders. For instance, p53 responds to DNA and plays a critical role in neuroprotection. It was also demonstrated that p53 controls synaptic genes, and, thus, synaptic function seems to be a central function of p53 and, thus, prevents neurodegeneration and Alzheimer’s disease and autism [207]. In lupus, bone marrow-derived mesenchymal stem cells (BM-MSCs) showed characteristics of senescence and significantly increased expressions of p53 and p21 with a concomitant decrease in the levels of cyclin E, cyclin-dependent kinase-2, and phosphorylation of retinoblastoma protein. These senescence features can be reversed by suppressing p21 expression [87]. In this context, Wnt/β-catenin signaling has a role in the senescence of BM-MSCs through the p53/p21 pathway [88]. Mdm2, a negative regulator of p53, expression is increased in lupus, whereas Mdm2 inhibition is of benefit in lupus by suppressing inappropriate inflammation [89], implying that inhibition of Mdm2 and enhancing the expression of p53 are of benefit in lupus. TNF production is increased in lupus that could be suppressed by p53 in macrophages. This suggests that p53 expression could be exploited in the treatment of inflammatory diseases including sepsis [208]. PTEN mutations are common in Alzheimer’s disease and autism. PTEN haplo-­ insufficiency in neural tissues increases mitochondrial complex activities (II–III, IV, and V)

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1  Introduction to Genes, Oncogenes, and Anti-oncogenes

proliferate, migrate, and form metastasis) and some neurological conditions such as autism (wherein there could occur defects in synapse formation, synapse transmission, and memory). Though PTEN was originally identified as a tumor promoter, it is also constitutively an active lipid phosphatase that opposes PI 3-kinase-dependent signaling by promoting dephosphorylation of PIP3 into PIP2. PTEN is highly expressed in neurons. Familial mutations that result in PTEN inactivation have been linked to neurological disorders such as ataxia, mental retardation, and seizures [114, 115]. Mice with a tissue-specific deletion of the mouse homolog PTEN (PtenloxP/loxP; Gfap-cre) develop seizures and ataxia and have brain enlargement due to primary granule-cell dysplasia in the cerebellum and dentate gyrus. PTEN mutant cells have elevated phosphorylation of Akt, suggesting that PTEN and Akt have a role in the regulation of cell size and their function that may be relevant to some neurological conditions [115]. Furthermore, loss of PTEN function at early stages of development resulted in widespread deficits in neuronal growth, synaptogenesis, and synaptic plasticity suggesting additional roles for PTEN in these processes. Sperow et al. [114] showed that PTEN controls hippocampal synapses and activity-dependent hippocampal synaptic plasticity. These and other studies [114– 118] demonstrated that PTEN is important for brain growth and development, hippocampal synaptic plasticity, and neuronal morphology. Leptin that can cross the blood-brain barrier (BBB) and play a key role in regulating food intake and body weight by virtue of its actions on hypothalamic nuclei and leptin receptors [119] also seems to have a critical role in hippocampal synaptic function, partly, by enhancing the phosphorylation of the lipid phosphatase of PTEN [120–124] and thus may have a role in autism and related disorders. These results [117, 119–124] are interesting in the light of the reports that plasma leptin levels are elevated in subjects with autism and other related disorders [125– 127], while those of adiponectin significantly decreased [128]. These findings are interesting to note that none of the patients studied were obese, suggesting that there could be a role for leptin and adiponectin in some, if not all, of the clinical manifestations in these subjects other than weight balance. Furthermore, ghrelin and growth hormone levels were found to be significantly lower in the autism group compared to control [129]. Since ghrelin is known to regulate neuroinflammation and apoptosis that are linked to autism, these results suggest a potential role for ghrelin in the pathogenesis of autism [130–139]. Furthermore, there is a potential link between ghrelin and leptin, molecules that seem to have a role in autism. Ghrelin and leptin have an inverse relationship. Leptin inhibits ghrelin transcription in a dose-dependent manner, thus reducing ghrelin levels [140], and leptin levels are known to be significantly higher in children with autism [125], suggesting that the elevated leptin levels may be due to or associated with decreased ghrelin levels in autism. Thus, there are several abnormalities in autism and related disorders such as changes in the expression of PTEN, p53, reduced levels of ghrelin, and reduced concentrations of leptin, all of which are likely to lead to alterations in the neuronal synaptic plasticity, neuronal transmission, and neuronal function that may ultimately reflect in the form of various clinical features seen in autism.

PTEN, Brain Function, and Autism

17

In instances where PTEN protein is deficient or insufficient, it could lead to an ineffective interaction with p53 that leads to defects or inappropriate actions of other proteins as is seen in those with learning disabilities including autism [111]. PTEN haplo-insufficiency leads to an increase in the activities of mitochondrial complexes (II–III, IV, and V) with an activation of the PI3K/Akt pathway. PTEN is known to be a negative modulator of PI3K/Akt pathway. PTEN haplo-insufficiency led to significant behavioral abnormalities or changes in mitochondrial outcomes by age 20–29 weeks but not earlier than this age. These animals show aberrant social behavior, microcephaly, increased oxidative stress, decreased cytochrome C oxidase (CCO) activity, and increased mtDNA deletions in the cerebellum and hippocampus that seemed to be a result of p53 signaling pathway downregulation resulting in lower protein expression of p21 and the CCO chaperone SCO2. SCO2 deficiency and/or CCO activity defects are also seen in autism, and autism is also associated with defects in p53 signaling pathways and PTEN abnormalities [141, 142]. These results imply that there is a close relationship between PTEN and energy metabolism. Does this mean a decrease in the expression of PTEN leads to suppression of energy expenditure/energy utilization by the neurons that lead to their dysfunction and features of autism? Similarly, an alteration in the expression of p53 as a result of decreased PTEN expression could cause mitochondrial dysfunction and oxidative stress that result in neuronal apoptosis. Thus, alterations in PTEN and p53 expression can cause neuronal loss leading to decrease/loss of neuronal synaptic plasticity and neuronal transmission that result in neuronal dysfunction that may ultimately reflect in the form of various clinical features seen in autism. This is supported by the observation that a reduction of Bcl-2 and increase in p53 expressions, markers of apoptosis, are seen in the parietal cortex of autism. Brain extracts obtained from the superior frontal cortex and cerebellum (at the time of postmortem of children with autism) showed decrease Bcl-2 levels in the superior frontal and cerebellar cortices compared to control, whereas p53 levels were found to be increased in the same brain areas. The ratios of Bcl-2/p53 values were also found to be decreased in autistic frontal and cerebellar cortices vs controls. These results indicate that levels of Bcl-2 and p53 are altered in frontal, parietal, and cerebellar cortices of autistic subjects, alluding to deranged apoptotic mechanisms in autism [143–145]. These results confirm that altered expressions of PTEN, p53, and Bcl-2 could lead to enhanced apoptosis of neurons especially in the affected regions of the brain of autistic children that may underlie the pathological and clinical features of autism. In this context, it is noteworthy that there may be a direct interaction between PTEN and p53. In general, it is believed that tumor suppressor genes (PTEN and p53) function in an autonomous fashion to prevent the development of cancer. Thus, only their homozygous loss is expected to lead to the onset of cancer [146]. But it needs to be understood that in the cells, no single gene or protein works in an autonomous fashion, and there are innumerable positive and negative regulators of their actions. Since it is known that there exists some sort of an indirect cross talk between PTEN and p53, it is likely that there could be a direct interaction between these two genes and their proteins. Though PTEN is mainly active in the cytoplasm, while p53 is

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1  Introduction to Genes, Oncogenes, and Anti-oncogenes

active in the nucleus, recent studies showed that these two genes/proteins can still interact with each other. In normal cells p53 levels are maintained low due to its regular proteasome-dependent degradation mediated by the mdm2 protein that acts as a p53 ubiquitin ligase. p53 undergoes several posttranslational modifications that include phosphorylation, acetylation, and sumoylation that are very tightly controlled, and these events are responsible for its stabilization and transcriptional activation upon cellular stress. PTEN gene dephosphorylates the plasma membrane lipid phosphatidylinositol 3- phosphate (PIP3) and converts PIP3 to PIP2 that inactivates Akt and PDK1 that, in turn, stimulates cell survival, proliferation, growth, and migration. PTEN heterozygous mice develop multiple tumors attesting to the fact that it is an important tumor suppressor [147–149]. p53 binds to the PTEN promoter site [150]. Conversely, PTEN regulates p53 function by antagonizing PI3 kinase [151, 152]. Akt phosphorylation sites are present in mdm2, a major negative regulator of p53. Phosphorylation of mdm2 results in translocation of p53 to the cell nucleus to ubiquitinate p53 leading to its nuclear export and degradation. PTEN prevents p53 degradation by inactivating Akt, and, thus, PTEN regulates p53 action. This is further supported by the observation that a p53 binding domain is present in PTEN [153, 154]. There is also evidence to suggest that PTEN can regulate p53 activity independently of its enzymatic activity, implying that there are multiple levels of cross talk between PTEN and p53. It appears PTEN activates p53-mediated transcription per se in addition to protecting it from degradation [154]. These observations may explain as to why, at least, in human breast cancers PTEN and p53 somatic mutations are mutually exclusive [155]. Since both PTEN and p53 mutations and dysfunction are known to occur in autism, the above findings are relevant to this clinical condition as well. This rises an intriguing possibility that at the molecular levels, there are similarities between autism and cancer. The ratio between Bcl-2 and p53 is known to be shifted in favor of p53 explaining as to why apoptosis occurs in parietal cortex neurons of autistic subjects. Bcl-2 expression is decreased and p53 is increased in superior frontal cortex and cerebellum extracts obtained from patients with autism compared to control. These results explain the deranged apoptotic mechanisms seen in autism [143, 144]. Cathepsin D, a lysosomal protease, a mediator of apoptosis induced by TNF-α and interferon (IFN)-gamma, has also been found to be significantly increased in the frontal cortex, pyramidal and granule cells of the hippocampus, and cerebellar neurons in autism. In addition, caspase-3, an executioner of apoptosis, has also been reported to have been increased in the cerebellum of autistic subjects [156]. These results in conjunction with the fact that Bcl-2 expression is decreased, and the BDNF-Akt-­ Bcl-2 pathway is decreased in the frontal cortex of autistic subjects [157], indicate that inappropriate increase in the process of apoptosis plays a crucial role in autism. In subjects with autism, deletion of the mitogen-activated protein kinase 3 (MAPK3) gene that encodes ERK1 has been described. Ras/Raf/ERK1/2 (extracellular signal-regulated kinase) signaling pathway is critical for neurogenesis of neural progenitors, learning, memory, and apoptosis of neural cells. BTBR mice, an

PTEN, Brain Function, and Autism

19

animal model of autism, show significantly increased Ras/Raf/ERK1/2 signaling activity. Significantly elevated expression of Ras is seen in the frontal cortex of autistic subjects. The frontal cortex showed increased C-Raf phosphorylation, whereas the cerebellum had an increase in both C-Raf and A-Raf activities in those with autism. On the other hand, significantly upregulated activities of ERK1/2 have been described in the frontal cortex of autistic subjects. In contrast, both the frontal cortex and cerebellum had upregulated expression of ERK5 protein in autism [158– 160], suggesting that upregulation of Ras/Raf/ERK1/2 signaling and ERK5 activities in the frontal cortex may have a critical role in autism. PTEN is a strong inhibitor of cell growth that may be relevant to both cancer and autism. PTEN deletion enhances nerve regeneration [161]. These and other evidences [141–161] suggest that both in autism and cancer, there could occur changes in the expression and action of p53, PTEN, BCL-2, and Ras/Raf/ERK1/2 and ERK5 [162] that lead to mitochondrial dysfunction, inappropriate apoptosis, and defects in synapse formation, synapse transmission, and memory that are relevant to the pathobiology of autism, whereas the same or similar abnormalities in cancer could lead to defects in apoptosis and cell-cell communication (especially between normal and tumor cells) and may alter growth, metabolism, proliferation, migration, and cell size by modulating PIP3 levels. Similarly, both leptin and ghrelin whose concentrations are altered in autism and cancer may alter inflammatory process and apoptosis and thus participate in these two diseases. Based on these evidences, it is not too farfetched to suggest that leptin and ghrelin may also have a regulatory role in the expression and action of p53, PTEN, Ras/Raf/ERK1/2, and PI3K/Akt pathway [163–166]. Thus, though both autism and cancer appear to be distinct disease entities, there seems to be several common fundamental abnormalities. It is noteworthy that the same gene/protein abnormalities are present in both diseases. For instance, changes in p53, PTEN, ghrelin, leptin, Ras/Raf/ERK1/2, and PI3K/Akt pathway seen in autism cause inappropriate apoptosis of neuronal cells and defects in synapse formation, neurotransmission, and memory. On the other hand, similar changes in p53, PTEN, ghrelin, leptin, Ras/Raf/ERK1/2, and PI3K/Akt pathway lead to mitochondrial dysfunction and alteration in the growth, metabolism, proliferation, migration, and cell size of tumor cells that results in continued proliferation of tumor cells and their migration to distant sites resulting in metastasis and cancer cachexia. Thus, at the more fundamental level, p53, PTEN, ghrelin, leptin, Ras/Raf/ ERK1/2, and PI3K/Akt and PIP3 are involved in mitochondrial function that is essential for energy generation and cell survival and function, growth, proliferation, migration, and cell size actions that seem to have been altered both in autism (in the brain) and cancer (in the tissue where it occurs). Thus, this implies that development of an effective therapeutic strategy for one condition (say autism) may be relevant to the other (say cancer) and vice versa. Similar to p53, PTEN (gene) is known to interact with several other genes that may also be relevant to their role in autism and cancer. In addition, alterations in the immune system are known to occur in cancer, and this aspect has been discussed in a separate chapter.

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1  Introduction to Genes, Oncogenes, and Anti-oncogenes

Immune Checkpoint Inhibitors in Cancer Despite many advances in the molecular pathobiology of cancer, it is still not clear how this knowledge can be exploited for the prevention and management of cancer in a very effective way. Methods designed to prevent cancer include avoiding risk factors that are known to enhance the development of cancer such as smoking, reducing intake of high-fat diet, avoiding or reducing exposure to environmental pollutants, etc. It is believed that almost 50–60% of cancers are due to exposure to environmental pollutants, some known and many unknown. Thus, the only preventive measures known at prsent are avoiding risk factors such as smoking, tobaccorelated products, etc. It is also well established that a defective immune response is responsible for the development of cancer in a given subject. It is believed that a very effective immune system is able to detect and mount adequate immune response to eliminate cancer cells as they arise and, thus, prevent their proliferation and development of cancer. But, more often than not, immune system fails to detect cancer cells, and so they proliferate and form detectable masses of cancer. This defect in the immune system can be because systemic immune response is defective, or the tumor cells are able to evade detection by the immune cells of the body or both. This immune evasion by the tumor cells is due to the ability of the tumor cells to mask their new antigens on their cell surface or synthesize and secrete certain immune suppressive molecules such as prostaglandin E2 (PGE2) or both. In order to prevent T cells and macrophages and other immunocytes to recognize normal cells as foreign and mount an immune response to eliminate them, certain regulatory systems are developed in the body. This capacity of T cells, macrophages, and dendritic cells to recognize foreign invading organisms is needed to protect the body from various infections. This is so since activated macrophages and T cells can mount an immune attack and destroy such microbial organisms and infected cells to prevent various diseases. In an occasional instance, when this checkpoint system fails, T cells, macrophages, and dendritic cells recognize body’s own cells as foreign and attack them to produce autoimmune diseases such as rheumatoid arthritis and lupus. This checkpoint inhibitory system is taken advantage by the cancer cells to evade the immune system and survive. Such checkpoint proteins include PD-1 (programmed cell death-1), PD-L1 (programmed cell death-1 ligand), PD-L2 (programmed cell death-2 ligand), and CTLA4 (cytotoxic T-lymphocyte-associated protein-4). Thus, PD-1, PD-L1, PD-L2, and CTLA4 are proteins that are expressed on the surface of cells that are designed to downregulate the immune system and promote self-tolerance. They do so by suppressing T-cell inflammatory activity. Though this is originally designed to prevent the development of autoimmune diseases by promoting self-tolerance, this is exploited by the cancer cells to downregulate the immune system and survive. For instance, PD-1 guarded against autoimmune diseases by promoting apoptosis of antigen-specific T cells in lymph nodes and reduces apoptosis of regulatory T cells that have anti-inflammatory action (that are also called as suppressive T cells). This

PD-L1 Inhibitors Include

21

suggests that by specifically blocking the immune checkpoint proteins (PD-1, PD-L1, PD-L2, and CTLA4), it is possible to enhance immune response against tumor cells and, thus, eliminate them. Thus, it is evident that when normal cells express PD-L1 and PD-L2 and T cells, macrophages, dendritic cells, and other immunocytes express PD-1 and PD-2 on their surface and when they come in contact with each other, the immune system is switched off so that the immune system is not activated against normal cells. It is known that some, if not all, cancer cells have large amounts of PD-L1 which allows them to evade the immune system since now the T cells, macrophages, and dendritic cells recognize them as self and does not mount an immune attack on the cancer cells. In such an instance, monoclonal antibodies that target PD-1, PD-L1, PD-L2, and CTLA4 are able to block the binding of PD-1 to PD-L1, PD-L2, and CTLA4 leading to mounting of an immune attack on tumor cells and eliminate them. Examples of drugs that target PD-1 include: • Pembrolizumab (Keytruda) • Nivolumab (Opdivo) • Cemiplimab (Libtayo) These drugs are useful in the treatment of melanoma of the skin, non-small cell lung cancer, kidney cancer, bladder cancer, head and neck cancers, and Hodgkin’s lymphoma.

PD-L1 Inhibitors Include • Atezolizumab (Tecentriq) • Avelumab (Bavencio) • Durvalumab (Imfinzi) These drugs are useful in the treatment of bladder cancer, non-small cell lung cancer, and Merkel cell skin cancer (Merkel cell carcinoma). Drugs that target CTLA-4 include: • Ipilimumab (Yervoy) This drug is useful in the treatment of melanoma of the skin. When these immune checkpoint inhibitors activate T cells, macrophages, and dendritic cells, these immunocytes release pro-inflammatory cytokines such as IL-6 and TNF-α that can cause side effects such as fatigue, cough, nausea, loss of appetite, skin rash, and itching. Some of the serious side effects of these immune checkpoint inhibitors include inflammation of the lungs, intestines, liver, kidneys, and other organs. Sometimes these side effects of immune checkpoint inhibitors are very serious due to cytokine storm (release of large amounts of IL-6 and TNF-α) leading

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1  Introduction to Genes, Oncogenes, and Anti-oncogenes

to death. Furthermore, checkpoint inhibitors are effective in only 20–30% of the patients. These results suggest that more suitable methods of delivery of immune checkpoint inhibitors are needed such that only tumor cells are eliminated with little or no side effects or very few side effects that are tolerable or can be managed easily.

Bioactive Lipids and Immune Response in Cancer In this context, it is interesting to note that prostaglandin E2 (PGE2) can suppress the activation of cytotoxic lymphocytes (CTLs) and their ability to secrete cytokines such as IL-6 and TNF-α [167, 168]. Since PGE2 can be produced by macrophages, T cells, and CTLs and tumor-infiltrating lymphocytes (TILs), it is reasonable to propose that there is negative and positive feedback regulation between secretion of cytokines by various immunocytes and PGE2 and other such lipid immunosuppressors. It is likely that whenever activated T cells, macrophages, TIL, and CTLs produce IL-6 and TNF-α, there will occur release of PGE2 also in order to regulate and suppress excess secretion of these toxic cytokines and, thus, regulate the immune response. Thus, it can be said that under normal physiological conditions, there is a balance maintained between cytokines and PGE2. When the secretion of IL-6 and TNF-α is adequate and its desired function is achieved, the same immunocytes secrete PGE2 to shut off the synthesis and secretion of IL-6 and TNF-α, so that excess production of these toxic cytokines would not occur. Similarly, when the secretion of PGE2 is high that is likely to suppress adequate production of IL-6 and TNF-α, then automatically the production of PGE2 is inhibited by the inhibitory action of cytokines on desaturases that decreases cell membrane lipid content of AA and other polyunsaturated fatty acids (PUFAs). This positive and negative feedback regulation is important and essential to regulate immune response under normal physiological conditions to prevent unwanted side effects of IL-6 and TNF-α. This proposal is supported by the fact that IL-6 and TNF-α stimulate phospholipase A2 (PLA2) that results in the release of arachidonic acid (AA) from the cell membrane lipid pool, the precursor of PGE2, a potent immunosuppressor [169, 170]. At the same time, both IL-6 and TNF-α inhibit the activities of delta-6 and delta-5 desaturases that are needed for the conversion of dietary linoleic acid and alpha-linolenic acid to form their long-chain metabolites such as AA and EPA/ DHA (eicosapentaenoic acid and docosahexaenoic acid), respectively [171]. In addition, both IL-6 and TNF-α activate cyclooxygenase-2 (COX-2) and lipoxygenase (LOX) enzymes that are needed for the formation of PGE2 and lipoxin A4 (LXA4) [172–177]. Thus, whenever the concentrations of IL-6 and TNF-α are high, it leads to enhanced formation of PGE2 and LXA4. But increase in the formation of PGE2 or LXA4 depends on the target tissue whether it is normal or tumor cells. Furthermore, tumor cells are known to have low concentrations of AA/EPA/

Bioactive Lipids and Immune Response in Cancer

23

DHA in their cell membrane lipid pool due to decreased activities of desaturases that, in turn, leads to increase in the synthesis and release of PGE2 as a compensatory mechanism [178–185]. But, for some unknown reasons, whenever there is a deficiency of AA/EPA/DHA (but mostly AA), there is also an increase in the activity of COX-2 that results in the formation of increased amounts of PGE2 but decreased formation of LXA4. This may, in part, be a compensatory mechanism due to PUFA deficiency. This is also, in part, due to the inhibitory action of IL-6 and TNF-α on desaturases and activation of COX-2. In contrast, in situations wherein there is increased amounts of IL-6 and TNF-α and enhanced activity of COX-2 and decreased amounts of AA/EPA/DHA (mostly AA) in the cell membrane lipid pool, supplementation of AA was found to enhance LXA4 formation with or without any change on PGE2 formation [186, 187] that ultimately results in suppression of IL-6 and TNF-α synthesis and suppression of inflammation induced by PGE2. It may be noted here that LXA4 has potent anti-inflammatory actions and suppresses the production of PGE2, IL-6, and TNF-α and has growth inhibitory actions on tumor cells [188–191]. There seems to be a delicate balance maintained between PGE2 and LXA4, though both are derived from AA. These results suggest that there is a selfregulatory system operating in the secretion and suppression of IL-6 and TNF-α. Thus, it is reasonable to suggest that as and when T cells, monocytes, TILs, and CTLs are exposed to tumor cells, there would occur their activation and release of IL-6 and TNF-α that are needed to kill the tumor cells. At the same time, the production and secretion of PGE2 are inhibited with or without a change in LXA4 production so that adequate production and secretion of IL-6 and TNF-α would occur. Once the desired function of secreted cytokines is over, both IL-6 and TNF-α would suppress the formation of PGE2 by inhibiting the activities of desaturases, and so PGE2 formation would decrease as a result of precursor deficiency (AA and EPA/DHA). Once the production of IL-6 and TNF-α is halted, then the activities of desaturases would return to normal, and production of normal physiological levels of PGE2 and LXA4 would be resumed to suppress excess production of IL-6 and TNF-α, and the balance between cytokines and PGE2 is restored to normal. This delicate balance among the precursors of PGE2, PGE2, LXA4, and cytokines is important in the prevention of cancer development and to eliminate tumor cells as and when they arise. Based on these results, it can be argued that tumor cells have developed an ingenious system to suppress immune response (by enhanced production of PGE2), enhance local inflammation to the extent needed (by increased secretion of PGE2 that is pro-­inflammatory in nature and inflammation is known to enhance the growth of tumor cells), and escape from the immune surveillance normally rendered by T cells, macrophages, dendritic cells, TIL, and CTLs (as PGE2 suppresses their immune surveillance ability). This physiological regulatory system is utilized by cancer cells to suppress the immune system and thrive. Tumor cells are known to secrete PGE2 and, thus, suppress the cytotoxic action of T cells, macrophages, TILs, and CTLs.

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1  Introduction to Genes, Oncogenes, and Anti-oncogenes

Prostaglandin E2 (PGE2) and Immune Checkpoint Inhibition It is known that PGE2 may have immunoregulatory action that is relevant to immunosuppression seen in patients with cancer and the ability of tumor cells to escape immunosurveillance in the body. Microsomal PGE2 synthase 1 (mPGES1) is an effector of the COX-2 pathway and is responsible for the immunosuppression seen in cancer. It was reported that in a syngeneic melanoma mouse model, PTGES knockout increases melanoma expression of PD-L1 and enhances infiltration of CD8a+T cells and CD8a+dendritic cells into the tumor tissue that led to suppression of tumor growth. In addition, it was noted that mice-bearing PTGES knockout tumors when were given anti-PD1 therapy showed enhanced response to the therapy. It is noteworthy that microarray analysis of the melanoma tumor tissue showed a significant association between high PGES1 expression and low CD8+infiltration that could be correlated with a shorter patient survival [192]. These results are in support of the contention that PGE2 is an immunosuppressor. Furthermore, inhibition of PGE2 formation reduced PD-L1 expression in tumorinfiltrating myeloid cells. Tumor-infiltrating PD-L1+cells obtained from tumorbearing mice expressed high levels of PGE2-forming enzymes: mPGES-1 and COX-2 [193]. Several other studies have emphasized the role of PGE2 in immunosuppression seen in cancer and its involvement in the expression of PD-L1 (and possibly, PD-1, PD-L2, and CTLA4) [194–197]. In summary, these results emphasize the role of the COX2/mPGES1/PGE2 pathway in the regulation of PD-L1 (possibly, PD-1, PD-L2, and CTLA4) expression implying that methods designed to suppress PGE2 metabolism in tumor microenvironment reduce immune suppression in the tumor host and aid in cancer therapy and in the induction of remission of various cancers.

LXA4 Versus PGE2 in Cancer It is possible that a balance is maintained between pro- and anti-inflammatory cytokines and eicosanoids under normal physiological conditions, and this may be tilted more toward pro-inflammatory molecules in cancer and other conditions. Thus, it is envisaged that an increase in the plasma or tissue levels of PGE2 would occur when the synthesis or action of LXA4, a potent anti-inflammatory metabolite of AA, is decreased. Thus, in cancer wherein an increase in the synthesis and release of PGE2 is known to occur (especially by tumor cells themselves, T cells, NK cells, macrophages, and dendritic cells), one would expect the levels to decrease or remain the same of LXA4 such that the balance is tilted more toward pro-inflammatory PGE2. Such a situation would cause immunosuppression and escape of the tumor cells from the immunosurveillance system rendering tumor cells to grow. It is

LXA4 Versus PGE2 in Cancer

25

paradoxical that both PGE2 and LXA4 are derived from the same precursor AA. Thus, the enzymes and cofactors that control the formation of PGE2 and LXA4 from AA are critical to the development or arrest of cancer. This proposal is supported by our recent studies where we found that LXA4 has growth inhibitor actions on tumor cells, whereas PGE2 does not have [198– 200]. What is interesting is that these studies showed that PUFAs (especially AA, EPA, DHA, and GLA) can selectively induce apoptosis of tumor cells with little action on normal cells by enhancing the accumulation of lipid peroxides specifically in tumor cells that, in turn, seem to suppress the expression of Ras, Myc, and Fos oncogenes. In addition, it was also found that both COX and LOX inhibitors are not able to suppress the pro-apoptotic action of PUFAs implying that PGs, LTs, and TXs do not have any role in the anti-cancer action of PUFAs. Thus, it appears as though tumor cells have evolved a mechanism to convert PUFAs to PGE2 and other eicosanoids (but not into LXA4) as an escape mechanism to prevent formation of lipid peroxides from the PUFAs. Furthermore, tumor cells have low amounts of PUFAs and relatively large amounts of antioxidants compared to normal cells that further prevent formation of lipid peroxides. These results suggest that tumor cells have cleverly fine-tuned their PUFA metabolism to form excess of PGE2 to suppress immune response against them, form low amounts of LXA4 that have growth inhibitory action on tumor cells, and form low amounts of lipid peroxides to escape from the pro-apoptosis action of peroxides [19, 20, 30, 185, 190, 191, 198–200]. In this context, it is interesting that tumor cells form relatively low amounts of LXA4, higher amounts of PGE2, and low amounts of lipid peroxides compared to normal cells. The sources of PGE2, LXA4, and lipid peroxides are tumor cells themselves, T cells, NK cells, macrophages, dendritic cells, and tumor milieu. It is pertinent to note that the cytolytic granules released by NK cells and other cells contain significant amounts of lipids (probably PUFAs) that can induce apoptosis of tumor cells [201]. Furthermore, studies showed that drug-tolerant tumor cells can be rendered sensitive to chemotherapeutic drugs by blocking GPX4 and that efforts made to enhance lipid peroxidation induce ferroptosis/apoptosis of tumor cells [202–204]. These results indicate that lipid peroxidation is the ultimate pathway by which tumor cells undergo apoptosis or ferroptosis [204]. Based on these evidences, it is proposed that PUFA metabolism has a central role in the pathobiology of cancer, and modulating their metabolism could form a novel approach to cancer therapy in the future (see Fig. 1.6). Further discussion about the role of PUFAs in cancer is discussed in Chap. 3. These results also indicate that a combination of immune checkpoint inhibitors and PUFAs may form a novel method of tackling cancer with a few side effects (see Fig. 1.6, [195–210]). This is further supported by the recent observation that immune checkpoint inhibitors enhance lipid peroxidation in tumor cells [206].

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1  Introduction to Genes, Oncogenes, and Anti-oncogenes Normal cell

Tumor cell

PUFAs ↔

PUFAs↓↓

PGE2 ↔LXA4 (1:1)

PGE2↑↑

Lipid peroxides ↔

Lipid Peroxides↓

Vitamin E ↔Lipid peroxides (1:1)

Vitamin E↑ (Vit E: LP ratio↑)

Desaturases ↔(N)

Desaturases↓

COX vs LOX enzymes ↔

COX-2↑↑; LOX↓

PUFAs Lactate-

Glucose Plasma membrane

Nucleotides NADPH

GSH-mediated antioxidant defense

GLUT1,3,4

NHE1

MCT4

2 Pyruvate

CPT1A

H+ Na+

Glucose HK1,2 2ADP (at VDAC) 2NAD* PPP G6P TLK1 2ATP PFK2 2NADH Aldolase Lactate- H+ PGM LDH-A M2-PK

PUFAs

Fatty acids

H+

NAD+

Amino acids

V-type H+ATPase F1F0 ATPase CA 9, 12

LAT1

Amino acids

Proteins

IsoCholesterol prenoids

Fatty acids

H+ FASN Mevalonate

Malonyl-CoA

PUFAs NADH PDK1 Acetyl-CoA ACL PDH Acetyl-COA Citrate Citrate Pyruvate OAA Malate Malate OAA b-oxidation

NADH

NAD+

NAD+

PUFAs

NADH

FADH2 OXPHOS Mitochondrion

Fig. 1.6 (a) Scheme showing differences in the metabolism of PUFAs in normal and tumor cells. For more reading, see Ref. [190–204]. (b) Metabolic reprogramming seen in cancer cells and instances where PUFAs can modulate the same to inhibit their growth/induce apoptosis. Blue arrows indicate the instances wherein PUFAs act to interrupt the altered metabolism of glucose seen in cancer cell. (For more reading, see Ref. [190–205]) These changes in the PUFA metabolism ultimately results in tumor cells having high PGE2, low LXA4, relatively high vitamin E content, and relatively low levels of lipid peroxides resulting in increased cell proliferation. Thus, cancer cells are vulnerable to apoptosis if their lipid peroxide content can be increased by supplementing with PUFAs (especially AA and GLA) and/or decrease the activity of GPX4 and other antioxidants.. (b) In normal cells, one glucose molecule is metabolized via pyruvate, acetyl-CoA,

LXA4 Versus PGE2 in Cancer

27

PUFAs

GLUT1 GLUT4 HK1/2 HK/VDAC interact. PFK PGM PKM2 LDHA MCT4 CA9, 12 NHE1 PDK SCO2 ACL FASN ChoK CPT1A

RTK NF1

Ras

PI3K PTEN

PUFAs

LKB1

AMPK

Nutrient depletion

TSC1/2

PIP3

Akt

Myc

RhebGTP

OXPHOS defects

mTOR PUFAs

ROS

p53 defect

PUFAs

SDH

PHDs

FH

Oncogenesis

VHL

HIF1

Transformation

Glucose transport

Glycolysis

Lactate productiom and lactate/proton extrusion Reduced OXPHOS Lipid synthesis Inhibited β-oxidation

Metabolic reprogramming

Fig. 1.6  (continued) TCA (tricarboxylic acid) cycle, and oxidative phosphorylation to CO2 and H2O generating 38 ATP molecules. In contrast, tumor cells’ glucose undergoes aerobic glycolysis (glucose converted into pyruvate which generates 2 ATP molecules and subsequently into lactate). In tumor cells, acetyl-CoA enters a truncated TCA cycle to be exported to cytosol to serve as the building block for cell growth and proliferation. Citrate that enters the truncated TCA cycle enters the cytosol via the tricarboxylate transporter that is cleaved by the ATP citrate lyase (ACL) to give rise to oxaloacetate and acetyl-CoA.  Oxaloacetate is reduced to malate and then reimported to mitochondria to be reconverted to oxaloacetate during which it generates NADH that represses the TCA cycle, and it (oxaloacetate) reacts with acetyl-CoA to complete the substrate cycle. Small arrows (up or down) indicate cancer-associated upregulation or downregulation/inhibition of enzymes, respectively. Highlights indicated in red are due to the activation of HIF-1 (hypoxiainducible factor-1). CA9 and CA12 Carbonic anhydrases 9 and 12, CPT Carnitine palmitoyltransferase, GLUT Glucose transporter, GSH Glutathione, IDO Indoleamine 2,3-dioxygenase, HK Hexokinase, OXPHOS Oxidative phosphorylation, LAT1 L-type amino acid transporter 1, LDHA Lactate dehydrogenase isoform, MCT Monocarboxylate transporter, PDH Pyruvate dehydrogenase, PDK Pyruvate dehydrogenase kinase, PFK Phosphofructokinase, PI3K Phosphatidylinositol 3-kinase, PGM Phosphoglycerate mutase, PKM2 Pyruvate kinase isoform M2, PPP Pentose phosphate pathway, SCO2 Synthesis of cytochrome c oxidase 2, TLK Transketolase, VDAC Voltagedependent anion channel. (c) Molecular mechanisms responsible for cancer-specific metabolic reprogramming and instances wherein these mechanisms could be acted upon by PUFAs to inhibit cancer cell growth/apoptosis. Blue arrows indicate the instances wherein PUFAs act to suppress cancer cell growth/metabolism. For more information, see Ref. [190–205]. As a result of oncogenic gain of function (pink boxes) or the loss of tumor suppressors (green boxes), PI3K/Akt/ mTOR/HIF axis activation and/or inactivation of the p53 system occurs. This results in a change in the metabolism of cancer cells. ACL ATP citrate lyase. AMPK AMP-activated kinase, CA9 and 12 Carbonic anhydrases 9 and 12, ChoK Choline kinase, CPT Carnitine palmitoyltransferase, FH Fumarate hydratase, GLUT Glucose transporter, HIF Hypoxia-inducible factor, HK Hexokinase, OXPHOS Oxidative phosphorylation, LAT1 L-type amino acid transporter, LDHA Lactate

28

1  Introduction to Genes, Oncogenes, and Anti-oncogenes

PUFAs

Akt

HK-VDAC association

Glycolysis

mTOR PI3K Anabolic reactions

Inhibited OXPHOS PUFAs

Selfsufficiency Evading apotosis in growth signals

VEGF HIF-1

Avoidance of immunosurveillance

Sustained angiogenesis

Ang-2

Kynurenins PUFAs

Lactate

PUFAs

Limitless replicative potential

F1F0ATPase

Intensitivity to antigrowth signals

Tissue invasion and metastasis

Inhibited OXPHOS

Oxygen independence

p53

SCO2

PGM Glycolysis

Extracelluar acidification

Proton extrusion

Anaerobic glycolysis

Lactate

HIF-1

PUFAs

Fig. 1.6 (continued) dehydrogenase isoform A, MCT Monocarboxylate transporter, mTOR Mammalian target of rapamycin, NF Neurofibromin, PDK Pyruvate dehydrogenase kinase, PFK Phosphofructokinase, PI3K Phosphatidylinositol 3-kinase, PIP3 Phosphatidylinositol triphosphate, PGM Phosphoglycerate mutase, PHD Propyl hydroxylase, PKM2 Pyruvate kinase isoform M2, SCO2 Synthesis of cytochrome c oxidase, TSC Tuberous sclerosis complex, VDAC Voltagedependent anion channel, VHL von Hippel-Lindau ubiquitin ligase. (d) The chief characteristics of cancer cells and their links to the altered tumor metabolism as known and instances where PUFAs can interfere with to inhibit tumor cell growth/induce their apoptosis. Blue arrows indicate the instances wherein PUFAs act to interrupt the altered metabolism and suppress cancer cell growth/induce apoptosis/ferroptosis. (b, c, and d) are taken from Ref. [205] and modified. For further information, see Ref. [190–205]. Ang-2 Angiopoietin-2, GLU Glucose transporter, HIF Hypoxia-inducible factor, HK Hexokinase, OXPHOS Oxidative phosphorylation, PGM hosphoglycerate mutase, PI3K Phosphatidylinositol 3-kinase, SCO2 Synthesis of cytochrome c oxidase, VDAC Voltage-­dependent anion channel, VEGF Vascular endothelial growth factor

References

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222. Das UN. Lipoxins, resolvins, protectins, maresins and nitrolipids and their clinical implications with specific reference to cancer: part I. Clin Lipidol. 2013;8:437–63. 223. Das UN. Lipoxins, resolvins, protectins, maresins and nitrolipids and their clinical implications with specific reference to diabetes mellitus and other diseases: part II.  Clin Lipidol. 2013;8:465–80. 224. Krishnamoorthy N, Burkett PR, Dalli J, Abdulnour RE, Colas R, Ramon S, Phipps RP, Petasis NA, Kuchroo VK, Serhan CN, Levy BD. Cutting edge: maresin-1 engages regulatory T cells to limit type 2 innate lymphoid cell activation and promote resolution of lung inflammation. J Immunol. 2015;194:863–7. 225. Serhan CN, Chiang N, Dalli J, Levy BD. Lipid mediators in the resolution of inflammation. Cold Spring Harb Perspect Biol. 2014;7:a016311. 226. Faragó N, Fehér LZ, Kitajka K, Das UN, Puskás LG. MicroRNA profile of polyunsaturated fatty acid treated glioma cells reveal apoptosis-specific expression changes. Lipids Health Dis. 2011;10:173. 227. Siddesha JM, Valente AJ, Yoshida T, Sakamuri SS, Delafontaine P, Iba H, Noda M, Chandrasekar B. Docosahexaenoic acid reverses angiotensin II-induced RECK suppression and cardiac fibroblast migration. Cell Signal. 2014;26:933–41. 228. Ghosh-Choudhury T, Mandal CC, Woodruff K, St Clair P, Fernandes G, Choudhury GG, Ghosh-Choudhury N.  Fish oil targets PTEN to regulate NFkappaB for downregulation of anti-apoptotic genes in breast tumor growth. Breast Cancer Res Treat. 2009;118:213–28. 229. Vasudevan A, Yu Y, Banerjee S, Woods J, Farhana L, Rajendra SG, Patel A, Dyson G, Levi E, Maddipati KR, Majumdar AP, Nangia-Makker P. Omega-3 fatty acid is a potential preventive agent for recurrent colon cancer. Cancer Prev Res (Phila). 2014;7:1138–48. 230. Das UN. Essential fatty acids and their metabolites as modulators of stem cell biology. Agro Food Ind Hi Tech. 2010;21:2–3. 231. Das UN. Influence of polyunsaturated fatty acids and their metabolites on stem cell biology. Nutrition. 2011;27:21–5. 232. Das UN. Essential fatty acids and their metabolites as modulators of stem cell biology with reference to inflammation, cancer and metastasis. Cancer Metastasis Rev. 2011;30:311–24. 233. Fillmore N, Huqi A, Jaswal JS, Mori J, Paulin R, Haromy A, Onay-Besikci A, Ionescu L, Thébaud B, Michelakis E, Lopaschuk GD. Effect of fatty acids on human bone marrow mesenchymal stem cell energy metabolism and survival. PLoS One. 2015;10:e0120257. 234. Katakura M, Hashimoto M, Okui T, Shahdat HM, Matsuzaki K, Shido O. Omega-3 polyunsaturated fatty acids enhance neuronal differentiation in cultured rat neural stem cells. Stem Cells Int. 2013;2013:490476. 235. Lee SH, Kim MH, Han HJ. Arachidonic acid potentiates hypoxia-induced VEGF expression in mouse embryonic stem cells: involvement of notch, Wnt, and HIF-1alpha. Am J Physiol Cell Physiol. 2009;297:C207–16. 236. Uversky VN, et al. Pathological unfoldomics of uncontrolled chaos: intrinsically disordered proteins and human diseases. Chem Rev. 2014;114:6844–79.

Chapter 2

Introduction to Free Radicals, Antioxidants, Lipid Peroxidation, and Their Effects on Cell Proliferation

Abstract  There is evidence to suggest that reactive oxygen species (ROS) have a role in cancer. ROS have important functions both in physiological processes pathological events. Hence, it is believed that balance between ROS and antioxidants is critical to cell health. ROS can damage DNA, produce mutations, and, thus, can have a role in carcinogenesis. It is paradoxical that the same ROS may also have a role in bringing about the actions of radiation, chemotherapeutic drugs, and immunocytes on cancer cells. ROS also have a role in inflammation and immune response. Hence, modulating the ROS-antioxidant balance can be exploited in the prevention and management of cancer. In this chapter, a brief background about ROS is given. Keywords  Reactive oxygen species · Free radicals · Antioxidants · Nitric oxide · Cytokines

Introduction It is desired that anti-cancer agents kill only tumor cells with little effect(s) on normal cells that is rarely achieved by the current modalities of therapy that include radiation and chemotherapy. Radiation and several chemotherapeutic drugs including doxorubicin and bleomycin bring about their tumoricidal action by augmenting free radical generation [1–3]. These modalities of therapy induce generation of significant amounts of free radicals even in normal cells that are responsible for various side effects seen in patients with cancer. Hence, efforts need to be made to generate free radicals specifically only in the tumor but not normal cells such that only tumor cells are eliminated with no action on normal cells, and hence, there will not be significant side effects seen in the patients. Free radicals (including superoxide anion, hydroxyl radical, hydrogen peroxide, carbon monoxide, and nitric oxide) also referred to as reactive oxygen species (ROS) have significant role and influence on cell growth and development, metabolism, and several physiological cellular functions. ROS have both positive and negative impact on tumor cells as well [4–6]. During the process of ATP generation by © Springer Science+Business Media, LLC, part of Springer Nature 2020 U. N. Das, Molecular Biochemical Aspects of Cancer, https://doi.org/10.1007/978-1-0716-0741-1_2

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2  Introduction to Free Radicals, Antioxidants, Lipid Peroxidation, and Their Effects…

mitochondria that involves electron transport steps, free radicals are generated due to leakage of electrons. Superoxide anion (O2-.) and hydroxyl (OH.) radical generated can lead to the formation/generation of hydrogen peroxide (H2O2) that, in turn, in the presence of Fe2+ions lead to the formation of more hydroxyl radicals [7]. ROS have both harmful and beneficial actions (reviewed in 4) depending on their concentration (lower concentrations may have physiological actions, whereas high levels may be toxic to cells and produce apoptosis), context, source of origin, and the target tissues/cells. ROS participate in signal-transduction pathways that have a regulatory role in cell growth and functions [8, 9] and various physiological reduction-­oxidation (redox) reactions ([7], see Fig. 2.1 outlining metabolism of arachidonic acid in which ROS are generated during the formation of its various

PGE2

PGF2

PGH2

PGE2 synthase

EET

HETE

PGI2

PGD2

ROS

TXA2

ROS

ROS Cytochrome P450

COX-1 (constitutional COX-2 (inducible)

ROS

12SHETE

Arachidonic Acid (AA, 20:4 n-6) 5-LOX

12/15LOX

ROS

8-LOX

15SHETE

5SHETE ROS

ROS

5-LOX

8SHETE

p12LOX

5- LOX PGE2 synthase

LXA4

LTA4

ROS

ROS

LTA4 Hydr olase

12/15LOX

12SHETE 15-oxo-HETE

LTC4 Hydrolase

ROS

LXB4 LTE4

LTD4

ROS

LTB4

LTC4

Fig. 2.1  Scheme showing metabolism of arachidonic acid (AA, 20:4 n-6) by cyclooxygenase, lipoxygenase, and P450 enzymes leading to the formation of its various products. Both eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (22:6 n-6) also follow the same metabolic pathways and form similar products

Free Radicals

43

products); ROS also serve as the first line of defense against various infections by polymorphonuclear leukocytes and macrophages [4]. These physiological functions of free radicals are generally performed by their physiological concentrations. In contrast, high or excessive amounts of free radicals are toxic and inactivate vital enzymes, proteins, and other subcellular elements that are needed for cell survival. As a result of these toxic actions, ROS may cause apoptosis of cells [4, 10–12]. ROS damage to DNA and various cell membranes including cell membrane, nuclear membrane, and mitochondrial membrane and lead to leakage of contents of various cell organelles resulting in apoptosis and other means of cell death including necrosis, ferroptosis, etc. In this context, it is noteworthy that selective generation of free radicals in tumor cells without little change in the intracellular concentrations of free radicals in normal cells may form a new approach to selectively kill tumor cells. Such an approach is a feasible strategy provided molecules either endogenous or exogenous that can selectively enhance free radical generation in tumor cells are identified to induce their apoptosis. There is now reasonable evidence available that such an approach is possible. To develop such an approach wherein free radicals are selectively enhanced in tumor cells such that they undergo apoptosis, one need to understand the physics, chemistry, and biology of free radicals. Free radicals (ROS) induce cell death by leading to the accumulation of toxic lipid peroxides. Free radicals react with polyunsaturated fatty acids (PUFAs) present in the phospholipids of the cell membranes leading to form toxic lipid peroxides and PUFA radicals that can produce apoptosis. It is noteworthy that in the presence of Fe2+, cells undergo ferroptosis, a form of cell death. Ferroptosis is especially seems to be a dominant form of cell death when F2+is available in free form.

Free Radicals In general, a free radical is an atom, molecule, or ion that has an unpaired electron that renders free radicals highly chemically reactive that are reasonably stable only at low concentrations in inert media or in a vacuum. Most of the free radicals have one or more “dangling” covalent bonds that actually render them highly reactive/ active. Examples of free radicals (ROS) include hydroxyl radical (HO•), superoxide anion (•O − 2), and nitric oxide (NO). The best method of generating free radicals is using ionizing radiation. In fact, free radicals are generated during several enzymatic reactions that occur under physiological conditions in the body and also during intermediate stages of many synthetic chemical reactions. One of the best examples of generation of free radicals in the body under physiological conditions is the formation of various prostaglandins (PGs) from their precursors such as arachidonic acid (AA) by the action of enzyme cyclooxygenase-2 (COX-2). Free radicals (such as superoxide anion and NO) can regulate vascular tone, neurotransmission, and cell mitosis.

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2  Introduction to Free Radicals, Antioxidants, Lipid Peroxidation, and Their Effects…

Free radicals are denoted by a dot placed immediately to the right of the atomic symbol or molecular formula as follows: UV



Cl 2 → Cl ⋅ +Cl ⋅

Chlorine gas can be broken down by ultraviolet light to form atomic chlorine radicals as shown above. Single-headed arrows are used to show the movement of single electrons in radical reaction mechanisms:



Free radicals participate in radical addition and radical substitution reactions as reactive intermediates. Chain reactions involving free radicals consist of three processes: initiation, propagation, and termination. These processes are self-­ explanatory as their names indicate.

Formation of Free Radicals Free radicals are formed due to breaking of covalent bonds, a process that needs energy. Radicals that need more energy to form are relatively less stable compared to those that need less energy.

Persistence and Stability of Free Radicals In general, free radicals are considered to be short-lived due to their high reactivity nature. But they can have significantly long periods and, hence, are categorized as stable radicals, persistent radicals, and diradicals. The best examples of stable radicals are the molecular dioxygen (O2) and nitric oxide (NO). Persistent radicals are those which, as a result of a steric crowding around the radical center are less likely to react with other molecules [4]. Examples of these include triphenylmethyl radical, TEMPO, etc. These are generated during

Combustion

45

combustion and are capable of causing oxidative stress. They seem to be involved in cardiopulmonary disease and cancer due to airborne fine particles.

Diradicals Diradicals are those that have two radical centers, and atmospheric oxygen is the best example of this (as shown below), and it has reactivity.

Free Radical Reaction A free radical reaction is any chemical reaction involving free radicals and is generally chain reactions that include an initiation step, propagation step, and a termination step. When two radicals react to form two different non-radical products, it is called as radical disproportionation. Since radical molecules are unstable, disproportionation proceeds rather rapidly and needs no or very little energy. Radical reactions can occur both in the gas phase and solution phase [5]. Free radicals generally attack double bonds so that the resulting α-radical carbonyl is stable and is capable of coupling with another molecule or be oxidized. Despite the extreme nature of reactivity of radicals, their precise control is possible. As a rule, radicals attack the closest reactive site though certain exceptions are possible.

Combustion Combustion is a common method of free radical reaction that depends on the concentration of free radicals. Complete consumption of the combustible material results in termination of the reactions, a stage at which the flame dies out. Flammability can be altered by promotion of propagation or termination reactions. Burning of the hydrocarbon involves a large number of different oxygen radicals in which, to start with, hydroperoxyl radical (HOO·) is formed that reacts further to generate hydroperoxides that break up into hydroxyl radicals (HO·).

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2  Introduction to Free Radicals, Antioxidants, Lipid Peroxidation, and Their Effects…

Polymerization Many polymerization reactions are due to free radicals, and many plastics, enamels, and other polymers are formed through this reaction. Radical cross-linking as a result of atmospheric oxygen leads to hardening of drying oils and alkyd paints.

Radicals Present in the Atmosphere Molecular oxygen is the most common radical in the atmosphere. Photodissociation of nitrogen dioxide into an oxygen atom and nitric oxide (see Eq. 2.1 below) is one example of this reaction that plays a key role in smog formation. The photodissociation of ozone gives rise to the excited oxygen atom O(1D) (see Eq. 2.2 below). The net and return reactions are also shown in Eqs. 2.3 and 2.4. hv



NO2 → NO + O

(2.1)



O + O 2 → O3

(2.2)



NO2 + O2 → NO + O3

(2.3)



NO + O3 → NO2 + O2

(2.4)

hv

Biological Significance of Free Radicals Free radicals have many biological and pathological processes. For instance, free radicals are needed for the intracellular killing of bacteria by leukocytes and macrophages. In addition, free radicals play a critical role in cell signaling processes [6, 7] called as redox signaling. Superoxide, hydroxyl radical, and nitric oxide (NO) are the three most important free radicals present in the body. Both superoxide anion and hydroxyl radical are derived from the molecular oxygen under reducing conditions, whereas NO is formed from combustion of petrochemicals, exhausts from the motor vehicles and in the body from the endothelial cells and macrophages, leukocytes, and dendritic cells. Thus, free radicals are present both in the atmosphere and in the body. NO was known to be present in the atmosphere since the 1930s and considered as

Biological Significance of Free Radicals

47

atmospheric pollutant and harmful chemical. NO is present in combustion of substances in the air, as in automobile engines, fossil fuel power plants, and is produced naturally during the electrical discharges of lightning in thunderstorms. This view changed dramatically after its (NO) presence was noted in the human body synthesized and secreted mainly by the vascular endothelial cells. Subsequent studied showed that NO is an important vasodilator, neurotransmitter, and memory molecule [8–10]. In addition, in view of its radical nature, NO can participate in unwanted side reactions that can cause cell damage. Excessive amounts of these free radicals including NO can lead to cell injury and death. Thus, excess of ROS (including NO) can participate in the pathobiology of cancer, stroke, myocardial infarction, diabetes mellitus, Alzheimer’s disease, cirrhosis of the liver, and various rheumatological conditions [6–29]. In general, it is believed that many forms of cancer are as a result of alterations in cellular DNA due to its interaction/reaction with free radicals leading to cell cycle changes and carcinogenesis. Some age-related diseases such as atherosclerosis, coronary heart disease, diabetes mellitus, and Alzheimer’s disease have also been linked to inappropriate generation and action of free radicals. Free radical-induced oxidation of cholesterol to 7-ketocholesterol [30, 31] may have a role in atherosclerosis. Free radicals have a contributory role in alcohol-induced liver damage. Free radicals produced by cigarette smoke can inactivate alpha-1 antitrypsin in the lung leading to the development of emphysema. It is believed that free radicals also have a role in the pathobiology of aging process. It is thought that continuous exposure to gradually increasing concentrations of various free radicals without the much-needed generation of relevant antioxidants may lead to inactivation of cellular enzymes, proteins, and other essential cellular elements leading to aging. At the same time, it is also being recognized that repeated exposure to optimal amounts of free radicals may render the human cells/ tissues to generate adequate amounts of antioxidants and other defense systems such that it will help the body to live longer with good health [32–40]. It is also interesting to note that hematopoietic stem cells (HSCs) that are needed for hematopoietic homeostasis and regeneration for the entire lifespan are also affected by ROS. ROS function as signal molecules to regulate several cellular functions, including proliferation, differentiation, and mobilization, and so any inappropriate increase in ROS production can result in inhibition of HSC selfrenewal and induce HSC senescence and consequent hematopoietic dysfunction. In particular, there appears to be significant role for the p38 mitogen-activated protein kinase (p38)-p16(Ink4a) (p16) pathway in ROS-induced HSC senescence [38]. In view of the toxic nature of free radicals, our tissues are endowed with several defense mechanisms such as superoxide dismutase (SOD), catalase, glutathione peroxidase, and glutathione reductase that can quench or neutralize them. In addition, our diet contains several antioxidant vitamins such as vitamin A, vitamin C, and vitamin E. Furthermore, bilirubin and uric acid also have antioxidant actions.

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2  Introduction to Free Radicals, Antioxidants, Lipid Peroxidation, and Their Effects…

I dentification and Diagnostic Techniques to Detect Free Radicals Techniques that could be used to detect free radicals include: (i) Electron spin resonance spectroscopy (ii) Nuclear magnetic resonance spectroscopy using a phenomenon called as chemically induced dynamic nuclear polarization (iii) Chemical labeling by DPPH (2,2-diphenyl-1-picrylhydrazyl), followed by spectroscopic methods like X-ray photoelectron spectroscopy (XPS) or absorption spectroscopy (iv) Measurement of lipid peroxidation products such as isoprostanes using thiobarbituric acid reactive substances (TBARS), amino acid oxidation products (meta-tyrosine, ortho-tyrosine, hydroxy-Leu, dityrosine, etc.), peptide oxidation products (oxidized glutathione–GSSG), and 2,2'-Azobis(2-­ amidinopropane) dihydrochloride (AAPH), a compound that can serve as free radical-generating azo compound. Certain indirect methods also can be employed to know the status of ROS, and one such method is to measure the decrease in the amounts of antioxidants such as SOD, catalase, and glutathione. Yet another method is to use trapping agents that can react with free radicals to form a stable product(s) that can then be readily measured (hydroxyl radical and salicylic acid). In the light of the various actions of ROS/free radicals in several physiological and pathological processes, it is pertinent to discuss in detail about the importance and actions of NO.

Nitric Oxide (NO) Nitric oxide (NO) is a free radical. NO is an important cellular signaling molecule participating in several physiological and pathological processes. It is a potent vasodilator with a short half-life of a few seconds in the circulation. The well-known age old drug nitroglycerin (NTG) and amyl nitrite traditionally used in the management of malignant hypertension and cardiac failure have been found to be precursors/ donors of NO. Both NTG and amyl nitrite and their derivatives are also used in the prevention and management of angina pectoris. These two drugs produce dilatation of coronary vessel and, thus, relieve pain in the chest due to cardiac ischemia. Constitutive generation of low levels of NO is essential to maintain the lumen of blood vessels, protecting organs such as the liver from ischemic damage. NO can react with several other molecules such as oxygen, fluorine, chlorine, iodine, and bromine to form respective products. Nitric oxide reacts with acetone and an alkoxide to form a diazeniumdiolate or nitroso-hydroxylamine and methyl acetate as shown below:

Methods of Preparation of NO

O

49

NO MeONa

Na

O N

O

O

N

N

MeONa N O

Na

O



Na

O N

O

O

N

N

O N O

O

Na

The importance of this reaction lies in the fact that this could be exploited in the development NO prodrugs.

Methods of Preparation of NO NO can be produced on a commercial scale by the oxidation of ammonia at 750–900 °C (normally at 850 °C) with platinum as catalyst.

4 NH 3 + 5O2 → 4 NO + 6 H 2 O

In the laboratory, NO can be generated by reduction of dilute nitric acid with copper:



8 HNO3 + 3Cu → 3Cu ( NO3 )2 + 4 H 2 O + 2 NO



or by the reduction of nitrous acid in the form of sodium nitrite or potassium nitrite:

2 NaNO2 + 2 NaI + 2 H 2 SO 4 → I 2 + 4 NaHSO 4 + 2 NO



2 NaNO2 + 2 FeSO 4 + 3H 2 SO 4 → Fe 2 ( SO 4 )3 + 2 NaHSO 4 + 2 H 2 O + 2 NO



3KNO2( l ) + KNO3( l ) + Cr2 O3(s) → 2 K 2 CrO 4(s) + 4 NO( g )





It is possible to generate NO using NONOate compounds. NO reacts with all transition metals to give complexes called metal nitrosyls.

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2  Introduction to Free Radicals, Antioxidants, Lipid Peroxidation, and Their Effects…

Methods of Measurement of NO NO concentration can be measured using a simple chemiluminescent reaction involving ozone [41], electroanalysis (amperometric approach) in which NO reacts with an electrode and produces a voltage change. NO present in biological tissues can be detected as its more stable metabolite nitrite. Spin trapping of NO with iron-­ dithiocarbamate complexes and subsequent detection of the mono-nitrosyl-iron complex with electron paramagnetic resonance (EPR) can also be used to measure NO [42, 43]. Another method of measuring intracellular NO is using the fluorescent dye 4, 5-diaminofluorescein (DAF-2) [44].

Biological Functions/Significance of NO Despite being a free radical (that are, in general, believed to be harmful), NO is one of the few gaseous signaling molecules; {others are hydrogen sulfide and carbon monoxide (CO). All three gases, NO, H2S, and CO, have anti-inflammatory and vasodilator actions. NO has a role in many cellular processes and is present in bacteria, plants, fungi, and animal cells. NO, the endothelium-derived relaxing factor or “EDRF,” is derived from the semi-essential amino acid, L-arginine, by the action of NO synthase enzyme. Reduction of inorganic nitrate may also give rise to NO. NO derived from endothelial cells relaxes smooth muscle and, thus, causes vasodilation to increase blood flow and reduce blood pressure. Thus, NO plays a critical role in the pathobiology of hypertension. In contrast to this, in sepsis low blood pressure or hypotension is due to excess production of NO.  NO is highly reactive and capable of diffusing across cell membranes rather easily and freely, which makes it (NO) to function as a paracrine and autocrine molecule. Nitrate-nitrite-nitric oxide pathway can elevate NO through the sequential reduction of dietary nitrate derived from plant-based foods [45–48] and is independent of NO synthase. Nitrate-rich leafy vegetables increase NO with a corresponding reduction in blood pressure in prehypertensive persons [49, 50]. Generation of NO through the nitrate-nitrite-nitric oxide pathway needs reduction of nitrate to nitrite in the mouth by commensal bacteria [51]. A rise in salivary levels of NO is indicative of consumption of leafy vegetables. The production of NO is elevated in those who are living at high altitudes that enable them to avoid pulmonary edema due to hypoxia as a result of its vasodilatory actions. Other well-known actions of NO include neurotransmission, modulation of the hair cycle [52] and production of reactive nitrogen intermediates. Of all the actions of NO that is well-known is its ability to produce penile erection. Hence, NO donors or drugs that enhance NO generation are widely used for the treatment of erectile dysfunction. Nitroglycerin and amyl nitrite release NO and, thus, bring about their vasodilator actions. Minoxidil contains a NO moiety, and, thus, it serves as an NO agonist. Sildenafil citrate stimulates erections by enhancing signaling

Biological Functions/Significance of NO

51

through the NO pathway. Sildenafil citrate is also useful in other conditions such as pulmonary hypertension and preeclampsia [53–56]. The beneficial action of NO in hypertension, preeclampsia, and coronary heart disease may be due to its ability to relax vascular smooth muscles, prevent platelet aggregation, and block leukocyte adhesion to the endothelium. This is supported by the observation that subjects with atherosclerosis, type 2 diabetes mellitus, and hypertension have impaired NO generation or impaired NO action [57–61]. It was reported that subjects with hypertension also have insulin resistance, and both of these defects seem to be due to NO deficiency suggesting that endothelial NO is needed for peripheral insulin action [60]. In fact, it was noted that impaired endothelial NO generation is common in essential hypertension [62, 63]. The decreased NO seen in hypertension and type 2 diabetes mellitus are due to: (i) Deficiency of dietary L-arginine, the precursor of NO (ii) Impaired NOS activity (iii) Presence of inhibitors of NO synthesis such as asymmetrical dimethyl arginine (ADMA) (iv) Excess production of superoxide anion that can inactivate NO (v) Decreased half-life of NO (vi) Deficiency of cofactors needed for NO synthesis such as folic acid, vitamin C, insulin deficiency or resistance, and/or tetrahydrobiopterin (H4B) (vii) Deficiency of polyunsaturated fatty acids (PUFAs) arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), gamma-linolenic acid (GLA), and dihomo-gamma-linolenic acid (DGLA) that possess the ability to activate NOS and, thus, enhance NO generation and/or serve as precursors to the generation of known or unknown vasodilators [23, 25, 64–89]. It is known that supplementation of GLA, DGLA, EPA, and DHA could reduce hypertension in patients and experimental animals, and their beneficial actions have been linked to their ability either directly or indirectly induce generation of NO [23, 25, 64–93]. These results emphasize the close relationship and interaction between PUFAs and NO.  The purported role of various cofactors that are needed for NO synthesis and/or inhibition such as vitamin C, folic acid, H4B and ADMA, and superoxide anion reiterates the complexities involved in the pathobiology of hypertension. Since hypertension is associated with insulin resistance (both peripheral and central hypothalamus), it also suggests that same factors that are involved in its pathobiology may have a role in insulin resistance. This implies that obesity, type 2 diabetes mellitus, Alzheimer’s disease, depression, schizophrenia, and dementia that show insulin resistance and are low-grade systemic inflammatory conditions similar to hypertension may also have abnormalities in NO generation and action. Furthermore, all these conditions show abnormalities in the metabolism of PUFAs [94–100]. Thus, at the cellular level, all these conditions have similar molecular abnormalities implying that their prevention and management could employ similar strategies such as enhancing NO generation; restoring PUFAs metabolism to normal; and/or resetting the inflammatory pathway to normal physiological level (see Fig.  2.2). These evidences suggest that for all practical purposes,

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2  Introduction to Free Radicals, Antioxidants, Lipid Peroxidation, and Their Effects…

n-6

n-3

Diet

Cis-LA, 18:2

α-ALA, 18:3 Δ Desaturase 6

GLA, 18:3 Vitamin C, B6, folic acid, insulin, glucose, Mg2+, B12, etc.

DGLA, 20:3 Δ5 Desaturase

PGs of 1 series AA, 20:4

EPA, 20:5

DHA, 22:6

Vascular Endothelial cells, PMNs, MФ, Dendritic cells, Kupffer cells, etc.

PGs of 2 series, TXs, LTs of 4 series

PGs of 3 series, TXs, LTs of 5 series

Lipoxins, Resolvins, Protectins, Maresins NO, PGI2, PGI3 Pro-inflammatory in Nature

Proinflammatory Cytokines IL-6, TNF-α, HMGB1

ROS

Anti-inflammatory in Nature

Anti-inflammatory Cytokines IL-4, IL-10, IL-12

Restoration of homeostasis LXR, FXR, RAR-RXR, Syntaxin, PPARs, eNO, Ras, GTPases, SREBPs, HMG-CoA reductase, NF-κB, UCPs, Phospholipases, ROS, Antioxidants, Cytokines, Neurotransmitters, Growth factors, cytokeratins, various genes, oncogenes and anti-oncogenes

Fig. 2.2  Scheme showing metabolism of essential fatty acids, their role in inflammation, and cytoprotection of endothelial cells

L-arginine-NOS-NO pathway, metabolism of PUFAs, inflammatory and anti-­ inflammatory cytokines, and insulin and its related PI3-K/Akt/eNOS signaling pathways behave as one self-regulated system to maintain homeostasis. In this context, it is known that high salt intake-induced hypertension is also due to decrease in NO production [101, 102] in patients with essential hypertension. In fact, our studies showed that in patients with uncontrolled hypertension, there is

NO in Inflammation, Immune Response, and Cancer

53

increased generation of free radicals including superoxide anion, and concomitantly there is reduced production of NO and a decrease in antioxidants [66]. These abnormalities reverted to normal after control of hypertension with various antihypertensive drugs suggesting that majority of antihypertensive medicines possibly enhance NO generation and reduce superoxide anion production. Thus, antihypertensive drugs may behave like antioxidants and/or have antioxidant actions.

NO in Inflammation, Immune Response, and Cancer NO is generated by phagocytes (monocytes, macrophages, and neutrophils) as part of the human immune response [103–105]. Phagocytes possess inducible nitric oxide synthase (iNOS), which is activated by interferon-γ (IFN-γ). On the other hand, TNF-α and other cytokines, generally, need IFN-γ to signal the production of NO or a combination of TNF, and ILs are necessary to induce iNOS activity [106– 109]. It is interesting to note that transforming growth factor-β (TGF-β), a known anti-inflammatory cytokine, inhibits iNOS, whereas interleukin-4 (IL-4) and IL-10 are much weaker inhibitory signals. Thus, there is a close interaction between various cytokines, and NO generation that show either enhancing or inhibitory influence on iNOS activity: pro-inflammatory molecules enhancing NO generation, whereas anti-inflammatory cytokines suppress its generation [110–118]. This positive and negative feedback regulation among T cells, PMNs, macrophages, pro- and anti-inflammatory cytokines, and NO shows the way the immune system may regulate itself for the benefit of the body to limit inflammation and control inappropriate immune response in response to infections and injury: at one end IFN-γ, IL-6, IL-1β, and TNF-α enhance free radical and NO generation so as to kill the invading bacteria, viruses, fungi, and intracellular parasites to induce inflammation and upregulate the immune response, and on the other hand, TGF-β, IL-4, IL-10, and IL-12 function as anti-inflammatory and immunosuppressive molecules to limit inflammation and suppress unwanted immune response. Though these cytokines are defined as promoters and suppressors of inflammation and immune response, it does not work in this fashion in all situations, implying that there are other molecules or downstream events that could alter their action and role in a given situation/ disease. Similarly, NO has both pro- and anti-inflammatory actions [119–126]. This is akin to the role of free radicals as both physiological signaling molecules and pro-­inflammatory and toxic chemicals in the body. This can be expanded to suggest that not just NO, oxygen-free radicals (or reactive oxygen species, ROS), and cytokines but several other physiologically existing endogenous molecules may have both beneficial and harmful actions depending on the site of their production, their concentration, and the tissue/cell that produces them. Thus, the same molecule may have both beneficial and harmful actions. Thus, both free radicals (ROS) and NO are needed to kill bacteria, intracellular parasites, and cancer cells, and their production could be induced by various cytokines, and their mechanism of action includes DNA damage [127, 128] and degradation of iron sulfur centers into iron ions and

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2  Introduction to Free Radicals, Antioxidants, Lipid Peroxidation, and Their Effects…

iron-nitrosyl compounds [129]. Another example of toxic action of NO is its involvement in ischemia-reperfusion injury wherein there is an excess of NO and ROS production during reperfusion. Superoxide reacts with NO to produce peroxynitrite that is toxic to tissues. On the other hand, inhaled NO is beneficial to recover from paraquat poisoning by quenching superoxide anion that can hinder NOS metabolism. Two important biological reaction mechanisms of NO include (i) S-nitrosation of thiols and (ii) nitrosylation of transition metal ions [130, 131].

NO Induces Apoptosis of Cancer Cells Macrophage-induced apoptosis of cancer cells is brought about by its ability to secrete NO. Sodium nitroprusside (SNP), which releases NO, showed a dose-­ dependent cytotoxic action on cancer cells by suppressing the expressions of c-myc mRNA and c-myb mRNA. These results indicate that NO has cytotoxic action on tumor cells via apoptosis by downregulation of c-myc and c-myb proto-oncogenes [132, 133]. These studies were further confirmed by the fact that a NO prodrug JS-K (O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-­1,2-­ diolate) induced apoptotic cell death of cancer cells by inactivating E1, a ubiquitin-­ activating enzyme. E1 activity induces apoptosis in p53-expressing transformed cells, possibly, due to the interaction between E1 with NO and due to an increase in p53 levels [134, 135]. These studies confirmed the apoptosis-inducing action of NO. In this context, it is interesting to note that (i) strain SL7838 of Salmonella typhimurium that kills cancer cells brings about its action by generating NO even under anaerobic conditions [136]; (ii) artesunate (ART), a semisynthetic derivative of antimalarial artemisinin, induced apoptosis of hepatoma cell line, HepG2, by NO [137]; and (iii) a dibasic hydroxamic acid derivative, viz., oxalyl bis (N-phenyl) hydroxamic acid (OBPHA) induced apoptosis of doxorubicin-resistant T-lymphoblastic leukemia (CEM/ADR5000) cells by generating ROS and NO. It was found that the presence of substituted hydroxamic acid groups (-CO-NH-OH) generates NO through auto degeneration. OBPHA downregulated HDAC3 expression by increasing ROS and NO production and a simultaneous decrease in cellular GSH level [138]. All these results [132–138] confirmed that NO has potent anti-­ cancer actions, and this could form the basis of decreased incidence of cancer in those who do regular exercise since exercise enhances NO generation but reduces ROS production and enhances not only in muscles but also other cells/tissues including macrophages, T cells, and PMNs [139–142]. These alterations observed due to regular strenuous exercise will render tumor cells to undergo apoptosis due to the increased generation of NO and protects normal cells from ROS (since NO can quench ROS), and increased antioxidant capacity will aid in this protection of normal cells from ROS. It is known that regular moderate exercise produces a disproportional upregulation of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase

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(GPX) expression (in response to exercise-induced ROS and NO generation) especially in cancer cells, which can lead to mitochondrial accumulation of H2O2. This, in turn, induces cancer cell apoptosis [143]. Such an exercise-induced disproportional upregulation of SOD, catalase, and glutathione peroxidase may be responsible for the decreased incidence of cancer seen in subjects who do regular exercise. The mechanisms of action of NO include oxidation of as ribonucleotide reductase and aconitase, activation of the soluble guanylate cyclase, ADP-ribosylation of proteins, protein sulfhydryl group nitrosylation, iron regulatory factor activation, and activation of NF-κB, an important transcription factor in iNOS gene expression in response to inflammation [144, 145]. NO stimulates soluble guanylate cyclase, which is needed for the formation of cyclic-GMP. Cyclic-GMP activates protein kinase G that leads to reuptake of Ca2+ and the opening of calcium-activated potassium channels, events that lead to relaxation of the smooth muscle cell [146]. These actions of NO remain to be exploited in the management of cancer. One of the factors that modulate ROS and NO generation in various cells is cytokines. Hence, a brief introduction to various cytokines and their actions is given in the next chapter.

 xidative Stress in Cancer Initiation and Progression O Including Apoptosis and Necrosis One of the major issues in cancer cell biology is whether oxidative stress (indirectly the balance between oxidative stress and antioxidant defenses) is the cause or effect of cancer. In other words, ROS has a role in the conversion of a normal cell into a malignant cell and in the induction of apoptosis/ferroptosis of cancer cell. Thus, there is evidence to suggest that ROS has a role both in the malignant transformation process and in the death of tumor cell. In general, many believe that ROS are always harmful. But this is not always true. ROS are involved in many cellular processes including physiology and pathology. Some of these include ischemia-reperfusion injury, pulmonary oxygen toxicity, atherosclerosis, radiation effects, chemotherapeutic effects, mutagenesis, carcinogenesis, and aging. In addition, ROS also are needed for some physiological processes especially in cell signaling. There is reasonable evidence to suggest that ROS participate in the biology of malignant neoplasia. ROS, when present in sufficient amounts, can induce damage to DNA and, thus, play an important role in tumor biology. ROS is known to induce several kinds of DNA damage, including strand breakage, base modification, and DNA-protein cross-linkage. 8-Hydroxy-2′-deoxyguanosine (8-OHdG, also known as 7,8-dihydro&oxo-2′-deoxyguanosine) is one of the oxidatively modified DNA base products that can be detected by high-performance liquid chromatography combined with an electrochemical detector. It is known that the hydroxyl radical (‘OH), singlet oxygen, or photodynamic action can induce the formation of 8-OH-dG.

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Table 2.1  Tumor and surrounding normal tissue content of 8-hydroxy-2′-deoxyguanosine. This data is taken from Ref. [147] Content of 8-hydroxy-2′-deoxyguanosine in human carcinoma (multiple case study) Organ Histology Control Tumor n; statistics Method Breast Invasive ductal carcinoma 4.13 ± 0.43 40.1 ± 11.1 (5; P  DHA) released from the membrane are capable of inducing apoptosis/ferroptosis/pyroptosis of cells containing micronuclei. Thus, those cells that contain micronuclei are ultimately eliminated and homeostasis is restored. This could be one of the ways of suppressing inflammation triggered by cytoplasmic DNA and eliminating cells that contain abnormal DNA received from bacteria, viruses, and other cells from the neighborhood

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Treg Cells and Autoimmune Diseases One of the important functions of the immune system is to discriminate between self and non-self so that body’s own cells/tissues are not recognized as foreign and mount an immune response against them. Such a recognition of self as foreign by the immune system may result in the development of autoimmune diseases. One of the major functions of the regulatory T cells is to suppress inappropriate activation of the immune system to the self-antigens and thus prevent the development of autoimmune diseases. But the exact molecular mechanism by which regulatory T cells bring about this important function is not clear. In general, it is thought that cytokines TGF-β and IL-10, prostaglandins, and other unidentified molecules seem to have a modulatory role in the regulatory T cell function. The putative role of bioactive lipids in this process, namely, suppression of autoreactive T cells and thus the development of autoimmune diseases, needs to be studied. Recent studies suggest that it is likely that bioactive lipids such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid and their anti-inflammatory metabolites lipoxins, resolvins, protectins, and maresins may have a role in the suppression autoimmune diseases. This seems to occur largely by acting on the cGAS-NF-kB system as discussed briefly below. It is obvious that autoimmune diseases are due to failure in self and non-self discrimination. Some of the pathogenic mechanisms involved in the development of autoimmune diseases include molecular mimicry, exposure of hidden antigens, loss of suppressor cell function, T- and B cell dysfunction, epitope spreading and epitope drift, and polyclonal B-cell activation by superantigens. Recognition of microbial nucleic acids by the host is an important strategy that is essential to respond to infectious agents. Microbial DNAs introduced into the host cells during infections need to be recognized and eliminated without triggering abnormal immune responses. The intracellular DNA that is introduced into cells during infections trigger inflammatory responses by the induction of antiviral type I interferons (IFNs), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-18. If nucleases such as DNase II and DNase III (Trex1) fail to clear cytosolic DNA, then accumulated DNA drives inflammatory responses leading to the development of autoimmune diseases. There are specific sensors that can couple cytosolic DNA recognition to immune signaling. One such pathway leads to the proteolytic activation of the cysteine protease caspase-1 that is associated with maturation and secretion of the IL-1β and IL-18. The second pathway could involve the transcriptional induction of type 1 IFN and pro-inflammatory genes (see Fig. 3.2). IL-1β activates neutrophils, macrophages, dendritic cells, and T cells, whereas IL-18 incites IFN-γ production by NK and T cells. All these events ultimately lead to the formation of inflammasome that occurs as a result of immune responses to intracellular DNA of bacterial or viral origin. These results suggest that while DNA-induced immune responses are critical to immunity, failure to recognize self-DNA can lead to inappropriate consequences, namely, autoimmune diseases such as lupus in which type I IFN and autoantibodies directed against dsDNA, RNA, and nucleosomes can be found. Thus, failure of the

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multiple fail-safe mechanisms employed by the host is subverted leading to DNA-­ induced immune responses and inflammation (see Fig. 3.3). One such regulation provided by the body includes cellular endonucleases such as DNase I, DNase II, and DNase III (also known as Trex1) that are involved in the clearance of extracellular, lysosomal, and cytosolic DNA, respectively. This suggests that functional defects in these enzymes can lead to the triggering of lupus and other autoimmune diseases. Recent studies showed that cytoplasmic chromatin (chromatin is a complex of DNA, RNA, and protein found in eukaryotic cells) activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway that can lead to the activation of type 1 IFN through IRF3 and pro-inflammatory response through NF-κB (see Fig. 3.3). cGAS-­ STING pathway is connected to the NF-κB-mediated senescent-associated secretory phenotype (SASP) that results in the secretion of pro-inflammatory cytokines (into the surrounding milieu and systemic circulation) and recruitment of immune cells, modulates their activity, and consequently alters tissue microenvironment. STING is essential for Ras-induced SASP and immunosurveillance and immune-­ mediated clearance of malignant cells. Short-term inflammation and senescence prevent tumorigenesis, whereas persistent inflammation produces tissue damage and enhances tumor growth that explains increased incidence of cancer (especially lymphomas) in those with lupus in which that extranuclear chromatin is seen in the form of micronucleus cells. The cytoplasmic and nuclear DNA/chromatin (micronuclei) induces the production of pro-inflammatory cytokines IL-1β, TNF-α, IL-18, IL-6, and IFN-γ that can spill over into the intercellular surrounding milieu and systemic circulation. IL-1, IL-6, TNF-α, and IFN-γ activate phospholipases, enhance ROS generation, and increase activity of COX-2 and LOX enzymes to augment the production of pro-­inflammatory PGE2, TXA2, and LTs. Once the inflammation reaches its peak, the same AA, EPA, and DHA released from the cell membrane lipid pool are utilized for the formation of anti-inflammatory lipoxins, resolvins, protectins, and maresins. Thus, inflammation is suppressed, and homeostasis is restored. When this switch over from pro-inflammatory PGE2, TXA2 and LTs to lipoxins, resolvins, protectins, and maresins fails to occur in an orderly and smooth fashion, inflammation persists. Thus, a delicate balance is maintained between pro- and anti-inflammatory cytokines and bioactive lipids. It is possible that bioactive lipids or their peroxidized products can bind to DNA and render it nonantigenic.

Methods to Analyze and Monitor Treg Cells In order to assess the role of Treg cells especially in the pathobiology of autoimmune diseases, there is a necessity for dependable methods of their identification, number, and function. Such methods available employ flow cytometric analysis using specific antibodies tagged with different dyes/fluorescent probes, gene expression

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analysis, knockout techniques, and chip-on-chip analysis and of late DNA methylation identification methods [17–20]. For instance, Treg cells show a specific region within the foxp3 gene (TSDR, Treg-specific demethylated region) to be demethylated that can be used to monitor the Treg cells by employing a PCR reaction or other DNA-­based analysis methods [17].

B Lymphocytes One of the main functions of B cells is to recognize pathogens following which in cooperation with matching helper T cells that release cytokines and activate B cells to proliferate and form plasma cells that are capable of secreting appropriate antibody(ies). Antibodies produced by these plasma cells enter the circulation and lymph are capable of binding to pathogens such that they are recognized for destruction/elimination by complement and/or for destruction by phagocytes. Antibodies are also capable of neutralizing the invading bacterial antigens directly by binding to them. These antibodies not only are specific to a given bacterial antigen but can also neutralize bacterial toxins or interfere with the receptors expressed by the viruses and bacteria that they use to invade the cells. Long-term active memory that is needed to prevent subsequent infections by agents such as polio virus, tuberculosis, measles, etc. is necessary for the activation of B and T cells. This long-term active immunity is achieved artificially by vaccination. In this instance, the antigen of the specific pathogen is inactivated, or its virulence is attenuated or reduced and then injected into the body such that the immune system is stimulated to optimal degree to enable the development of specific and long-lasting immunity against the specific pathogen (bacteria or virus) without causing disease. It is possible that in this instance, occasionally, mild infection may occur. Thus, vaccination is nothing but inducing sub-clinical infection with the desired bacterial or viral attenuated antigen but is sufficient to activate the immune system in order to enable it to produce sufficient antibodies that can produce long-­ lasting immunity. Thus, vaccination is effective because it is able to exploit successfully the natural specificity of the immune system. The specificity of the immune system and its successful exploitation led to the development of many useful vaccines that helped to save millions of people from many infectious diseases and reduced to a significant degree many infectious disease-associated morbidity and mortality. In general, viral vaccines use live attenuated viruses, whereas many bacterial vaccines use acellular components of microorganisms. The same principle is being explored in the development of vaccines/immunotherapeutic strategies against cancer, Alzheimer’s disease, and other diseases. Development of B cells occurs in the bone marrow or the fetal liver. During this process of development, recombination of genes occurs in the “naïve” cells such that self-reactive B cells are removed or suppressed so that autoimmune disease does not occur. Despite this effort on the part of the body, about 20–40% of B cells remain “autoreactive” that have the potential to elaborate self-reactive antibodies if given a suitable environment. When B cells are exposed to antigen(s), many B cells undergo

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“isotype switching” due to DA deletion and recombination that results in generation of isotypes of IgG1, IgG2, IgG3, IgA1 IgA2, IgA3, IgD, and IgE from the initial IgM that have distinct functional characteristics and play a very specific role in disease(s). Based on this information, it is logical to propose that delineation of isotypes would give clues to the BCR (B-cell receptor) repertoire. Further diversification of BCRs is expected to occur in specialized germinal centers wherein somatic hypermutation (SHM) of genes that encode the variable (V) regions of antibodies occurs, leading to the enhancement of BCR affinity and specificity. Such a diversification of B-cell clones that occurs after exposure to antigen(s) is tempered by tolerance checkpoints that are meant to reduce the risk of autoimmunity. Thus, the peripheral BCR repertoire consists of a combination of both the native repertoire and that generated as a result of exposure to antigen(s). When the BR repertoire in lupus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Crohn’s disease, Behcet’s disease, eosinophilic granulomatosis with polyangiitis, and immunoglobulin A (IgA) vasculitis were compared by analyzing BCR clonality, it was noted that lupus and Crohn’s disease were dominated by IgA isotype suggesting a microbial contribution to their pathogenesis. Furthermore, B cells that persisted after treatment with rituximab (a chimeric monoclonal antibody against the protein CD20 that is found on the surface of B cells) were found to be predominantly isotype switched and clonally expanded type, whereas the inverse was true for B cells that persisted after treatment with another immunosuppressive drug, namely, mycophenolate mofetil. These results suggest the complex nature of the autoimmune diseases and the BCR repertoire.

Toxic Shock Syndrome Despite the fact that the immune system is tuned to respond to a variety of different antigens as described above, some pathogens can hijack the immune system. Staphylococcus aureus (S. aureus) and Streptococcus pyogenes (S. pyogenes) and some viruses release superantigens that are capable of overactivating the immune system. Some of these superantigens have been linked to severe effects of bacterial infections, such as toxic shock syndrome and rheumatic fever.

Superantigens and the Immune System Superantigens interfere with the adaptive immune system. In general, antigen-­ presenting cells, such as dendritic cells and phagocytes, take up extracellular pathogens and process them into individual antigen fragments that are displayed on the outside of the cell by the major histocompatibility complex II (MHC-II). The antigen-­carrying MHC-II complex is then recognized by receptors on circulating T cells. These TCRs (T-cell receptors) are specific to certain MHC-II/antigen combinations, and they bind very briefly to the T cell to activate and initiate the immune response that results in the activation of only about 0.001–0.01% of all T cells are activated.

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In contrast to this, superantigens are not processed by the antigen-presenting cell (APC); instead, they bind directly to the MHC-II, and another binding site of the superantigen interacts with the Vβ subunit of the TCR. This results in the bridging of the TCR and MHC-II leading to superantigens connecting these complexes in contact with each other for a much longer time than usual (normal). This triggers a much less specific process compared to the normal antigen-specific immune response. As a result, around 20% of T cells get activated since a large fraction of the T cells have a Vβ subunit in their receptor (since the superantigen connects them to MHC-II). This activation of T cells results in the release of a large number of cytokines: IL-2, interferon-γ (INF-γ), and TNF-α resulting in cytokine storm that could be lethal. It is now being thought that superantigens may have other roles as well (i.e., in addition to inducing cytokine storm). It is interesting to note that production of antibodies against one superantigen can have protective immunity against other superantigens. T follicular helper (Tfh) cells are a type of CD4+ cell that help in the production of the antibody response, and superantigens seem to induce higher frequencies of cytolytic Tfh cells infected with the superantigen such as SpeA superantigen of S. pyogenes. As a result, children who had recurrent tonsillitis with superantigen SpeA had their B cells undergo apoptosis due to the action of these aberrant Tfh cells. In contrast, children who did not have recurrent tonsillitis, but had been exposed to S. pyogenes, had higher levels of antibodies against SpeA, and their tonsils showed larger germinal centers, suggesting that the immune response to SpeA can alter the course of the disease. These results imply that SpeA can be used as a possible vaccine to strengthen the immune response against S. pyogenes infection. If methods can be devised wherein tumor-specific antigens (or viruses and bacteria that are responsible for cancer) can be converted into superantigens, then, perhaps, they could be used to immunize against certain cancers.

Immunological Aspects of Cancer It is known that a close interaction exists between tumor cells and the immune system of the body. Tumor cells do express some specific antigens on their cell surface that are recognized by the immune system of the body but are not (tumor antigens) strong enough to elicit adequate immune response so that the tumor cells can be eliminated. But of late it is being recognized that the recognition of cancerspecific antigens could be enhanced such that targeted therapy and tumor markerbased diagnostic tests can be developed [21]. The basis for this approach has come from the observation that tumor-infiltrating lymphocytes and macrophages play a significant role the prognosis of cancer in a given patient. It was noted that in an instance wherein there was a significant infiltration of tumor tissue with inflammatory cells had a better chance of survival, implying that to some extent anti-tumor immunity is present in patients with cancer but is probably insufficient to eliminate the tumor cells completely. There is now sufficient evidence and knowledge that is enabling us to develop potentially useful vaccines against some cancers (such as HPV for the prevention of

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papilloma and carcinoma of cervix) based on the concept of cancer immunosurveillance [21]. Identification of specific antigens of some cancers is enabling us to develop specific monoclonal antibodies that can be used to target specific cancer cells and/or being used to carry toxins or chemotherapeutic drugs to be delivered to the cancer cells. These principles form the basis of current excitement about immunotherapy of cancer such as checkpoint inhibitors for cancer. Recent developments of checkpoint inhibitors of PD-1 (programmed cell death 1 protein), PD-1 ligand (PD-L1), cytotoxic T-lymphocyte-associated protein 4 {CTLA-4 also called as CD152 (cluster of differentiation) 152}, and adoptive cell transfer (ACT) are based on these principles.

Immunosurveillance and Immunoediting Burnet proposed the theory of cancer immunosurveillance in 1957 in which they suggested that lymphocytes act as sentinels in recognizing and eliminating continuously arising, nascent transformed cells [22, 23]. This theory suggested that cancer immunosurveillance is an important host protection process that constantly recognizes tumor cells that are arising every day in the body and eliminates them by successfully mounting an immune response and thus prevents the occurrence of cancer [24]. This function of immune system is essential to maintain cellular homeostasis and prevent the incidence of cancer. This process of tumor immunosurveillance consists of three phases: (i) The phase of recognition of the cancer-specific antigens and the initiation of an antitumor immune response that involves recruiting innate immune system’s cells such as NK cells and natural killer T cells, macrophages, and dendritic cells to the site of tumor. These cells while infiltrating the tumor get stimulated and produce IFN-γ. (ii) During the second phase of immunosurveillance, IFN-γ induces tumor cell apoptosis/ferroptosis/lysis/pyroptosis and simultaneously promotes the production of chemokines that can further promote the death of more number of tumor cells by their anti-angiogenic action. These chemokines promote further recruitment of immune cells to the site of the tumor by their chemokine action and inducing tumor cell death that, in turn, leads to inflammation (that is stimulated by the tumor cell debris). (iii) The infiltrating immunocytes including T cells, NK cells, and macrophages produce IFN-γ and IL-12 which can activate these cells to produce other pro-­ inflammatory cytokines ILs and TNF-α, ROS, and RNS (reactive nitrogen species) that produce tumor cell apoptosis. During this phase of immunosurveillance, dendritic cells present in the tumor-draining lymph nodes trigger differentiation of TH1 cells that facilitate the development of CD8+ T cells (killer T cells). Following this, tumor-specific CD4+ and CD8 + T cells home to the tumor site and the cytolytic T cells eliminate the remaining tumor cells. Though this process of tumor cell recognition by the immune system and their elimination has been described in simple terms, this is not so simple nor is effective in many instances.

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In order to escape the body’s immunosurveillance system, tumor cells continuously mutate and evolve and escape their detection and elimination by the immune system rather effectively and thus survive at the cost of the host. One of the important functions of the immune system is to identify the specific antigens that are expressed by the tumor cells and mount an immune response and eliminate them. In general, the tumor cells express antigens that are unique to them that are not found on normal cells. The immune system recognizes these tumor-­ specific antigens as foreign which enables them to recognize them. The antigens expressed by tumors are derived from three sources: (i) oncogenic viruses; (ii) normal proteins that occur at low levels in normal cells but reach high levels in tumor cells such as growth factor(s) and/or their receptors; and (iii) normal proteins that are needed for regulating cell growth and survival that are mutated into cancer-­ causing molecules such as oncogenes, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) [25]. Similar to the viral antigens, tumor antigens are presented on MHC class I molecules that are supposed to be recognized by the killer T cells as foreign and mount an appropriate immune attack and eliminate them. In an occasional instance, antibodies that are generated against tumor cells use complement system to kill the tumor cells. But, paradoxically, despite the presence of a robust immunological system in the body, in some instances, it fails to kill the tumor cells, and tumor cells evade the immune system by producing immunosuppressive molecules such as TGF-β, prostaglandin E2 (PGE2), and certain blocking antibodies. This makes the immune system ineffective in eliminating the tumor cells and so they grow. Studies have also shown that sometimes macrophages may actually promote tumor growth by secreting some cytokines and growth factors that may enhance the growth of the tumor cells. For instance, it was reported that TNF-α, though is meant to kill tumor cells, may actually enhance tumor growth [26–28]. In this context, it is noteworthy that macrophages participate not only in inflammation and immune response but also possess ability to elicit anti-­ inflammatory action and suppress immune reactions. M1 macrophages encourage pro-inflammatory events (profibrotic), whereas M2 macrophages decrease inflammation and encourage tissue repair (pro-tissue repair or pro-regenerative). This difference in the actions of M1 and M2 macrophages can be attributed to changes in their metabolic pathways. For instance, macrophages (M1) convert L-arginine to NO and thus induce inflammatory events, whereas M2 macrophages convert L-arginine to ornithine to produce anti-inflammatory actions [29]. The M1 “killer” phenotype of macrophages is induced by lipopolysaccharide (LPS) and IFN-γ that render macrophages to secrete high levels of  IL-12  and low levels of  IL-10. In contrast, the M2 “repair” phenotype macrophages secrete anti-inflammatory cytokine IL-10. IL-4 cytokine encourages macrophages to become M2 type. M2 macrophages secrete high levels of IL-10 and TGF-β and low levels of IL-12. Tumor-associated macrophages (TAM) are of the M2 phenotype and encourage tumor growth [30].

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Though the exact mechanism by which M2 macrophages contribute to tumor growth and progression is not clear, it is opined that TNF-α secreted by these macrophages activate nuclear factor-kappa B (NF-kB) that, in turn, induces the production of monocyte chemotactic protein 1 (MCP1) by tumor cells. MCP1 is expressed mainly by tumor cells, fibroblasts, monocytes, macrophages, and vascular endothelial cells in the tumors. There is a close association between the expression of MCP1 and the accumulation of TAM. The expression of MCP1 also correlated significantly with the levels of VEGF, TNF-α, and IL-8. Increased expression of MCP1 and VEGF was found to be an early indicator of tumor progression and recurrence of tumor. Thus, MCP1 is an early indicator of tumor angiogenesis and growth, and, hence, it can be considered as a pro-tumorigenic protein [31]. These results are supported by the observation that TAM promote tumor cell proliferation, angiogenesis, and tumor cell metastasis through NF-kB that seems to be needed for macrophage polarization from M1 type to M2 type. Inhibition of NF-kB activity of macrophages in the tumor microenvironment (M2) in vitro led to a significant decrease in IL-10 secretion, a TH2 cytokine, and a significant increase in IL-12, TNF-α, and IL-6, which are TH1 cytokines. In addition, inhibition of NF-kB expression resulted in suppression of VEGF production and matrix metalloproteinase-9 mRNA expression in M2-macrophages associated with a reduction of arginase mRNA expression and an increase in NO production [32]. These results suggested that NF-κB activity in M2 macrophages plays a key role in the growth of tumor cells by elaborating factors that are needed for tumor growth. In this context, the ability of macrophages to influence arginine metabolism via NOS and arginase pathways is another mechanism by which M1 and M2 macrophages are able to reduce and enhance tumor cell proliferation, respectively. The increased production of TGF-β produced by M2 macrophages downregulate the NOS pathway and upregulates arginase activity by reducing Km value of arginase. TGF-β-induced upregulation of arginase activity enhances the release of more polyamines, mainly putrescine, and reduces NO production. Thus, the ability of TGF-β to upregulate arginase activity leads to a decrease in the availability of L-arginine and so there will be reduced production of NO, a cytostatic molecule, by macrophages, which is yet another mechanism by which M2 macrophages enhance tumor cell proliferation [33]. In contrast, M1 macrophages enhance NO generation and thus inhibit the proliferation of tumor cells. TAM serve as a source for pro-­ angiogenic factors including VEGF, TNF-α, granulocyte macrophage colony-­ stimulating factor (GM-CSF), and IL-1 and IL-6 that can enhance tumor growth. Macrophages avidly infiltrate tumors, and there is a close correlation between their number and their prognosis especially of cancers such as breast, cervix, bladder, and brain: the higher the number of macrophages infiltrating the tumor, the poorer the prognosis. Thus, it looks very paradoxical that the same molecule and mechanisms that are needed for normal wound healing and regulation of inflammatory process are exploited by the tumor cells to escape the immunosurveillance system and proliferate. For instance, for normal wound healing to occur and restore homeostasis, initially a well-designed and orchestrated inflammatory process and secretion of pro-inflammatory molecules and local factors are needed at the initial stages of injury. Once the wound is formed, for its healing to occur in a well-orchestrated fashion, anti-inflammatory cytokines and cell proliferation-inducing molecules are

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Mutagens & Carcinogens

Treg cells Helper T cell

Normal Cell ROS, RNS, IFN-γ, TNF-α

Genetic Damage

Tumor antigens

M1Ф

TNF-α NF-kB↑

(-)

Apoptosis

Tumor Cell

Killer T Cells

PUFAs

Proliferation & Metastasis IL-4, IL-10, TGF-β, VEGF, IL-8, MMP-9, GM-CSF, Arginase ↑

MCP-1

NO↓

M2Ф↑

NF-kB↑

Fibroblasts, monocytes, macrophages and vascular endothelial cells

Fig. 3.4  Scheme showing the role of various molecules in normal wound healing and cancer cell proliferation and metastasis and their modulation by PUFAs. This scheme also shows interaction(s) among various immunocytes and macrophages and their products and growth factors (including cytokines) in response to mutagens and carcinogens

Fig. 3.5  (continued)  IL-17A production from Vγ4+ γδ T cells and promotes psoriatic dermatitis in experimental animals [38]. In psoriatic inflammation, PD-1 is overexpressed on CD27(−) Vγ1(−) γδ T cells. In the CD27(−)Vγ1(−) γδ T-cell population, Vγ4(−) γδ T cells with Vγ6 mRNA expression showed a high level of PD-1 expression. These PD-1(hi)Vγ4(−) (Vγ6(+)) γδ T cells produced anti-CD3-­induced IL-17A production, which is inhibited by PD-L1-Fc treatment. PD-L1-Fc not only suppressed psoriatic inflammation but also enhanced the therapeutic effect of anti-p40 [39]. These results emphasize the anti-inflammatory nature of PD-1 that seems to have a dominant role in the escape of cancer cells from immunosurveillance system of the body. Lipoxins, resolvins, protectins, and maresins suppress inflammatory events and are useful in the suppression of psoriasis, rheumatoid arthritis, and autoimmunity and inhibit tumor cell proliferation with no action on normal cells [40–42]. Lipoxins, resolvins, protectins, and maresins suppress IL-17 and IL-23 production and their receptors expression, whereas PGE2 and possibly TXA4 have opposite actions [43, 44]. But, paradoxically, in an occasional instance, PGE2 has been shown to inhibit the production of IL-23 and IL-12 and thus bring about its anti-inflammatory action [45]. But, in general, PGE2 and leukotrienes (LTs) induce and maintain inflammatory status within the tumor microenvironment by enhancing the production of IL-17 and IL-23 [46, 47], whereas lipoxins, resolvins, protectins, and maresins oppose these actions [48, 49]. This data emphasizes the complex nature of interaction(s) among PUFAs, their metabolites (especially PGE2/LTs/TXA2 and lipoxins, resolvins, protectins, and maresins), T cells, and their cytokines. It is likely that local production of PUFAs and their metabolites and cytokines within the tumor microenvironment and sites of inflammation such as joints in rheumatoid arthritis, in the dermis (and surrounding area of the skin cells) in psoriasis, and colon cells in colitis may ultimately determine the outcome of the disease (proliferation of tumor cells or remission or exacerbation or continuation of the inflammatory process). The release of PUFAs from the cell membrane lipid pool and their subsequent metabolism to their respective metabolites and their local concentrations depends on the activities of phospholipases, COX, LOX, and degradation enzymes such as PGDH

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?

PUFAs

TH1 T-bet

? IFN-γ

Clearance of intracellular pathogens, Immunopathology, Autoimmunity

Tolerance ?

iTreg FOXP3

TGF-β

?

T naive

IL-4

TH2 GATA3

Clearance of extracellular pathogens, Allergy, Atopy

TGF-β + IL-6 + IL-23 + PGE2 LXs, RSvs, PRTs, MaRs

TH17 RORγt PD-1

Clearance of certain classes of extracellular pathogens: Klebsiella and fungi, Tissue inflammation Immunopathology Autoimmunity

Tumor cell

Fig. 3.5  Factors controlling formation of different subsets of Helper T cells. LXs lipoxins, RSvs resolvins, PRTs protectins, MaRs maresins. Naive CD41 T cells differentiate into subsets of Helper T cells: TH1, TH2, and TH17. TGF-β converts naive T cells into FOXP3-expressing induced Treg (iTreg) cells. Each Helper T cell differentiation program needs specific transcription factors as master regulators (T-bet, GATA3, and ROR-γt). Terminally differentiated Helper T cells produce specific combination of effector cytokines that bring about specific and distinct effector functions of the adaptive immune system. TGF-β, retinoic acid, or cytokines: IL-6, IL-1, IL-23, or IL-27 provided by cells of the innate immune system (immature or activated dendritic cells (DCs), respectively) and dictate whether a naive T cell develops into a FOXP31 Treg cell, a TH17 cell, or otherwise. Prostaglandin E2 (PGE2) through its receptor EP4 on T cells and dendritic cells facilitates TH1 cell differentiation and amplifies IL-23-mediated TH17 cell expansion and EP4-selective antagonist decreases accumulation of both TH1 and TH17 cells and suppresses progression of autoimmune encephalomyelitis or contact hypersensitivity in experimental animals. Though the role of PUFAs and their various metabolites is not discussed in detail, it is known that GLA, AA, EPA, DHA, lipoxins, resolvins, protectins, maresins, and prostaglandins, leukotrienes, and thromboxanes can influence macrophage and other immunocytes’ phagocytosis, motility, and ability to alter ROS generation and final outcome of inflammation and immune response. Several studies revealed that EPA/DHA can restore IL-17 and Treg balance in experimental animals that have collagen antibody-induced arthritis, type 1 diabetes mellitus (in NOD mice), and depression in a lipopolysaccharide (LPS)-induced animal model (by balancing M1 and M2 phenotypes and normalizing BDNF function) [34–36]. It was opined that PGE2 and IL-23 are the key regulators of TH17 development [37]. PGE2 produced during the autoimmune conditions induces the expression of IL-23R in naïve CD4+ T cells. IL-23 released from APCs binds to IL-23R and activates signal transducer and activator of transcription factor 3 (STAT3). The activated STAT3 translocates to the nucleus and initiates RoRγ expression. The activated STAT3 and retinoid-related orphan receptor gamma (RoRγ) complexes binds to IL-17 promoter region to initiate the IL-17 production. In addition, TXA4, a pro-inflammatory molecule derived from AA, has also been found to facilitate

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needed to restore homeostasis. The initial stage of wound development is a pro-­ inflammatory process, while the wound healing process needs anti-inflammatory mechanisms (See Figs. 3.2, 3.4, and 3.5) Despite tremendous advances in our understanding of the immune system and molecular biological aspects of cancer, we are yet to exploit this knowledge for successful elimination of cancer cells from the body. This suggests that our understanding of both immune system and cancer biology is incomplete especially, with regard to how the switching of M1 to M2 macrophages and vice versa occurs in the body during inflammation, sepsis, cancer, and autoimmune diseases. It is clear that this switching of M1 to M2 macrophage status and vice versa is essential for proper healing and recover from injury, surgery, and other inflammatory conditions and also to restrict the growth of tumor cells. It is obvious that for some reason the endogenous regulatory mechanisms fail to activate the M1 macrophages, and hence tumor cells are not eliminated, while in autoimmune diseases the switching of M2 macrophage process does not occur at the right time and for sufficiently long time, and hence the inflammatory process continues, and autoimmune diseases set in and continue. Recent studies have suggested that lipids could play a significant role in the generation of TH17–Treg cells, and yet other investigations showed that blocking PD-1, programmed cell death protein 1, also known as PD-1, and CD279 (cluster of differentiation 279), a cell surface receptor that belongs to the immunoglobulin superfamily and expressed on T cells and pro-B cells, could be exploited to activate the immune system and treat cancer.

PD-1 (Programmed Cell Death Protein 1) Programmed cell death protein 1 (PD-1), encoded by the PDCD1 gene, is a cell surface receptor of the immunoglobulin superfamily expressed on T cells and pro-B cells. PD-1 has two ligands, PD-L1 and PD-L2, to which it can bind and downregulate the immune system by preventing the activation of T-cells. Thus, PD-1 promotes self-tolerance and suppresses autoimmunity and thus plays a significant role in autoimmune diseases. At the same time, overexpression of PD-1 and its ligands renders cancer cell escape from immunosurveillance and promotes tumor cell growth. PD-1 promotes apoptosis of antigen specific T-cells and inhibits apoptosis of regulatory T cells (suppressor T cells), and hence blocking PD-1 and its ligands activate the immune system to attack tumors and so are useful in the treatment of cancer. PD-1 is expressed mainly on the surface of activated T cells, B cells, and macrophages compared to CTLA-4, implying that PD-1 may have a broader negative regulatory action immune responses. The two ligands of PD-1, PD-L1 and PD-L2, belong to the B7 family and are upregulated on macrophages and dendritic cells when stimulated by LPS and GM-CSF and on T cells and B cells upon TCR and B-cell receptor signaling. PD-L1 mRNA has been detected in the heart, lung, thymus, spleen, and kidney [50–52] and is expressed on many tumor cell lines including myeloma, mastocytoma, and B16

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melanoma especially on stimulation with IFN-γ. In contrast, PD-L2 expression is expressed by DCs and very few tumor cells. Both PD-1 and cytotoxic T lymphocyte-associated protein 4 (CTLA4) are capable of actively modulating T-cell responses upon activation [53]. Both PD-1 and CTLA4 assist the tumor cells to escape from immunosurveillance, as they can impair T-cell functions, often leading to exhaustion; decreasing secretion of IL-2, IFN-γ, and TNFα; and dampening the proliferation of T cells, which would lead to their (T cells) reduced cytotoxic action. PD-1, which is expressed on effector T cells, has a negative regulatory action on T cell functions by itself. PD-1 binds to two different ligands, PD-L1 and PD-L2, that are expressed by several tumor cells including but not limited to breast, kidney, ovarian, pancreatic, bladder, and gastric cancer cells. Since PD-1 has an immunosuppressive role, it stands to reason that blocking PD-1 signaling with specific anti-PD-1 or anti-PD-L1 antibodies can reverse T-cell exhaustion and lead to tumor regression [54]. PD-1 and several co-­ stimulatory molecules such as CD28 are needed for the full activation and effector activity of naïve T cells. These cognate ligands such as the B7/CD28 family or the TNFα receptor (TNFR) family convey TCR-independent intracellular signals and help in T-cell expansion and in the acquisition of effector functions. This suggests that the balance between co-stimulatory and co-inhibitory signals is needed to regulate the response, function, and expansion of T cells. This evidence indicates that combination treatment with anti-CTLA-4 plus anti-PD-1 or anti-PD-L1 will enable the creation of an immunogenic tumor microenvironment that will lead to regression of tumors [54, 55]. Even CAR T (chimeric antigen receptor therapy) cell therapy may work in this manner.

TH17–Treg Cell Balance On exposure to different stimuli, naïve T cells are activated, show proliferation, and differentiate into distinct functional subsets. Based on cytokine phenotypes, three distinct effector TH subsets have been identified. As already discussed previously, TH1 cells produce IFN-γ that protects against intracellular pathogens; TH2 cells produce IL-4, IL-13, and IL-25 that are effective against extracellular pathogens; and the third subset of TH cells known as TH17 cells produce IL-17 that are needed to clear extracellular pathogens, which are not effectively handled by either TH1 or TH2 cells. TH17 cells also produce IL-21 and IL-22 and are capable of inducing the production of pro-inflammatory cytokines, chemokines, and metalloproteinases from various tissues and cell types and help in the recruitment of neutrophils to tissues. Both IL-21 and IL-22 are not necessarily exclusive to TH17 cells but are preferentially expressed in TH17 cells. It is believed that TGF-β, IL-6, IL-21, and IL-23 contribute to TH17 formation and IL-1β seems to have a role in the formation of TH17 cells. Other proteins involved in the formation/differentiation of TH17 cells are STAT3 (signal transducer and activator of transcription 3) and retinoic acid receptor-­ related orphan receptors α and γ (RORα and RORγ, respectively). It is noteworthy

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that high concentrations of TGF-β inhibit IL-6-induced IL-22 expression, whereas a combination of TGF-β plus IL-6 induces generation of IL-17 by TH17 cells. On the other hand, secretion of IL-22 by TH17 cells required the cooperation of IL-23. Thus, IL-22 is the end point effector cytokine secreted by TH17 cells ([56], see Fig. 3.5). IL-17 or IL-17A, a pro-inflammatory cytokine secreted by T helper 17 cell in response to stimulation with IL-23 and encoded by IL17A gene, is also referred to as CTLA8. IL-17 interacts with type I cell surface receptor IL-17R that has at least three variants referred to as IL17RA, IL17RB, and IL17RC. Binding of IL-17 to its receptor results in the induction of chemokines that recruit other immune cells including monocytes and neutrophils to the site of inflammation. IL-17 acts, in general, with TNF and IL-1 produce its actions. IL-17 is involved in the pathogenesis of autoimmune disorders including psoriasis. The IL-17 family comprises IL17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. IL-17E is also known as IL-25. IL-17 is commonly associated with allergic responses. IL-17 induces the production of other cytokines such as IL-6, G-CSF, GM-CSF, IL-1β, TGF-β, TNF-α, chemokines (including IL-8, GRO-α, and MCP-1), and prostaglandins such as PGE2 from many cell types including fibroblasts, endothelial cells, epithelial cells, keratinocytes, and macrophages. IL-17 seems to play a significant role in immune−/autoimmune-­ related diseases including rheumatoid arthritis, asthma, lupus, allograft rejection, anti-tumor immunity, psoriasis, and multiple sclerosis. TH17 cells defend the body against Gram-positive Propionibacterium acnes; the Gram-negative Citrobacter rodentium, Klebsiella pneumoniae, Bacteroides spp., and Borrelia spp.; the acid-fast Mycobacterium tuberculosis; and fungi such as Candida albicans acting as an early responsive immunocytes to a variety of pathogens that are not handled appropriately by TH1- or TH2-type immunity especially when the body needs a robust tissue inflammation to clear infection [56]. This suggests that TH17 cells bridge the gap between innate and adaptive immunity. IL-17 producing T cells produces severe experimental autoimmune encephalomyelitis (EAE) as a result of its profound pro-inflammatory action and induces tissue damage; IL-17 promoted cartilage and bone destruction, and IL-17-deficient mice show less severe collagen-induced arthritis and EAE; and increased plasma and tissue levels of IL-17 have been observed in patients with rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and psoriasis. This supports the contention that IL-17 and TH17 cells play a significant role in autoimmune disorders [56–65]. In addition to the role of IL-17 and TH17 cells in inflammatory diseases especially autoimmune diseases, TH1 cells also seem to have a role in autoimmune diseases. This is so since TH17 cells facilitate the migration of TH1 cells into the target tissues that would accentuate inflammation and tissue damage. These data suggest that TH17 cells are inducers of autoimmunity by promoting tissue inflammation and the mobilization of the innate immune system. As already discussed above, it appears that IL-6 and TGF-β that have opposite actions – one is pro-inflammatory and the other anti-inflammatory, respectively  – seem to be necessary to induce the development of TH17 cells ([56, 66, 67]; see Fig. 3.5). In this process of TH17 cell development, there seems to be a role for IL-23 cytokine also [56].

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TH17 development is dependent on the lineage-specific transcription factor, the orphan nuclear receptor ROR-ct that is selectively expressed in TH17 cells differentiated in the presence of TGF-β plus IL-6. On the other hand, loss of ROR-ct in T cells suppresses the generation of myelin-specific TH17 cells and is constitutively present in the intestinal lamina propria. Furthermore, Treg cells and TH17 cells seem to have a reciprocal relationship between them. IL-6 dictates the balance between these two cell populations. This reciprocal relationship between Treg and TH17 cells is supported by the observation that IL-2, which is a growth factor for Treg cells, inhibited the generation of TH17 cells, whereas mice that lack IL-2 have reduced numbers of Treg cells and an increased proportion of TH17 cells, and these mice develop inflammatory diseases that can be prevented by the passive transfer of Treg cells [68, 69]. IL-4, IL-25, and IFN-γ inhibit the expansion of TH17 cells [70– 72]. Similarly, IL-27, a member of the IL-12 heterodimeric family of cytokines, suppresses the development of TH17 responses. Despite the fact that IL-23 is needed for the generation of TH17 cells from naïve T cells and IL-17 may play a central role in the pathogenesis of inflammatory bowel disease, surprisingly antibodies against IL-17 showed low efficacy and increased infections in Crohn’s disease. It was noted that genetic deficiency of IL-17 did not suppress initiation of colitis but limited colitis progression and inhibition of IL-17 by monoclonal antibodies are ineffective in reducing the severity of colitis. In contrast to this, antibodies against IL-23 significantly alleviated both emerging and established colitis, downregulated TH17 pro-inflammatory cytokine expression, and diminished neutrophil infiltration. These results support clinical studies that showed that IL-17 neutralization is not of much therapeutic value in IBS (inflammatory bowel disease), while targeting IL-23 suppressed intestinal inflammation [73]. These results raise the interesting possibility that using monoclonal antibodies against IL-23 is of use in the management of IBS and its action could also include inhibition of suppression of conversion of naïve T cells to TH17 cells. In a recent study, it was found that blocking the protein IL-23 which is produced by granulocytic myeloid-derived suppressor cells can restore tumor cell sensitivity to hormone therapy. PUFAs (especially EPA/DHA), lipoxins, resolvins, protectins, and maresins that can inhibit IL-23 and IL-17 not only are anti-inflammatory but are also capable of enhancing the sensitivity of tumor cells to the action of chemotherapeutic drugs and radiation (see Fig. 3.5).

IL-17, Synaptic Plasticity, and Memory γδ Th17 cells that are resident in the meninges of the brain, called the meninges, seem to have an impact on short-term memory. TH17 cells that are usually found in the peripheral immune system and secrete cytokine IL-17 are known to play a significant role in multiple sclerosis. It was reported that healthy mice, in the socalled “steady state”, γδ Th17 cells are generated in the embryonic stage in the thymus, and at birth, migrate to the meninges, and colonize the tissue and their

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numbers peak in the first week of life, a stage when learning and cognitive functions are developing. It has been suggested that γδ Th17 cells reside in the meninges coming from the lymphatic vessels and produce soluble factors such as BDNF (brain-­derived neurotrophic factor) and thus influence synaptic plasticity and memory formation by specifically acting on the hypothalamus. It was reported that mice lacking γδ T cells or IL-17 showed deficient short-term memory while retaining long-term memory, which was found to be due to reduced plasticity of glutamatergic synapses in the hippocampus. The observation that IL-17 has the ability to enhance glial cell production of BDNF, an important neurotrophic factor, suggests a close interaction between IL-17 and BDNF. It is noteworthy that exogenous administration of BDNF rescued the synaptic and behavioral phenotypes of IL-17-deficient animals which is in support of the critical role played by BDNF in memory formation. These results suggest the close interaction between immune system and nervous system at least with regard to memory. Both astrocytes and microglial cells constitutively express IL-17RA. In contrast to these results, excess production of IL-17 has been shown to be associated with neuronal death and behavioral abnormalities by triggering systemic inflammation as seen in multiple sclerosis and some viral infections. Maternal IL-17 has been linked to autism-like phenotype in their offspring suggesting a detrimental impact of high IL-17 levels. Putting these results together, it can be argued that one has to make a distinction between physiological and pathological actions of IL-17 based on the context. It is likely that steady-state (physiological) production of IL-17 by meningeal γδ T cells promote learning and memory, whereas excess production of IL-17 under pathological conditions that trigger inflammation may actually cause neurodegeneration due to neuroinflammation as seen in Parkinson’s disease and other conditions. It is possible that such a dual role may be seen with other cytokines, neurotransmitters, and neurotrophic factors. This argument is supported by the observation that TNF-α produced by glial cells enhances synaptic efficacy by increasing surface expression of AMPA receptors. Thus, it can be argued that continual presence of TNF-α is needed for preservation of synaptic strength at excitatory synapses. Similar to IL-17, excess production of TNF-α is known to induce apoptosis of neurons. This data suggests that there could possibly be an interaction between IL-17 and TNF-α in neuronal plasticity, synapse formation, and synaptic strength and finally in memory formation and cognitive function. In this context, it is noteworthy that both IL-17 and TNF-α interact with each other and have the ability to activate phospholipase A2 and induce the release of various PUFAs from the cell membrane lipid pool. These PUFAs, especially AA, can be utilized by the COX-2 to form PGE2, a known immunosuppressor and pro-­ inflammatory molecule. It is relevant to know that anti-inflammatory metabolite of DHA such as resolvin D1 suppresses the production of IL-17 and TNF-α and thus is able to restore homeostasis. It is likely that other pro-inflammatory cytokines IL-6, HMGB1, IL-1, and IL-2 may have actions similar to IL-17 and TNF-α in synaptic plasticity, synaptic strength, and memory formation. It is possible that circulating monocytes, macrophages, and glial cells produce the much-needed IL-6, TNF-α,

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and HMGB1 to induce synaptic plasticity, synaptic strength, and memory formation. Since all these pro-inflammatory cytokines are able to activate phospholipase A2 and induce the release of AA (and other PUFAs such as EPA and DHA) and enhance the formation of PGE2 that has the ability to suppress the production of IL-6, TNF-α, and other cytokines. In addition, the released AA, EPA, and DHA may be utilized to synthesize anti-inflammatory bioactive lipids such as lipoxin A4, resolvins, protectins, and maresins that antagonize the actions of pro-inflammatory cytokines (IL-6, IL-17, TNF-α, HMGB1, etc.). Thus, there is a cross talk among cytokines, PUFAs, and anti-inflammatory and pro-inflammatory bioactive lipids such that the delicate balance that is present under physiological conditions is maintained in order to help and sustain synaptic plasticity, synaptic strength, and memory formation. It is interesting to note that PUFAs (especially AA, DHA, and to a limited extent EPA) are needed for neuronal cell membrane extension by acting on syntaxin 3. For proper neuronal development and normal brain function and memory, it is important that growth of neurite processes from the cell body occurs and is a critical step that involves a large increase in cell membrane surface area. Cell membrane architecture is dependent on its lipid content – the higher the PUFA content the better for increasing the cell membrane surface area, its fluidity, and expression of various receptors that are needed for proper growth and development of brain. In this context, it is worthwhile to note that AA and other PUFA-releasing phospholipases are highly enriched in nerve growth cones and are believed to be involved in neurite outgrowth. For cell membrane expansion to occur, the fusion of transport organelles with the plasma membrane is needed. Studies revealed that syntaxin 3 (STX3), a plasma membrane protein that plays an important role in the growth of neurites, is activated by AA, DHA, and alpha-linolenic acid. Thus, PUFAs are needed for the activation of syntaxin 3 that, in turn, is essential for membrane expansion at the growth cones. Since TNF-α and IL-17 are needed for synapse formation, synaptic strength, and memory and as these cytokines activate phospholipase A2 to induce the release of PUFAs from cell membrane lipid pool, it is likely that there is a close interaction among these factors that ultimately determine synapse formation, synaptic plasticity, synaptic strength, and memory. It is relevant to note here that PUFAs have been thought to improve memory and are essential for brain growth and development. Furthermore, PUFA metabolites such as PGE2 and lipoxins, resolvins, protectins, and maresins have the ability to regulate IL-17, IL-6, and TNF- α production and action and have a negative and positive feedback control on their formation and release; it can be argued that there is a close connection among these molecules in the regulation of synapse formation, synapse strength, and memory (see Fig. 3.7 and accompanying references). The cross talk among cytokines (including IL-17/IL-23), bioactive lipids, and BDNF suggests that to maintain homeostasis, this positive and negative feedback is needed. For instance, IL-6 and TNF-α at (possibly) low doses (or at physiological concentrations) enhance synapse formation and strength, whereas at higher concentrations (as it happens during infections, trauma, etc.) they are harmful and induce

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apoptosis of neurons and could lead to memory loss, possibly, by enhancing the formation of pro-inflammatory PGE2. On the other hand, the purpose of AA/EPA/ DHA and their anti-inflammatory metabolites such as LXA4, resolvins, protectins, and maresins is to restrict or have a negative control on the formation of IL-6, TNFα, and HMGB1 and negate their harmful actions on neurons and memory. Both pro-inflammatory cytokines (IL-6, TNF-α, and HMGB1 and PGE2) and anti-­ inflammatory bioactive lipids (AA/EPA/DHA, LXA4/resolvins, protectins/maresins) seem to act on neurons by modulating the production and action of BDNF. Thus, it will not be surprising if BDNF has both positive and negative actions on neuron survival, synapse formation, synaptic strength, and brain development and growth. Thus, it is the question of the delicate balance between these two critical factors that ultimately determines memory formation and brain growth and development (see Fig. 3.7).

 ancer and Autoimmune Diseases Are Two Sides C of the Same Coin As already discussed above and depicted in Figs. 3.3, 3.4, 3.5, and 3.8a, both cancer and autoimmune diseases are pro-inflammatory conditions. Despite this, it is clear that there are some very distinct differences in the way the inflammatory process operates in these two conditions. In autoimmune diseases such as rheumatoid arthritis (RA), lupus, multiple sclerosis, and other diseases, there are distinct injury to the involved tissues: bones and synovial membrane in RA; skin, blood vessels, and kidney (sometimes brain) in lupus; and neurons in MS. But what is not clear is why joint deformities occur in RA but not in lupus, why renal involvement is more common in lupus but not in RA, and why in MS there is no involvement of tissues other than the brain. These facts suggest that local inflammatory events are more important than systemic inflammatory changes despite the fact that there are systemic inflammatory signs and symptoms such as fever, leukocytosis, loss of appetite, etc. that occur in all these conditions. In contrast to this, in cancer both local and systemic manifestations are not uncommon and yet times systemic events such as cachexia and immunosuppression are more dominant that may ultimately result in significant morbidity and mortality. In cancer, local events may lead to dislodgment of tumor cells and cause metastasis that may be fatal in some. These similarities in local and systemic manifestations in autoimmune diseases and cancer are quite striking. But, in autoimmune diseases, local manifestations are more dominant such as arthritis in RA and lupus and neurological manifestations such as headache, cranial nerve palsies, gait abnormalities, and muscular weakness (such as cerebellar manifestations, paraparesis, quadriparesis, monoparesis, etc.) in MS. But, paradoxically, in both autoimmune diseases and cancer, inflammation seems to be the root cause. In autoimmune diseases, the inflammatory events seem to be more dominant as a result of recognition of self as foreign and mounting of an immune attack, whereas in cancer there is failure on the part of the

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immune system of the abnormal cells (cancer cells) as foreign and so an appropriate immune attack is not mounted. Despite the failure of recognition of cancer cells as foreign, some amount of inflammation occurs at the site of cancer. Thus, it can be said that in autoimmune diseases there is predominantly systemic inflammation with local inflammation as well, whereas in cancer local inflammation predominates with systemic inflammatory events being seen in the late stages (advanced cancer). Despite these seemingly striking differences between autoimmune diseases and cancer, it is interesting to note that subjects with autoimmune diseases have higher incidence of cancer especially those with RA and lupus who develop predominantly lymphomas. With the recent development of immune checkpoint inhibitor (ICI) therapy for cancer, it is becoming evident that some of these patients are likely to develop autoimmune diseases. Thus, both autoimmune diseases and cancer can be considered as two sides of the same coin (see Fig. 3.8a). There is a significant amount of evidence to suggest a role for IL-17, IL-6, and TNF-α and PGE2, LTs, and TXs in autoimmune diseases such as RA and lupus. Similarly, there is evidence to propose a role for IL-17, IL-6, TNF-α, and other pro-­ inflammatory cytokines and PGE2 in cancer. Thus, same molecules seem to participate in both cancer and autoimmune diseases suggesting that similar approaches in their management are possible. In Table 3.1, similarities and contrasting features between cancer and autoimmune diseases are given. It is clear from this table that there are certainly some overlapping features between autoimmune diseases and cancer especially with regard to the pro-inflammatory cytokines. In both diseases, increased levels of IL-6, TNF-α, and IL-17 are seen; though in the case of autoimmune diseases, increased plasma levels of these cytokines are seen, in cancer they are predominantly seen at the site of cancer. This suggests that autoimmune diseases are predominantly systemic diseases, whereas cancer is a more localized disease (at least in the initial stages). But it needs to be noted that lupus, RA, and other autoimmune diseases may start locally in a specific tissue or organ and later spread to the whole organ/system or whole body. For instance, RA may start in one joint to start with and later may involve several joints. Similarly, lupus may start initially as nonspecific skin rash or arthralgia and later show more systemic manifestations. Thus, at the molecular/biochemical level, there seems to be a role for same cytokines in both these diseases. Theoretically, it is expected that in autoimmune diseases, there will be decreased expression of PD-1 and PD-L1, whereas in cancer their expressions are increased to escape the immunosurveillance system. It is possible that PD-1 expression may be increased, but PD-L1 expression may be decreased that ultimately results in the development of autoimmune diseases. For instance, it was reported that PD-1 is expressed on infiltrating lymphocytes, while PD-L1 is expressed on synovial lining cells and the expression of PD-L1 on synovial lining cells significantly correlated with the active state of the disease in RA. It is evident from the details given in Table 3.1 that there are many similarities between autoimmune diseases and cancer, implying that same therapeutic strategies could be useful in the prevention and management of these diseases as briefly outlined under legend to Table 3.1. This aspect is further elaborated in a subsequent section of this book.

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Table 3.1  Comparison between autoimmune diseases and cancer with regard to their biochemical, immunological, and management issues Parameter Systemic inflammation

Autoimmune diseases More common↑↑↑

Local inflammation

Common↑↑↑

Systemic manifestations of disease such as loss of appetite, loss of weight, fever, etc., Plasma PGE2 levels Plasma IL-17 Plasma IL-6 Plasma TNF-α Plasma LXA4

More common↑↑↑

Plasma PUFAs (especially AA, EPA and DHA) Autoantibodies Immunosuppression

↓↓↓

↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↓↓

+++ Unlikely except when administered immunosuppressive drugs as part of treatment Immune system attacks Self and self-antigens and produces non-self-recognition: PD-1 and PD-L1 expression disease PD1 and PD-L1 expression decreased (abnormal) Management

Immunosuppressive drugs used include anti-cancer drugs methotrexate, cyclophosphamide, etc.

Cancer Less common expect in late stages↑ More common than systemic inflammation↑ Less common but seen in late stages of disease↑

↑ ↑ ↑ ↑ ↓local levels at the site of cancer is more common than systemic levels ↓ ± Common

Immunosurveillance fails PD-1 and PD-L1 expression is increased. Immune checkpoint inhibitors use may lead to the development of autoimmune diseases Most anti-cancer drugs are immunosuppressors

Plasma, synovial fluid, and urinary levels of IL-6, TNF-α, and IL-17 are increased in those with active RA and lupus [125, 126]. Simultaneously these patients may have low plasma concentrations of anti-inflammatory cytokines such as IL-10. Patients with RA and lupus also have increased plasma, urinary, and synovial fluid levels of PGE2 and TXA2 levels [127–136] implying an increase in pro-inflammatory eicosanoids. In addition, decreased plasma levels of DGLA, AA, EPA, and DHA in RA and lupus have been described [128–132]. Recent studies suggested that patients with lupus and RA and other rheumatological (and autoimmune) conditions may have low plasma and urinary levels of lipoxin A4 (LXA4) [137–143], and restoring LXA4 levels to normal may resolve arthritis (especially in RA). In this context, it is interesting to note that resolution of active arthritis needs cyclooxygenase-2 (COX-2) activity. COX-2 and its metabolite, PGE2, are present in the joints during resolution. Blocking COX-2 activity and consequently reducing PGE2 formation led to continuation of inflammation in contrast to the expectation that reducing PGE2 levels will resolve inflammation. Subsequent studies revealed that repletion of PGE2 attenuated inflammation by enhancing the formation of LXA4, a lipoxygenase metabolite formed from (continued)

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Table 3.1 (continued) AA. These results imply that there is a close link between cyclooxygenase-lipoxygenase pathways and PGE2 serves as a feedback inhibitor essential for limiting inflammation. This may explain as to why use of COX-2 inhibitors alone is not of much use in the management of RA and other inflammatory conditions. This is supported by the finding that inhibition of 15-PGDH (15-prostaglandin dehydrogenase) enzyme results in a twofold increase in PGE2 levels in several tissues such as bone marrow, colon, and liver responds to partial hepatectomy with a greater than twofold increase in hepatocyte proliferation and are resistant to chemical-induced colitis. Furthermore, 15-PGDH inhibition accelerated recovery of erythropoiesis after bone marrow transplantation [144]. These results imply that 15-PGDH and possibly PGE2 may serve as negative regulators of tissue regeneration and repair in several tissues including the bone marrow, colon, and liver. It is not clear whether 15-PGDH inhibition and consequent increase in PGE2 levels will lead to enhanced formation of LXA4, since in these studies [144] the authors did not measure LX4 levels in various tissues. It is possible that acute increase in PGE may cause inflammation, whereas enhanced levels persisting for long periods of time as it would happen when 15-PGDH is inhibited may have anti-inflammatory actions. Thus, depending on the context, PGE2 may have both proand anti-inflammatory actions. Based on these evidences, it can be proposed that enhanced levels of PGE2 may serve as a signal for an increase in the formation of LXA4. Since both PGE2 and LXA4 are formed from AA, these studies imply that once the tissue levels of PGE2 reach a certain critical level, it leads to formation of LXA4 from AA that not only resolves inflammation but also enhances tissue regeneration. This proposal is supported by the observation that oral supplementation of AA does not affect PGE2 levels but enhances the formation of LXA4 [145, 146]. It was observed by us that such oral supplementation of AA suppresses inflammation by inhibiting the formation of IL-6 and TNF-α and the expression of NF-kB [87, 147]. It is noteworthy that anti-­ inflammatory cytokines IL-4 and IL-10 trigger the conversion of AA, EPA, and DHA to lipoxins, resolvins, protectins, and maresins suggesting a mechanism by which they are able to suppress inflammation [137, 148]. In both autoimmune diseases and cancer, an increase in IL-17 levels has been described [85, 149– 156]. It is noteworthy that IL-17 not only promoted lung cancer growth but also contributed to the resistance to PD-1 blockade and promoted inflammation, factors that worsen prognosis of cancer [156]. As already described above, IL-17 interacts with PGE2, IL-23, IL-6, TNF-α, and immune checkpoint inhibitors (PD-1 and PD-L1) and thus may facilitate tumor cell growth (see Fig. 3.8a). Thus, there are many overlapping features between autoimmune diseases (especially RA and lupus) and cancer implying that both could be managed by same, if not identical, therapeutic strategies. In this context, the role of PUFAs (especially AA, EPA and DHA) and their anti-inflammatory metabolites LXA4, resolvins, protectins, and maresins in these diseases may prove to be relevant. It is possible that oral or intravenous administration of AA/EPA/DHA/GLA/DGLA and vitamin C, B1, B6, and B12 in conjunction with immunosuppressive drugs such as corticosteroids and cyclophosphamide/methotrexate/cyclosporine may be effective against both RA/lupus and cancer. Both PUFAs and vitamin C may serve as antioxidants with regard to autoimmune diseases and as pro-­ oxidants in cancer to eliminate tumor cells. This aspect has been discussed in more detail in another section of this book.

IL-17 and Fibrosis One of the consequences of long-standing inflammation (chronic inflammation as seen in TB, RA, chronic hepatitis) is the development of fibrosis in the target organ/ tissues. In some instance, development of fibrosis may prove to be more harmful than the underlying disease. This is particularly true of lung fibrosis seen in those with TB and cirrhosis of the liver after hepatitis infection. Thus, understanding the

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molecular basis of fibrosis is clinically relevant so that appropriate interventions could be developed to prevent end-organ/tissue damage and functional incapacity. Recent studies suggested that IL-17 may have a role in the development of fibrosis. One of the cell types that plays a major role in tissue repair and fibrosis could be macrophages that are known to be involved in inflammation, removal of debris, and tissue regeneration and repair. Traditionally macrophages have been classified as M1 and M2 types depending on their ability to induce inflammation and/or tissue repair and regeneration. For instance, M1-type macrophages are believed to be pro-­ inflammatory in nature and are activated by IFN-γ, whereas M2 macrophages promote a pro-regenerative tissue microenvironment that leads to induction of tissue repair by increased expression of IL-4 and its secretion. This action of M2 macrophages seems to need TH2 cell response that shows a reduced CD86 expression. The pro-inflammatory M1 macrophages not only produce inflammation at the site of injury but may also be associated with TH1 response. IL-17A signaling via IL-17 receptor A (IL17ra) induces inflammation by recruiting innate immune cells and has a role in neutrophilic granulocyte regulation. It has been shown that IL17ra is needed for tissue macrophage formation and fibrosis to occur recruiting IL-36γ macrophages (alternatively IL-36γ macrophages drive IL-17 to produce fibrosis). In contrast to this, downregulation of IL-17 prevents fibrosis. Bone marrow-derived stem cells when transplanted are able to suppress the production of IL-17 and thus prevent fibrosis. In addition, both IL-6 and TNF-α seem to cooperate with IL-17 in inducing inflammation and consequent development of fibrosis in various tissues. IL-17 enhances fibroblast proliferation and augments the deposition of collagen that ultimately leads to fibrosis (see Figs. 3.6, 3.7, and 3.8a). In this context it is noteworthy that anti-inflammatory bioactive lipids LXA4, resolvins, protectins, and maresins are capable of preventing fibrosis, at least, in part by suppressing the production of IL-6, TNF-α, and IL-17 (see Figs. 3.6, 3.7, and 3.8a and references given in the legends to these figures).

Macrophages in Tissue Repair (Regeneration) and Fibrosis Despite the fact that macrophages appear or look similar under microscope and are seen in all tissues at the time of infection, injury, repair or regeneration, and fibrosis, it is likely that at the molecular level, there are several subtypes of macrophages that have distinct and unique functions depending on the subtype, context, time taken to appear from the onset of infection or injury, and type of tissue that is under observation (it is possible that there are subtypes of macrophages for each type of tissue or organ). It was reported that the regenerative condition is marked by the presence of two clusters of macrophages, R1 (CD9  +  CD301b  +  MHCIIhi) and R2 (CD9 − CD301b + CD206+), whereas cells present in the profibrotic microenvironment are characterized by the presence of the two terminal clusters F1 (CD9  −  CD301b  −  MHCIIhi) and F2 (CD9hiCD301b  −  MHCII−IL36g+). I

a

LPS and other Stimuli Activation of PLA2

Release of Arachidonic acid COX EP2-EP4PI3K

TH1 cells↑

EP2-EP4cAMPPKA

PGE2 GPCRs

cAMP↑

DCs

TH17 Cells

EP4cAMP -Epac

TH1 cells↓

IL-23

CRPC

MDSCs

b

Lipoxins AA NO

IL-10↑ p53↓

Treg cells↑

PGE2

IL-17

IL-6, TNF-α↓

Resistance to Anti-VEGF

CTL↓

Tumor cell proliferation, angiogenesis, metastasis↑

IFN-γ↓ CSC↑

↓

Fig. 3.6 (a) Scheme showing the role of PGE2 and its receptors in the regulation of TH1 and TH17 cell differentiation and proliferation. Recent studies revealed that IL-23 produced by myeloid-­ derived suppressor cells (MDSCs) act as a driver of castration-resistant prostate cancer (CRPC) in mice and patients with CRPC. IL-23 secreted by MDSCs activate the androgen receptor pathway in prostate tumor cells, promoting cell survival and proliferation in androgen-deprived conditions. Intratumor MDSC infiltration and IL-23 concentration are increased in blood and tumor samples from patients with CRPC. Inactivation of IL-23 using antibody to IL-23 restored sensitivity to androgen-deprivation therapy in mice, suggesting that MDSCs promote CRPC by acting in a non-­ cell autonomous manner. Blocking IL-23 opposes MDSC-mediated resistance to castration in prostate cancer (Nature 2018 Jun 27. doi: https://doi.org/10.1038/s41586-018-0266-0). (b) Actions of PGE2 on various cytokines and immunocytes and its relationship to tumor cell behavior. CSC cancer stem cells. EPA and DHA may have actions similar to AA and so also their metabolites such as resolvins, protectins, and maresins

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IL-17 Exercise

Exercise

IL-6, TNF-α, HMGB1

PLA2

BDNF

? PUFAs, especially AA and DHA

(+)

Syntaxin 3

LXA4, RSVs, PRTs, MaRs

PGE2

(-) Exercise

(+)

Synapse formation and strength Exercise

? Memory

Fig. 3.7 Scheme showing possible interaction(s) among cytokines, bioactive lipids, syntaxin 3, synapse formation and strength and memory. LXA4 lipoxin A4, RSVs resolvins, PRTs protectins, MaRs maresins, PLA2 phospholipase A2. IL-17, IL-6, TNF-α, and HMGB1 are known to activate PLA2 and induce the release of PUFAs (especially AA, EPA, and DHA) from the cell membrane phospholipid pool [74–79]. The released PUFAs, especially AA, can be utilized for the formation of pro-inflammatory PGE2 and/or anti-inflammatory LXA4. Both PGE2 and LXA4 can inhibit the release and actions of IL-7, TNF-α, and HMGB1. PGE2 is needed for the initiation of TH17 CD4+Tcell development in collaboration with IL-23. EPA/DHA can attenuate IL-17-induced inflammation by increasing the expression of FoxP3 and Treg cell differentiation and thus reduce IL-17 production [34, 80]. In contrast, LXA4, resolvins, protectins, and maresins seem to have the ability to suppress L-17 production, partly, by inhibiting IL-23 production and suppressing migration of dendritic cells and γδ T cells (that produce IL-17) [40, 42, 48, 49, 81]. IL-17 can enhance the production of TNF-α and possibly other pro-inflammatory cytokines such as IL-6 and HMGB1 [82–86]. In contrast, AA/ EPA/DHA can suppress the production of pro-inflammatory IL-17, TNF-­α, and HMGB1 cytokines [87–92]. Both IL-17 and TNF-α have the ability to enhance synaptic plasticity and synapse strength [93–95]. This coupled with the observation that IL-6, TNF-α, and IL-17-induced PLA2 activityrelated release of PUFAs activate syntaxin 3, a plasma membrane protein, that is essential for cell membrane expansion, and the growth of neurites [96–98] suggests that cytokines and PUFAs work in a coordinated fashion to regulate synapse formation, synapse strength, and memory. By acting on syntaxin 3, PUFAs are able to enhance brain growth and ultimately memory enhancement.

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propose that profibrotic macrophages secrete IL-36, IL-17, and IL-23 and cooperate with cells that secrete IL-6 and TNF-α, whereas R1 and R2 macrophages are capable of secreting anti-inflammatory cytokines IL-4, IL-10, LXA4, resolvins, protectins, and maresins. But these proposals need to be verified and confirmed.

PUFAs, Ion Channels, Cell Proliferation, or Apoptosis Yet another action of PUFAs that is relevant to their cytotoxic action on tumor cells is their ability to modulate the properties of ion channels as shown in Fig.  3.8b. PUFAs can modify the properties of TRPV group of transient receptor potential family of ion channels and Piezo1 and Piezo2 channels. It is known that there could occur a close interaction between Piezo channels and TRPV ion channels. Under the legend to Fig. 3.8b, a detailed discussion on the role of PUFAs in modulating

Fig. 3.7  (continued)  The inhibitory action of PUFAs and their anti-inflammatory products (LXA4, resolvins, protectins, and maresins) on IL-6, IL-17, TNF-α, and HMGB1 speaks of the positive and negative feedback regulation among all these factors to maintain homeostasis not only in the regulation of immune response but also in brain growth and development and memory formation. In addition, it was shown that IL-17 enhances the production of BDNF to produce its action of enhancing synaptic plasticity and short-term memory [95] is interesting since we observed that PUFAs can enhance the formation of BDNF and BDNF and lipoxin A enhance each other’s synthesis and secretion [99] implying that cytokines, PUFAs, LXA4, and other similar anti-­inflammatory bioactive lipids and BDNF act in a coordinated manner to regulate dendritic growth of hippocampal neurons. There could be other actions of PUFAs that can also influence neuronal growth and synapse formation and its strength such as their action on Piezo-1 and TRPV1 channels, etc., and this has been discussed elsewhere in this book. It is noteworthy that regular exercise improves memory [100]. Exercise is known to enhance brain and plasma BDNF levels [100], increase the formation of LXA4 (lipoxin A4) and possibly resolvins, protectins, and maresins that are anti-inflammatory in nature [101] and reduce plasma IL-6 and TNF-α levels [102]. LXA4, resolvins, protectins, and maresins reduce IL-17 generation, in part, by inhibiting IL-23 generation and action [48]. Thus, exercise is anti-inflammatory in nature. During exercise there is an increase in plasma IL-6 and TNF-α levels (derived from muscles), and generation of ROS (reactive oxygen species) that triggers generation of endogenous MnSOD, and thus antioxidant capacity is enhanced. This could be one of the reasons for the anti-inflammatory nature of exercise. Once the exercise is completed, there would occur a precipitous fall in IL-6, TNF-α, and ROS levels below the pre-­exercise level. The increase in the synthesis and release of LXA4 (seen as increased excretion of LXA4 in the urine following exercise) are also anti-inflammatory in nature. As a result of an increase in LXA4 secretion, there will occur a fall in the formation of PGE2 (a pro-inflammatory molecule). Thus, ultimately regular exercise serves as an anti-inflammatory event. Increase in the levels of BDNF and LXA4 may enhance synapse formation and strength that finally results in improvement in memory. Exercise is known to decrease the incidence of cancer. This could be related to the anti-inflammatory nature of exercise. The spike in IL-6, TNF-α, and ROS seen during exercise may be yet another reason for the decreased incidence of cancer. It is likely that the short duration of spike in cytokines and ROS seen during exercise results in apoptosis of small number of cancer cells that arise on a day-to-day basis in our body. Furthermore, LXA4 is known to inhibit the growth of tumor cells (unpublished data). Thus, changes in the concentrations of bioactive lipids, ROS, and BDNF seen may explain many of the beneficial actions of exercise

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IL-17 IL-23

PGE2

R1 and R2 MØ

PLA2

F1 and F2 MØ IL-36γ MØ

Release of PUFAs DGLA

PD-1 & PD-L1

PGE1

Arachidonic acid

Fibrosis

LXA4

EPA

Resolvins of E series

DHA

Resolvins of D series. Protectins and Maresins

TRPV1 IL-6, TNF-α, HMGB1

? BDNF Piezo1

(+) Cancer

TRPV1

Autoimmune diseases

Fig. 3.8 (a) Scheme showing potential relationship and interactions among cytokines, bioactive lipids, BDNF, and PD-1 and PD-L1 and their potential role in cancer and autoimmune diseases. IL-17, IL-23 and PGE2 act together to induce pro-inflammatory status in autoimmune diseases. Cytokines IL-17, IL-23, IL-6, TNF-α, and HMGB1 activate phospholipase A2 to induce the release of PUFAs (especially DGLA, AA, EPA, and DHA) that form precursors to PGE1, PGE2/LXA4, resolvins, protectins, and maresins as shown in the figure. DGLA, AA, EPA, and DHA suppress the production of IL17, IL-23, IL-6, TNF-α, and HMGB1 and thus have a negative feedback control on the formation of pro-inflammatory cytokines. IL-17 enhances resistance to PD-1 and PD-L1 blockade. LXA4, resolvins, protectins, and maresins inhibit inflammatory process and thus are useful in protections against autoimmune diseases such as rheumatoid arthritis, lupus, inflammatory bowel disease, and multiple sclerosis. In addition, LXA4, resolvins, protectins, and maresins inhibit proliferation of tumor cells. Similar and more potent anti-cancer action is shown by DGLA, AA, EPA, and DHA and induces apoptosis of various types of tumor cells. PUFAs may also suppress the expression of PD-1 and PD-LI and thus may assist in overcoming immunosuppression seen in cancer. Furthermore, these PUFAs can act on Piezo1 channel that is capable of mediating mechanoelectrical transduction that, in turn, regulates several crucial cellular processes including cell migration [103]. This action of PUFAs on Piezo1 could be attributed to their ability to change cell membrane fluidity. Similarly, PUFAs can regulate the other ion channel, namely, the TRPV1. There seems to be an interaction between Piezo1 and TRPV1 channels. Thus, PUFAs by their ability to alter the properties of Piezo1 and TRPV1 channels can regulate membrane voltage changes that, in turn, would alter the cell adhesion, cell volume, apoptosis, and angiogenesis [104]. Since many cancer cells overexpress K+, Na+, Ca2+, and Cl− channels [105], it is likely that incorporation

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= PUFAs; LP

= Lipid peroxides;

= Calcium; G

= Saturated fatty acid;

= Glucose.

Fig. 3.8 (continued)

= Potassium;

= TRPV1; = Sodium;

= Piezo1 = Chloride;

= Magnesium ions; PLA2 = Phospholipase A2;

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Fig. 3.8  (continued)  of various PUFAs into the cell membrane can effectively alter these channels leading to changes in their mitotic and other properties (see Fig. 3.8b). This could be one of the many actions of PUFAs to result in the arrest of growth of cancer cells and their eventual apoptosis. Not many studies have been performed on the action of PGs, LTs, TXs, lipoxin A4, resolvins, protectins, and maresins on ion channels, especially on Trpv 1 and Piezo1. But it is likely that these bioactive lipids can also alter the behavior of various ion channels. For instance, it has been shown that PGE2 activates Ca2+channels [106]. It is likely that other bioactive lipids may also have similar actions on various ion channels and Trpv1 and Piezo1. It may be mentioned here that IL-17 may also have a role in fibrosis that is seen in chronic inflammatory conditions such as TB, RA, and post-hepatitis cirrhosis of the liver [107–116]. In this pro-fibrotic action of IL-17, there seems to be a role for IL-36γ-­producing macrophages, IL-6, and TNF-α by exacerbating inflammation and regulating the action of IL-17 (by enhancing the formation and release and possibly stabilizing and enhancing the half-­life of IL-17). IL-17 enhances the proliferation of fibroblasts and deposition of collagen. In contrast to this, PUFAs and LXA4, resolvins, protectins, and maresins suppress IL-6, TNF-α, and IL-17 production and action, inhibit fibroblast proliferation, and thus prevent fibrosis [117–122]. It is important to note that fibrosis is common in RA and almost not seen in lupus and cancer when regresses/regressed heals by local fibrosis. Thus, there are both positive and negative aspects of fibrosis. When healing occurs, it does so by fibrosis (especially in TB), and this could alter the architecture of the lung and lead to post-fibrotic sequelae. In the case of cancer, local fibrosis occurs in order to restrict the growth of the tumor. In the case of RA, fibrosis that occurs in the affected joints leads to ankylosis that restricts the movement of the involved joints leading to significant immobilization and morbidity. Thus, IL-17 seems to have both beneficial and adverse actions – beneficial since it improves memory and adverse actions in the form of fibrosis that could lead to significant mobility disorders or able to restrict tumor growth. Ultimately, the balance between bioactive lipids and IL-17 and IL-36 is important and determines the final outcome of the disease. It was reported that the regenerative condition is marked by the presence of two clusters of macrophages, R1 (CD9 + CD301b + MHCIIhi) and R2 (CD9 − CD301b + CD206+), whereas profibrotic microenvironment is characterized by the presence of the two terminal clusters F1 (CD9 − CD301b − MHCIIhi) and F2 (CD9hiCD301b − MHCII−IL36g+) [123]. It is not known but likely that profibrotic macrophages secrete IL-36, IL-17, and IL-23 and cooperate with cells that secrete IL-6 and TNF-α, whereas R1 and R2 macrophages are capable of secreting anti-­ inflammatory cytokines IL-4, IL-10, and LXA4 and resolvins, protectins, and maresins. But these proposals need to be verified and confirmed. Hence, employing strategies that enhance the formation of LXA4, resolvins, protectins, and maresins at the most appropriate time (before fibrosis sets in) from their precursor AA, EPA, and DHA is important [103–122]. (b) Scheme showing possible relationship among ion channels, fatty acids, and cell proliferation or apoptosis. (Modified from Ref. [3] given below). Fatty acids form an important constituent of cell membrane. These fatty acids can be saturated and unsaturated fatty acids. Depending on the amount of these fatty acids, the cell membrane can be fluid or rigid. The nature of the cell membrane determines the expression and function of various membrane receptors. Thus, in general, if the membrane is more fluid, the number of receptors (such as insulin) will be more, whereas if the membrane is more rigid, the number of insulin receptors may decrease. Cell membrane also contains several ion channel voltage-­gated ion channels (VGIC) that allow the diffusion of ions such as K+, Ca2+, Cl−, and Na+. These ion channels play a significant role in controlling rapid bioelectrical signaling including action potential and/or contraction. These classes of proteins can also contribute to cell mitotic signaling, cell cycle progression, as well as cell volume regulation. Thus, they are critical for cancer cell proliferation. Phosphatidylserine (PS) is a phospholipid and is an important constituent of the cell membrane. PS plays a key role in cell cycle signaling, especially in relation to apoptosis. PS consists of two fatty acids attached in ester linkage to the first and second carbon of glycerol and serine attached through a phosphodiester linkage to the third carbon of the glycerol. Most phospholipids have a saturated fatty acid on C-1 and an unsaturated fatty acid on C-2 of the glycerol backbone. The fatty acid distribution at the C-1 and C-2 positions of glycerol within phospholipids is continually in flux, owing to its continuous degradation and remodeling. PS typically carry a net charge of −1 at physiological pH. PS mostly has palmitic or stearic acid on carbon 1 and a

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Fig. 3.8  (continued)  long-chain unsaturated fatty acid (such as 18:2, 20:4 and 22:6) on carbon 2. But this composition of PS is amenable to alteration depending on the diet, supplementation, state of the cell, environment, and stimuli to which the cell is exposed. In addition to these VGIC, there are two other ion channels, namely, TrpV1 and Piezo1. The transient receptor potential cation channel subfamily V member 1 (TrpV1) is a member of the TRPV group of transient receptor potential family of ion channels. In general, the function of TRPV1 is detection and regulation of body temperature and provides a sensation of scalding heat and pain. Piezo1 and Piezo2 are nonselective Ca2 + −permeable cation channels that interact with Trpv1 [124]. Changes in the cell membrane lipid composition can alter the activities of all these channels that may have profound effect on cell proliferation, cell volume, and cell metastasis (cancer metastasis, [103, 104]). Recent studies suggested that plasma membrane depolarization induces reorganization of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. This results in K-Ras plasma membrane organization leading to amplification of K-Ras-dependent mitogen-activated protein kinase (MAPK) signaling, whereas plasma membrane repolarization disrupts K-Ras nanoclustering and inhibits MAPK signaling. Changes in cell membrane composition thus can lead to changes in VGIC, TrpV1, and Piezo1 that can either enhance mitosis (as seen in cancer) or suppress mitosis or cause apoptosis of cell. It is suggested that under normal physiological conditions, the cell membrane will contain a balanced ratio between saturated fatty acids and polyunsaturated fatty acids (see Fig. 3.8bi) resulting in PS to occur in small clusters that localize to K-Ras that leads to low activation of RAF-MAPK pathway. Cancer cell may contain more saturated fatty acids and less number (amount) of polyunsaturated fatty acids that results in changes in membrane fluidity (more rigid) leading to clustering of PS and K-Ras that promotes RAF-MAPK signaling, and consequently uncontrolled proliferation occurs (see Table 3.1 for fatty acid composition of cancer cells). When tumor cells are supplemented with PUFAs, it could result in cell membrane to become more fluid and formation of excess of lipid peroxides that can disrupt PS and K-Ras clustering and their inactivation and results in arrest of cell proliferation and apoptosis. Changes in cell membrane fluidity and its composition can affect PS composition, changes in the expression, and function of various ion channels including Trpv1 and Piezo1 as shown in the figure. This may result in perturbed ion transmission across the channels and the membrane resulting in cell apoptosis. Lipid peroxides that accumulate in the cell as a result of PUFA supplementation may inactive various ion channel receptors, block K-Ras and MAPK pathway, or suppress it (see Table 3.1 for lipid peroxides levels in normal and tumor cells). Furthermore, changes in lipid composition of the cell membrane may also alter T cell proliferation activation and local response of T cells to the tumor cells [157]. It is likely that under normal physiological conditions, a normal cell would contain balanced amounts of saturated and polyunsaturated fatty acids (this could be say ~1:1) so that normal fluidity of the membrane is maintained. On the other hand, tumor cells have more amounts of saturated fatty acids, and so the cell membrane is more rigid (the ratio between saturated and PUFAs could still be ~1:1 but with an increase in oleic acid content and drastic reduction in AA content; see Table 3.1 for fatty acid composition of normal and tumor cells). This change in the membrane fluidity will make the Piezo1 and Trpv1 channels and other ionic channels behave abnormally and activate oncogenes resulting in abnormal mitosis. In contrast, when tumor cells are supplemented with PUFAs, the PUFA content of the membrane is increased resulting in an increase in membrane fluidity that can also render Piezo1 and Trpv1 and other ionic channels abnormal resulting in accumulation of excess of Ca2+ and other ions such as K+, Ca2+, Cl−, and Na+ and suppression of expression of oncogenes that can result in arrest of mitosis and/or lead to apoptosis of the cells. It is possible that K+ and other ions may leak from the cells into the surrounding milieu that may act on infiltrating macrophages and T cells and suppress immune response against tumor cells and thus aid in the escape of tumor cells from immunosurveillance system [158]. AA and other PUFAs activate potassium channels [159, 160] and thus may aid in enhancing the T-cell response by removing excess of potassium that accumulates in the tumor cell milieu. In addition, KATP channels are inactivated by high glucose concentrations [161], a key factor that may explain why tumor cells have aerobic glycolysis. In contrast, GABA (gamma-aminobutyric acid) inhibits KATP channels [161] that explains the regulatory role of neurons/local nerves in tumor growth [162, 163]. Cancer cells form synaptic connections with neurons that are facilitated by cell adhesion proteins such as

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these ion channels has been outlined. It is possible that this action of PUFAs (and possibly for various PGs, LTs, TXs, LXA4, resolvins, protectins, and maresins including lipid peroxides) on various ion channels may explain several actions of these bioactive lipids including their role in inflammation (such as leukocyte, macrophage, and T-cell chemotaxis, proliferation, and secretion of their various products), resolution of inflammation, immune response, fibrosis, tissue regeneration, epithelial to mesenchymal transition, and induction of apoptosis, ferroptosis, and necrosis of tumor cells.

Prostanoids in Inflammation PGD2 (prostaglandin D2), PGE2, PGF2α, PGI2, and thromboxane A2 derived from arachidonic acid (AA, 20:4 n-6) in response to various stimuli by the action of cyclooxygenase and respective synthases are present in significant amounts at the sites of inflammation [168–179]. PGE2 is known to have immunomodulatory action, and it has been shown to suppress TH1 differentiation by increasing intracellular cyclic AMP (cAMP) concentrations [177–179]. PGE2 acts on four G protein-­ coupled receptors: EP1, EP2, EP3, and EP4. EP2 and EP4 signaling is coupled to a rise in cAMP concentration. Immunosuppressive action of PGE2 is mediated by both EP2 and EP4, and selective blockade of PGE2-EP4 suppresses progression of autoimmune disease such as experimental autoimmune encephalomyelitis (EAE), a

Fig. 3.8  (continued)  neurexins and neuroligins [164]. When such synapse is formed, neurotransmitters such as glutamate may be released that can bind and activate AMPA and NMDA receptors that cause positively charged ions to enter the cells through the receptors to cause depolarization leading to a rise in intracellular positive charge that results in cancer cell migration and proliferation that is in tune with the previous studies [158–163]. In addition, potassium leakage from cells activates Ca2+independent phospholipase A2 which, in turn, facilitates the cleavage of pro-IL-1β by the IL-1 converting enzyme caspase-­1 [165, 166], and this action of potassium on IL-1-converting enzyme can be overcome by other monovalent cations such as sodium. High intracellular concentrations of potassium suppress apoptosis [167], a fact that is in tune with the evidence that intracellular potassium concentration in many cells, in general, is higher compared to its extracellular concentration. Thus, the high potassium in the tumor microenvironment suppresses immune response [158] and provokes further apoptosis of tumor cells such that immunosuppression against tumor cells persists for a longer time. In addition, it will also lead to apoptosis of T cells (since the concentration of potassium is higher in the tumor microenvironment compared to intracellular levels of T cells). Furthermore, phospholipase A2 can induce the release of PUFAs from the cell membrane lipid pool and PUFAs can activate potassium channels [159, 160]. Thus, there is a close interaction among local (especially extra and intracellular) concentrations of Ca2+ and other ions such as K+, Ca2+, Cl−, Na+; phospholipase A2 activity, IL-1 and possibly other cytokines; glutamate, GABA and other neurotransmitters and tumor growth. Thus, tumor milieu (including intra- and extracellular glucose concentration) contributes to tumor cell proliferation. It may be noted here that there could be a close interaction among various ions within themselves and with intracellular and extracellular glucose concentrations. Glucose can activate or suppress the activity of phospholipase A2 depending upon its local concentration. Thus, glucose can influence lipid peroxides formation

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mouse models for multiple sclerosis. PGE2 acts through EP2 and EP4 to facilitate TH1 differentiation and enhances IL-23-induced TH17 expansion that is mediated by its action on cAMP. In contrast, TH1-differentiating action of EP2 and EP4 is mediated by PI3K and not cAMP. Thus, PGE2 actions are exerted through two different signaling modules of EP2 and EP4. Furthermore, PGE2-EP4 pathway is needed for the production of IL-23 by DCs when are activated by antibody to CD40. PGE2 itself cannot induce IL-23 production. It was reported that activation of bone marrow-­derived DCs with lipopolysaccharide when exposed to PGE2 could lead to increase in IL-23 production through the EP4-cAMP-Epac pathway since activated DCs contain enhanced activity of PG synthase enzyme. It is intriguing as to how this EP4-cAMP-Epac (Epac = exchange protein directly activated by cAMP) signaling regulates IL-23 production, how EP2- and EP4-PI3K signaling regulates TH1 differentiation, and how EP2- and EP4-cAMP-PKA signaling is involved in TH17 expansion, and it is likely that this may, partly, depend on the way G protein-­coupled receptor signaling interacts with cytokine and co-stimulation signaling (Fig. 3.6a, b). In general, it is believed that PGE2 has little role in adaptive immunity. But it is evident from the preceding discussion [168–180] that prostanoid receptors do regulate immune response at various steps [168–183]. Based on this data, it is evident that selective manipulation of E1, EP2, EP3, and EP4 receptor’s signaling could be used to manipulate and control immune response. Of all, EP4 is a promising drug target for immunomodulation. This is supported by the observation that stimulatory action of PGE2 on TH17 differentiation or expansion in  vitro is noted in human peripheral blood mononuclear cells implying that it is possible to manipulate EP4 signaling to control pathological conditions in various immune diseases in humans [184, 185].

PGE2 and PD-1 and CTL (Cytotoxic T Lymphocyte) Function In the light of the relationship of PGE2 to the development of TH17 cells, it is a surprise that PGE2 also has a role in the impairment of CTL function in coordination with PD-1. PGE2 has pro-inflammatory actions and is also a potent immunosuppressor [169–180, 186, 187]. The immunosuppressive function of PGE2 may be responsible for the immunosuppression seen in cancer and its ability to limit the functions of NK cells, CD4, and CTLs [188, 189]. In this context, it is noteworthy that PGE2induced IL-10, Treg cells, and myeloid-derived suppressor cells in the tumors suppressed the proliferation and cytotoxicity of CTLs and their ability to produce IFN-γ [186–192]. COX-2 inhibitors are used to inhibit PGE2 synthesis and have been shown to restore T-cell proliferation [193]. In view of the pro-­inflammatory and immunosuppressive actions, it is reasonable to expect that in situations where in there is increased production of PGE2, there could be an increase in the incidence of infections and persistence of infections and the inflammatory events would not resolve that may lead to chronic inflammation. This is especially so in those with cancer. As already discussed above, cancer may represent a non-resolving/non-­healing wound

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that could be due to increased production of PGE2. This is supported by the fact that in many cancers there is indeed an increase in the production of PGE2 both by the tumor cells and monocytes/macrophages infiltrating the tumor that has been held responsible for the defective cellular immune response, hypercalcemia, tumor cancer cell proliferation, tumor angiogenesis, and resistance of tumor to anti-VEGF therapy at least indirectly seen in these patients [194–212]. These data indicate that increased production of PGE2 by tumor cells and infiltrating macrophages will enable tumor cells to avoid immunosurveillance, enhance their proliferation, augment tumor angiogenesis, and ultimately render them to grow faster and also metastasize. Furthermore, PGE2 is an inhibitor of TNF-α and IL-6 production [213–219] and also that of IFN-γ [220], which are pro-inflammatory molecules and are known to possess tumoricidal actions. This is yet another action of PGE2 that renders tumor cells to avoid immunosurveillance. In addition, PGE2 may have the ability to modulate NO generation [221], and NO, in turn, may alter PGE2 synthesis [222–230]. This close interaction between PGE2 and NO depends on the tissue and the stimulus that is provided in a given situation. It was found that PGE2 can increase or decrease NO generation and similarly, NO may enhance or decrease PGE2 generation. Thus, sometimes PGE2 and NO may augment each other’s action and yet times may work against each other [222–230]. Thus, it is difficult to anticipate what could be the interaction between PGE2 and NO in a specific instance since there are many other factors that influence their interaction. For instance, both PGE2 and NO are modulated by TNF-α and other cytokines, and they (PGE2 and NO), in turn, influence the production and action of TNF-α and NO. In this context, it is interesting to note that PGE2 can enhance IL-10 production [231, 232], an anti-­inflammatory cytokine. Thus, PGE2 has actions on IL-17, TNF-α, IL-6 and IFN-γ, Treg cells, CTL, NO and may mediate the resistance of tumor cells to anti-VEGF therapy through its ability to enhance IL-17 secretion [231–245] that may ultimately result in tumor cell proliferation, angiogenesis and metastasis (see Figs. 3.5, 3.6, 3.7, and 3.8a). Despite these evidences, as outlined above, that PGE2 may augment tumor cell growth, our own studies showed that it (PGE2), in fact, may inhibit the growth of IMR-32 (human neuroblastoma) cells in vitro [246]. Of all the prostaglandins (PGE1, PGE2, PGF2a, and PGI2) and leukotrienes (LTD4 and LTE4) tested, PGE1, PGE2, and LTD4 inhibited the growth of IMR-32 cells to a significant degree at the doses used (10, 50, and 100 ng/ml of PGs and LTs when added to 0.5 × 104 cells). Previously, it was reported that PGA and PGD2 may have growth inhibitory action on erythroleukemia cells in vitro [247–249]. These results indicate that different types of PGs and LTs may show growth inhibitory or growth-promoting action on tumor cells depending on the type of tumor cells that are being tested. In contrast to the previous results, we also noted that both COX and LOX inhibitors (indomethacin and nordihydroguaiaretic acid when used as 60 and 20 mg/ml, respectively) enhanced the growth of IMR-32 cells. Though this growth-promoting action of indomethacin and NDGA could be attributed to their non-specific antioxidant actions, it is equally possible that some unknown products have been generated from PUFAs that have growth-promoting actions. In order to verify this possibility, we also studied the effect of LXA4, resolvins, and protectins formed from AA, EPA, and DHA and noted that these antioxidant

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metabolites inhibited the growth of IMR-32 cells [246]. Thus, PGE1, PGR2, LTD4, LXA4, resolvins, and protectins seem to possess growth inhibitory action on IMR-32 cells. These results [246] imply that the balance between various eicosanoids formed from their precursors and the cellular content and possibly, in the surrounding milieu, content of various PUFAs determines the final outcome whether tumor cells are induced to proliferate or inhibited from further growth. This is so since we and others noted that GLA, DGLA, AA, EPA, and DHA have potent growth inhibitory action on several types of tumor cells both in vitro and in vivo [250–267]. But, it needs to be noted that there are some occasional reports suggesting that fish oil, a rich source of EPA and DHA, may actually enhance tumor growth [268]. But, even in this study, it was found that (i) dietary MaxEPA (a rich source of EPA and DHA) had a dose-related enhancing effect on the development of atypical acinar cell nodules (AACNs) but not on development of carcinomas in the pancreas of azaserine-treated rats; (ii) dietary MaxEPA inhibited the conversion of LA to AA, as well as the conversion of AA to TXB2 or PGF2, in non-tumorous pancreatic tissue; and (iii) PGs may play a role in the growth/development of pancreatic adenocarcinomas, but not in the growth of AACNs. This study highlighted the total composition of dietary fatty acids and reported that presence of LA may modulate the effect of EPA/DHA on tumorigenesis.

Tumor Cells and Lipid Peroxidation An inverse relationship is known to exist between the concentrations of lipid peroxides and the rate of cell proliferation. The higher the concentrations of lipid peroxides, the lower the rate of cell division and vice versa [269–273]. In general, tumor cells have low concentrations of lipid peroxides and are resistant to lipid peroxidation compared with normal cells [271–273]. It has been shown that hepatomas that have a higher growth rate have lower microsomal phospholipid content and decreased concentrations of unsaturated fatty acids ( [274–278], see Table 3.2). Previously, I reported that PUFAs augment free radical generation (especially superoxide anion and hydrogen peroxide) and formation of lipid peroxides in the tumor but not normal cells (Table 3.3) despite the fact that fatty acid uptake is at least two to three times lower in tumor cells compared to tumor cells [279, 280]. This may explain the high degree of sensitivity of tumor cells to PUFA-induced cytotoxicity. Resistance to lipid peroxidation is seen even at the premalignant stage of the carcinogenesis process [281].

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Table 3.2  Data showing fatty acid content of normal and tumor cells and their NADPH-­ cytochrome C reductase activity, MDA formation and stimulated lipid a formation A. Content of total lipid, vitamin E, and fatty acids in normal liver, Yoshida hepatoma cells, and microsomal suspensions from normal liver and Yoshida hepatoma cells Normal Intact Yoshida Normal liver Yoshida Measurement intact liver cells microsomes microsomes 6 Protein mg/10 cells ND 0.133 ± 0.088 ND 0.028 ± 0.002 Total lipid mg/mg protein 0.41 ± 0.08 0.26 ± 0.9 0.37 ± 0.03 0.21 ± 0.05 Cholesterol μg/mg of lipid 96.0 ± 8.1 215.2 ± 6.2 110.0 ± 12.3 266.85 ± 13.5 Fatty acid (%) 16:0 18.5 ± 0.2 18.7 ± 2.0 18.9 ± 1.1 18.5 ± 0.5 18:0 17.5 ± 0.5 13.3 ± 1.1 22.0 ± 3.0 13.7 ± 0.2 18:1 (OA) 12.1 ± 1.0 21.5 ± 0.8 8.6 ± 1.0 18.1 ± 0.3 20:4 (AA) 16.7 ± 2.4 8.7 ± 0.7 19.1 ± 2.4 9.6 ± 0.8 22:5 – 2.9 ± 0.1 – 2.4 ± 0.1 24:0 – 1.2 ± 0.1 – 2.9 ± 0.3 22:6 (DHA) 6.3 ± 0.2 5.2 ± 0.6 6.1 ± 0.3 5.3 ± 0.4 All values are means ± standard deviations. The numbers of estimations are given in parentheses. AA: Arachidonic acid; DHA: Docosahexaenoic acid; ND: Not determined; OA: Oleic acid B. Activities of NADPH-cytochrome C reductase and 7-ethoxycoumarin de-ethylase in microsomal suspensions derived from normal rat liver and Yoshida hepatoma cells Normal liver Yoshida hepatoma Measurement microsomes microsomes NADPH-cytochrome C reductase (mmol/min per mg) 80.5 ± 4.2 9.13 ± 1.4 7-Ethoxycoumarin de-ethylase activity (mmol/min per 0.14 ± 0.04 ND mg) Cytochrome P-450 (mmol/mg) 0.81 ± 0.06 ND All values are means ± standard deviations. The numbers of determinations are given in parenthesis. ND: Not detectable. C. Malondialdehyde production stimulated by NADPH plus ADP plus iron (2.5 mM ADP/0.1 mM iron) in suspensions of isolated normal rat hepatocytes and Yoshida hepatoma cells Malondialdehyde Incubation time (min) production (nmol/107 cells) Isolated hepatocytes Yoshida hepatoma cells 5 11.6 ± 6.51 0.010 ± 0.002 30 27.93 ± 10.32 0.08 ± 0.01 60 37.87 ± 9.26 0.91 ± 0.02 Values are means ± standard deviations with the numbers of determinations shown in parenthesis. (continued)

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Table 3.2 (continued) D. Lipid peroxidation in microsomal preparations from normal rat liver and Yoshida hepatoma cells. Ascorbate plus iron-induced lipid peroxidation was measured as malondialdehyde production, and NADPH plus ADP plus iron-stimulated lipid peroxidation was measured as oxygen uptake. Normal liver Yoshida microsomes Pro-oxidant Peroxidation (nmol/min per mg) Ascorbate + iron 1.29 ± 0.16; 0.24 ± 0.10 NADPH + ADP + iron 82.4 ± 9.21; 1.95 ± 0.51 Values shown are means ± standard deviations with the numbers of determinations shown in parenthesis. It is evident from this data that tumor cells have low amounts of PUFAs such as AA and higher amounts of OA (oleic acid) and little or no change in the saturated fatty acid content. This tilts the balance between saturated fatty acids and PUFAs more towards saturated fatty acids (SFAs). In normal cells, the PUFAs/SFAs ratio is: 0.46 whereas in tumor cells it is: 0.27. In the microsomes of normal cells the ratio is: 0.46 and tumor cells it is: 0.30. It is interesting to note that the PUFAs/ SFAs ratio in the cells and microsomes are almost same in both normal and tumor cells. Lipid peroxides formation is much higher in normal cells and is significantly low in tumor cells (this includes not only the formation of lipid peroxides but also the activity of NADPH-cytochrome c reductase (see the highlighted data in the Table 3.1b–d). This data is taken from: Cheeseman et al. [276] All values are means ± standard deviations. The numbers of estimations are given in parentheses. Values are means ± standard deviations with the numbers of determinations shown in parenthesis. Values shown are means  ±  standard deviations with the numbers of determinations shown in parenthesis. AA arachidonic acid, DHA docosahexaenoic acid, ND not determined, OA oleic acid

Tumor Cells Are Relatively Rich in Vitamin E The tumor cells are known to have low concentrations of γ-linolenic acid (GLA, 18:3 n-6), dihomo-γ-linolenic acid (DGLA, 20:3 n-6), arachidonic acid (AA, 20:4 n-6), eicosapentaenoic acid (EPA, 20:5 n-3), and docosahexaenoic acid (DHA, 22:6 n-3) due to decreased activity of δ-6- and δ-5-desaturases [282–284] that convert dietary linoleic acid (LA, 18:2, n-6) and alpha-linolenic acid (ALA, 18:3 n-3) to their respective long-chain metabolites (see Fig.  3.9 for metabolism of essential fatty acids). AA and EPA are the precursors of prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LTs). AA, EPA, and DHA also form precursors to lipoxins (LXs), resolvins (RSVs), protectins, and maresins. PGs, TXS, and LTs are pro-­ inflammatory in nature, whereas LXs, RSVs, protectins, and maresins are potent anti-inflammatory compounds [285–290]. In general, elevated levels of PGE2 and LTs have been in reported in many cancers [203, 291–300] and are responsible for immunosuppression, increased rate of cell proliferation, and metastasis in cancer. Non-steroidal anti-inflammatory drugs can prevent colon cancer to some extent, but

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Table 3.3  Effect of various PUFAs on MDA-eq formation in normal (CV-1: normal monkey kidney) and tumor (ZR-75-1: human breast cancer) cells in vitro on day 7 of supplementation with fatty acids Cell line CV-1 (normal monkey kidney cells)

pmol MDA-eq In the cells 0.5 ± 0.3 6.2 ± 1.1 7.1 ± 0.4 15.0 ± 2.7 11.4 ± 3.0 0.2 ± 0.3 26.5 ± 2.7 35.9 ± 10.4 16.7 ± 1.7 11.3 ± 3.9

Treatment Control GLA AA EPA DHA Control GLA AA EPA DHA

ZR-75-1 (human breast cancer cells)

In the medium 0.3 ± 0.1 1.1 ± 0.3 2.8 ± 1.0 3.3 ± 0.5 10.1 ± 0.3 0.9 ± 0.2 10.6 ± 2.4 3.5 ± 1.6 10.2 ± 0.6 14.4 ± 0.4

It is clear from this data that all PUFAs enhance formation of lipid peroxides in normal and tumor cells in the presence of GLA and AA but, the formation of lipid peroxides is significantly higher in tumor cells compared to normal cells. This indicates that GLA and AA differentially enhance the formation of lipid peroxides more in tumor compared to normal cells that may explain their (GLA and AA) cytotoxicity to tumor cells (see the highlighted data) This data is taken from Das [253]

n-3

n-6

Diet

Cis-LA, 18:2

ALA, 18:3 Δ6 Desaturase

GLA, 18:3

DGLA, 20:3

PGs of 1 series Δ5 Desaturase

AA, 20:4 PGs of 2 series, TXs, LTs of 4 series

EPA, 20:5

LXA4 Resolvin E1 and E2

PGs of 3 series, TXs, LTs of 5 series

Fig. 3.9  Scheme showing metabolism of essential fatty acids

DHA, 22:6 Resolvin D1, Protectins, Maresins

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results are not satisfactory. Tumor cells may also have elevated levels of α-tocopherol, a potent antioxidant [270–272, 277]. Tumor cells may have low amounts of superoxide dismutase (SOD), glutathione peroxidase, and catalase [301–303]. Thus, PUFA deficiency, a higher amount of vitamin E, and increased levels of PGs seen in cancer (cells) may suppress the rate of lipid peroxidation seen in tumor cells [303, 304].

SOD and Tumor Cells Superoxide dismutase: Mn superoxide dismutase (MnSOD) and cytoplasmic CuZn superoxide dismutase (CuZnSOD) are needed to protect tissues from the cytotoxic action of superoxide anion. Inactivated MnSOD gene (Sod2) in mouse leads to their death within the first 10 days of life as a result of dilated cardiomyopathy, accumulation of lipid in the liver and skeletal muscle, metabolic acidosis and ketosis, and a reduction in succinate dehydrogenase (complex II) and aconitase (a TCA cycle enzyme) activities in the heart and other organs. These results suggest that MnSOD is needed for the integrity of mitochondrial enzymes [305] as uncontrolled action of superoxide and other radicals induces cell death by apoptosis and necrosis [306– 310]. This implies that free radical scavengers can delay apoptosis, though not always [311, 312] since Cu/Zn SOD-overexpressing cells showed higher activation of caspase 3. This suggests that intracellular superoxide anion modulates tumor cell response to drug-induced apoptosis/ferroptosis/necrosis via a direct or indirect effect on the caspase activation pathway [312, 313]. These results imply that prolonged or excessive oxidative stress can prevent caspase activation. This is exemplified by the fact that NADPH oxidase-derived oxidants generated by neutrophils prevent caspase activation. This dual role of reactive oxygen species in induction and inhibition of caspases needs to be carefully considered while attributing apoptosis or preventing apoptosis by various chemotherapeutic drugs on tumor cells [311]; in which instance, the critical role of Bcl-2 and Bax protein levels needs to be considered. This is especially important while studying the role of hepatocyte growth factor (HGF), donors of nitric oxide (NO), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), GM-CSF, and hematopoietic growth factors (HGFs) on apoptosis and their putative neuroprotective actions (especially of IGF-1 and bFGF) and in the growth and differentiation of progenitor cells [305– 317]. Based on these results [305–317], it is apparent that free radicals have both beneficial and harmful actions on both normal and tumor cells and participate in the differentiation, growth, and prevention of apoptosis of hematopoietic cells in response to growth factors. Thus, the actions of free radicals may depend on the type of free radical produced, site of their production, amount of the free radicals generated, duration and the rate with which they are produced, stimulus for their production (whether it is physiological or pathological stimulus), and the rate with which the free radicals are produced in normal and tumor cells. It is possible that anti-cancer drugs and radiation induce generation of excess of free radicals that

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Stimulus

PUFAs, lipoxins, resolvins, protectins, maresins

PGs, LTs, TXs, etc.

Physiological (Haematopoietic growth factors)

Pathological (Anti-cancer drugs, Radiation, TNF-α) PUFAs

Progenitor cells/ Stem cells

Free Radicals especially, H2O2↑

ROS↑

Tumor cells/ Virus infected cells

ROS↑↑↑ NO↑↑

Bcl-2↓ Bax↑

Cell membrane and Mitochondrial membrane disruption

Tyrosine phosphorylation, STAT5, c-FOS↑ Activation of Caspases

Transition from G1 to S phase

Differentiation and Growth of cells

Lipid peroxidation↑↑

Apoptosis and/or Necrosis of cells

Fig. 3.10  Scheme showing role of free radicals in normal precursor/stem cell differentiation and growth and tumor cell apoptosis

cause apoptosis, whereas physiological stimuli such as GM-CSF, IL-3, steel factor (SF), and thrombopoietin (TPO)-induced free radical generation occur in a controlled fashion and hence are not toxic to cells and are likely to induce tyrosine phosphorylation, STAT5, and other signaling proteins and enhance expression of the early response gene c-FOS that lead to G1- to S-phase transition and do not activate caspases (see Figs.  3.10, 3.11, and 3.12). It may be mentioned here that these three figures need to be seen from a comprehensive perspective as there are

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Irradiation Mitomycin C Doxorubicin PUFAs

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ROS↑ Translocation of P53↑

DNA damage (-)/(+)

PUFAs

Mitomycin C Doxorubicin

(-)/(+) No change in Bcl-2 and Bax

Bak, p21(WAF1/CIP1) and GADD45 ↑

MnSOD↓

ATP↓

PARP (poly-ADP-ribose-polymerase) ↑

Intermediate filaments

DNA fragmentation and Chromatin condensation ↑

Intermediate filaments

Apoptosis Fig. 3.11  Scheme showing interaction between ROS and p53 and consequent induction of apoptosis in tumor cells. For further details see the text. Our studies revealed that depending upon the cell type (normal or cancer cells), the action of PUFAs (especially AA) on BCL-2, BAX, p53 and pro- and antioxidants will vary. In a normal cell, the expression of BCL-2 is increased whereas in a cancer cell BCL-2 expression is decreased by PUFAs. The fatty acid composition of normal and tumor cells is different and thus, the membrane composition may be one factor that determines the response of BCL-2, BAX and p53 to PUFAs. Similarly, PUFAs may enhance or decrease SOD, p53 and ROS generation, depending on the target tissue/cell. (−)/(+) Indicates both negative and positive control/inhibition or enhancement of action/expression of concerned genes. Depending on the conditions, PUFAs may have both positive and negative action

many overlapping features and events depicted in these figures. For the sake of simplicity, all the events have been broken into three separate events and given as three separate figures. One endogenous molecule that could selectively generate free radicals in tumor cells to induce their apoptosis seems to be PUFAs such as γ-linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) and their anti-inflammatory metabolites: LXA4, resolvins, protectins, and maresins. In this context, the interaction between tumor necrosis factor and PUFAs is interesting.

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(-)

p53 deficiency

Free Radicals MnSOD activity↑

(-)/(+) PUFAs

(-)

Lipid peroxides↑↑

(-)

(-)/(+)

(-)/(+)

Resistant to Cytotoxic treatments

Fig. 3.12  Scheme showing feedback regulation between p53 deficiency and resistance of tumor cells to apoptosis. For details see text. (−) Indicates negative control, decrease in activity or synthesis or no resistance to cytotoxic treatments. (+) Indicates positive control, increase in activity or synthesis/expression. Cells lacking p53 are generally resistant to the cytotoxic effect of pro-­ oxidants. But, paradoxically, PUFAs and lipid peroxides decrease p53 expression but yet enhance cytochrome C release and enhance the expression of caspases 3 and 8 and Fas expression and thus, induce apoptosis of tumor cells (see Ref. [87]). MnSOD activity is increased in hepatic cells that are p53-deficient in comparison with wild type. Transient transfection of cells with p53 induced a significant reduction in MnSOD mRNA levels and enzymatic activity, suggesting that expression of the antioxidant enzyme MnSOD is negatively regulated by p53. Increased expression of MnSOD renders cells resistant to p53-dependent cytotoxic treatments and, in co-transfection experiments, counteracted the growth inhibitory effect of p53, indicating that p53 inhibits the expression of MnSOD, and overexpression of p53 decreases the levels of SOD. Tumor cells that have impaired p53 activity have relatively higher SOD activity and relatively ow lipid peroxides and thus, anti-­ oxidant capacity to these cells will be high, suggesting that p53 has a pro-oxidant type of activity. Tumor cells exposed to free radicals would have enhanced SOD activity and are likely to show enhanced expression of p53 due to free radical induced stress. The increase in the expression of p53, in turn, would suppress SOD, tilting the balance more towards a pro-oxidant state and thus, leads to apoptosis. It is likely that increased formation of lipid peroxides in tumor cells due to supplementation of PUFAs, exposure to radiation and chemotherapeutic drugs (provided tumor cells have adequate amounts of PUFAs) could lead to formation and accumulation of critical amounts of lipid peroxides and increased expression of caspases 3 and 8 and induction of apoptosis by activation of extrinsic apoptotic pathway. Thus, at times p53 expression becomes irrelevant for apoptosis of tumor cells to occur. It is likely that ultimately it is the balance between lipid peroxides and antioxidants that seems to determine whether the tumor cells will undergo apoptosis or not. It is interesting to note that normal and tumor cells (when supplemented with same amount of PUFAs) form and accumulate same amounts of lipid peroxides till the concentrations of lipid peroxides reach a critical point. Once this critical concentration is reached, normal cells are able to degrade/detoxify lipid peroxides and remove them whereas tumor cells fail to do resulting in continues accumulation of toxic lipid peroxides that ultimately lead to their apoptosis

Tumor Necrosis Factor (TNF) and PUFAs TNF induces tumor cell death, and its toxicity to normal cells can be related to its capacity to induce free radical generation [318]. In addition, high levels of intracellular glutathione induce tumor cell resistance to recombinant human TNF (rhTNF), whereas low glutathione levels augment sensitivity to rhTNF. IL-1 and TNF stimulate phospholipase activity and induce the release of AA [319] and stimulate collagenase and PGE2 production explaining TNF role in tissue destruction, inflammation, and cancer [320, 321]. Studies revealed that arachidonate metabolism plays a

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significant role in the cytolysis of tumor cells by TNF [322–336]. Thus, activation of phospholipase A2 with consequent AA release and its conversion to prostaglandins and leukotrienes and concomitant release of free radicals is essential for the tumoricidal action of TNF [318–336]. These results imply that AA by itself could function as an anti-cancer molecule [253, 337–339].

Anti-cancer Drugs, ROS, and SOD In general, majority of anti-cancer drugs produce DNA damage by virtue of their ability to enhance free radical generation, especially in the presence of copper and ferrous (Fe2+) ions, and thus are capable of inducing apoptosis and/or ferroptosis of tumor cells [340]. In contrast, increased levels of SOD [341, 342] decrease macrophage-­mediated tumor cytotoxicity. Thus, macrophage cytotoxicity on tumor cells is dictated by the level of O2− produced [342]. Furthermore, increase in protein levels of Mn-superoxide dismutase (Mn-SOD) is associated with TNF resistance [343]. It is noteworthy that the resistance of ADRR (adriamycin-resistant) breast tumor cells to adriamycin is due to the development of tolerance to superoxide [344], because of a two-fold increase in SOD activity, and a decreased susceptibility to hydrogen peroxide due to a 12-fold augmented selenium-dependent glutathione peroxidase activity. Thus, SOD and glutathione peroxidase induce resistance to adriamycin by decreasing the formation of hydroxyl radical [345]. Yet there are instances wherein decreased MnSOD-specific activity is not a characteristic of some tumors [346]. In addition, it was observed that the rate of lipid peroxidation is generally low in tumor cells [347]. Paradoxically, some tumors have higher SOD activity compared to normal [348, 349], and their malignant phenotype could be suppressed by overexpressing MnSOD [350]; this seems to be especially true of some gastric and colorectal adenocarcinomas [351, 352]. The activities of MnSOD and CuZn SOD may vary greatly among both human and rat glioma and other tumor cells [353, 354]. These apparently paradoxical results on the relationship between free radicals and SOD and other antioxidants in tumor cell proliferation and apoptosis could be related to the interaction between the caspases and free radicals since the SH groups of the caspases are essential for their catalytic activity that could be inactivated by free radicals. For instance, M14 melanoma cells survive due to the inactivation of caspases by O2− and H2O2 [355]. It is possible that enhanced levels of SOD reduce intracellular ROS that results in reduced formation of lipid peroxides. This could enhance cell proliferation due to the absence of negative feedback control exerted by lipid peroxides on cell proliferation. Thus, the final impact of ROS and antioxidants on cell proliferation depends on the interaction among ROS (including NO), antioxidants, caspases, lysophosphatidic acid signaling [356], Bcl-2 and Bax protein levels, poly-(ADP-­ribose)-polymerase and phospholipase A2 activity and the cellular content of PUFAs and eicosanoids formed, mitochondrial membrane integrity, and mTORC1 signaling in the tumor cells [357–367].

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Free Radicals, Lipid Peroxides, p53, Caspases, and Apoptosis Many genes and their products that control apoptosis are also regulators of cell cycle progression. p53 protein is an example of a gene product that affects both cell cycle progression and apoptosis [358, 367]. The p53 tumor suppressor is essential for the maintenance of the integrity of the genome, and its nuclear localization is critical to its action [358, 368–370], whereas free radicals modulate p53 activity and thus produce apoptosis. Furthermore, p53 inhibits the expression of MnSOD, and overexpression of p53 decreases the levels of SOD.  Tumor cells that have impaired p53 activity have higher SOD activity and so the antioxidant capacity of these cells will be high, implying that p53 has a pro-oxidant type of activity [371]. Thus, tumor cells exposed to free radicals have higher SOD activity and show enhanced expression of p53 due to free radical-induced stress [358, 369, 370]. This increase in the expression of p53, in turn, would suppress SOD, tilting the balance more toward a pro-oxidant state and thus leads to apoptosis. This feedback regulation among free radicals, p53, and SOD is crucial for cellular homeostasis. Free radicals and lipid peroxides inactivate various enzymes, denature proteins, and deplete cellular ATP content leading to their apoptosis [269, 270, 358, 372]. Free radicals cause ATP depletion in the cells by activating PARP (poly-ADP-ribose-polymerase), the substrate of caspase-3 [358, 372], though this is controversial [373]. Thus, free radicals induce apoptosis by enhancing the expression of p53, decreasing SOD levels, activating PARP and inactivating several cellular enzymes, denaturing cellular proteins, and depleting cellular ATP content (see Figs. 3.11 and 3.12). In this context, it is noteworthy that oxidative stress shortens telomere [358, 374– 379]. It is believed that in tumor cells telomere is longer compared to normal cells that is considered to be responsible for their longer life or immortality.

Oxidant Stress, Bcl-2, and Apoptosis Bcl-2 opposes the pro-oxidant action of p53 by its antioxidant action [380] suggesting that a balance exists between p53 and Bcl-2. Increased expression of p53 that occurs on exposure to radiation and free radicals results in inhibition of Bcl-2 expression that, in turn, augments free radical generation and apoptosis. Thus, Bcl-2 is downregulated by p53 [358, 381, 382]. Bcl-2 blocks lipid peroxidation [383] and thus opposes apoptosis [358, 383, 384]. Tumor cells when supplemented with PUFAs form increased amounts of lipid peroxides that cause apoptosis [358, 385, 386]. At the same token, there is evidence to suggest that BCL-2 may have pro-oxidant actions as well [387]. It was reported that expression of BCL-2 in SOD-deficient (SOD-) Escherichia coli leads to an increase in the transcription of the KatG catalase-­peroxidase, a 13-fold increase in KatG activity, and a 100-fold increase in

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resistance to hydrogen peroxide. In addition, mutation rate in E. coli was increased threefold; and katG and oxyR, transcriptional regulators of katG induction, were required for aerobic survival, indicating that in this instance BCL-2 acts as a pro-­ oxidant. Thus, in E. coli under some specific experimental conditions, BCL-2 generates reactive oxygen intermediates. Furthermore, a 73% increase in SOD activity in a murine B-cell line overexpressing BCL-2 was reported with an increase in reduced glutathione and in oxyradical damage to DNA [387]. Thus, BCL-2 seems to have both pro- and antioxidant actions under some conditions and thus is capable of influencing levels of reactive oxygen intermediates and endogenous cellular antioxidants that ultimately determine apoptosis process. Furthermore, it was reported that primary hippocampal cultures, with overexpression of BCL-2: (i) BCL-2 protected against glutamate; (ii) BCL-2 did not alter the levels of lipid peroxides, which is primarily responsible for glutamateinduced oxidative damage; but (iii) BCL-2 only partially protective against the prooxidant adriamycin and did so without any changes in superoxide, hydrogen peroxide or lipid peroxides levels; and paradoxically (iv) BCL-2 protected against anoxia, an insult that does not involve ROS generation. Put together, all these evidences suggest that BCL-2 may have antioxidant actions but that may not be crucial to its cytoprotective actions observed and may protect even in the absence of ROS generation [358, 388]. In this context, it is interesting to note the relationship between BCL-2 action and cell membrane fatty acid content and composition. It was reported that in a cultured cell line of immortalized keratinocytes (HaCaT), overexpressing the BCL-2 when exposed to tert-butyl-hydroperoxide (t-BOOH) (defined as peroxide treatment), the formation of reactive oxygen species was lower in BCL-2-expressing cells, suggesting that these BCL-2-expressing cells have a better capacity to withstand oxidative stress. These cells also showed higher SOD activity that was induced by oxidative t-BOOH treatment (by BCL-2 transfected cells) and showed decreased damage to membrane lipids and proteins. In contrast, formation of 4-hydroxy-­nonenal, a specific marker of peroxidative damage to PUFAs, was found to be higher in BCL-2transfected cells compared to control, implying that BCL-2 overexpression produces significant changes in the fatty acid composition of cell membranes. It was noted that BCL-2-transfected cells have a higher proportion of monounsaturated fatty acids and n-6 PUFAs and a lower proportion of pentaenoic PUFAs that results in a higher unsaturation index with respect to control cells. Changes in protein kinase C activity were also associated to BCL-2 expression, which may be secondary to changes in membrane fatty acid composition [358, 389]. These results suggest that the interaction(s) among BCL-2 expression, fatty acid composition of cell membrane, and the expression of SOD and other antioxidants play a significant role in the pro- and antioxidant actions of BCL-2 that ultimately results in apoptosis or anti-apoptosis of the cells. It is possible that in this context the metabolism of PUFAs to their pro- and anti-inflammatory metabolites (such as prostaglandins, leukotrienes, thromboxanes vs lipoxins, resolvins, protectins, and maresins) also plays a role in this process/event. This complex interplay among BCL-2, p53, pro- and antioxidants, and PUFAs is further evident from our recent study [87, 147, 390, 391]

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wherein we noted that AA could have differential action on the expression of BCL-2, BAX, p53, and NF-kB genes. It was noted that AA is cytoprotective against the cytotoxic action of alloxan and streptozotocin action on RIN5F (rat insulinoma cells) cells and enhances the expression of BCL-2 and suppresses that of NF-kB and thus shows anti-inflammatory, antioxidant, and anti-apoptotic actions, whereas the same AA enhanced the cytotoxic action of bleomycin (an anti-cancer drug) on IMR-32 cells (human neuroblastoma cells) in vitro by suppressing the expression of p53 and BCL-2 [87, 147, 390, 391]. These results emphasize the differential action of PUFAs (especially that of AA) depending on the target cell (whether it is normal or tumor cell). These results are interesting, if one takes into consideration the fatty acid composition of normal and tumor cell membranes. As already discussed above, tumor cells have low concentrations of AA, EPA, and DHA (especially of AA) compared to normal cells. Thus, it looks as though if the cell membrane content of AA and other PUFAs is low and if these PUFAs are supplemented, it leads to apoptosis, whereas if the cell membrane content of PUFAs is normal, PUFAs when supplemented seem to prevent apoptosis (see Figs. 3.8b, 3.10, 3.11, and 3.12). These results suggest that the expression of genes concerned with apoptosis, inflammation (such as NF-kB and IkB and COX-2 and LOX), antioxidants (such as SOD, catalase, glutathione), and oxidative stress (such as NADPH oxidase) depends on the cell membrane composition implying that there is a cross talk between membrane (and its fatty acid composition) and genes.

Connecting the Plasma Membrane to the Nucleus How exactly this cross talk occurs between the cell membrane and the genes is not very clear and needs further evaluation. One possibility is that membrane fluidity could influence the structure and composition of intermediate filaments and their multiple binding partners and thus regulate both cellular mechanics and gene regulation. The intermediate filaments, actin and microtubules, could form distinct cytoskeletal filament systems and are involved in a dynamic interplay between these networks. It is likely that intermediate filaments provide structural support for cells and play a major role in the cell’s responses to external mechanical forces. It has been shown that tensional force-induced reinforcement of actin stress fibers requires the interaction of the RhoA-targeting Rho-guanine nucleotide exchange factors Solo/ARHGEF40 with keratin intermediate filaments to activate RhoA signaling, which promotes stress fiber formation and keratin network organization. These results illustrate the importance of keratins in enabling cells to adapt to mechanical stress [392]. It is possible that interaction of desmoplakin with keratin filaments at desmosomes supports intercellular force transmission, traction force generation, and cell stiffness and ultimately alters expression of several genes concerned with mitosis and apoptosis [358, 392, 393]. 2-Methoxyestradiol (2-ME), PUFAs, thalidomide, TNF, ILs (interleukins), and many anti-cancer drugs, radiation, and protoporphyrin derivatives (used in

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photodynamic therapy) enhance free radical generation and augment lipid peroxidation process and thus lead to accumulation of lipid peroxides in the cells resulting in apoptosis of tumor cells [358, 394–398]. PUFAs are cytotoxic to tumor cells, have anti-angiogenic properties, and augment free radical generation in the tumor cells and inhibit the growth of human gliomas with few side effects [249– 251, 358, 399–403]. It is likely that various PUFAs and lipid peroxides can alter/ disrupt intermediate filaments, actin and microtubules, and cytoskeletal filament systems and thus produce their cytotoxic action on tumor cells.

 UFAs Enhance Lipid Peroxidation and Inhibit P Cell Proliferation GLA, AA, EPA, and DHA have selective cytotoxic action on tumor cells with little or no effect on normal cells [252, 254–258, 358, 404, 405] that does not appear to be dependent on the formation of cyclo-oxygenase and lipoxygenase products [252, 254–256, 358, 404]. It was noticed that GLA, AA, and EPA (irrespective of the form in which these fatty acids were supplemented: free acid, ethyl, methyl ester form) treated tumor cells but not normal cells produced almost a two–three-fold increase in free radicals and lipid peroxidation products despite almost 50% decrease in their uptake [279, 280, 358, 406–408]. These results attest to the proposal that low rates of lipid peroxidation seen in tumor cells are, in part, due to deficiency of PUFAs and a relative increase in their antioxidant content [276–278, 282–286]. Since there is a direct correlation between the rate of lipid peroxidation and the degree of deviation in hepatomas [270–273, 276, 277, 358] and as the rate of lipid peroxidation is low in several tumors (as discussed above), it is possible that lipid peroxidation is a physiological inhibitor of mitosis and regulator of cell multiplication. Similarly, TNF-α induces apoptosis of tumor cells by augmenting free radical generation and enhancing the release of endogenous AA [358, 409, 410]. Thus, the cellular level of unesterified AA (and so are EPA, DHA, GLA, and DGLA) could be a mechanism by which apoptosis is induced in tumor cells. Hence, methods designed to enhance the cellular content of PUFAs may form a new approach to kill tumor cells [358, 411] that may explain the beneficial action of EPA- and DHA-rich fish oils in the prevention of colon and other cancers [267, 412–414]. Furthermore, tumor cells exposed to PUFAs show low levels of various antioxidants [255, 256, 358], and thus, the balance is tilted more toward pro-oxidant status leading to oxidative stress and subsequent apoptosis of tumor cells. PUFAs, including n-3 fatty acids and AA, suppress carcinogen-induced ras activation [87, 267, 413] and Bcl-2 expression [87, 414] and inhibit the activity of cyclo-oxygenase enzyme and VEGF expression [415–418] that contributes to their anti-cancer action. In addition, anti-­inflammatory metabolites of PUFAs such as lipoxins, resolvins, protectins, and maresins also possess anti-cancer and anti-angiogenic action [246, 419–423]. In this context, it is interesting to note that lipoxins, resolvins, protectins, and maresins have little or no

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action on normal cell proliferation but seem to possess significant inhibitory action on the growth of tumor cells (at least in vitro) [246, 358, 420]. Our in vitro studies also revealed that prostaglandins and leukotrienes may have slight or no growth-­ promoting effect on tumor cells. These results suggest that excess prostaglandins secreted by tumor cells as a result of increase in COX-2 expression is meant to suppress immune response and incite inflammatory events locally (in the tumor microenvironment) in order to enhance tumor growth even though prostaglandins and leukotrienes themselves do not have any direct growth-promoting actions. Based on these results, it is proposed that one of the mechanisms by which various PUFAs, especially AA, EPA, and DHA, the precursors of anti-inflammatory lipoxins, resolvins, protectins and maresins and pro-inflammatory prostaglandins, leukotrienes and thromboxanes; regulate tumor cell growth is by rendering normal cells to produce anti-inflammatory and tumor inhibitory lipoxins, resolvins, protectins and maresins. Since lipoxins, resolvins, protectins, and maresins suppress the production of prostaglandins, leukotrienes, and thromboxanes, it is likely that the balance between these pro- and anti-inflammatory metabolites of PUFAs plays a crucial role in the growth of tumor cells. Thus, instances wherein tumor cells fail to grow or are restrained from growing can be ascribed to increased production of lipoxins, resolvins, protectins, and maresins by normal cells surrounding the tumor cells. When the normal cells fail to produce adequate amounts of lipoxins, resolvins, protectins, and maresins, it is only then tumor cells grow. Thus, it is envisaged that normal cells have an important action in restraining the growth of tumor cells. At the same time, if adequate amounts of PUFAs reach the microenvironment of the tumor and enter the tumor cells, they would undergo peroxidation and inhibit the growth of tumor cells or even induce their apoptosis. It is also envisaged that normal cells may peroxidize PUFAs to produce lipid peroxides and deliver them to tumor cells (since under in the body tumor cells are surrounded by normal cells) to restrict their growth. Thus, the way PUFAs are handled by normal and tumor cells, their availability in the tumor microenvironment, and their influx into the tumor cells ultimately determines whether the tumor cells are able to grow or undergo apoptosis. In this context, the role of PUFAs, especially GLA and AA, to activate macrophages and other immune cells (TH1 and TH2 and Treg and Teff) to inhibit the growth of tumor cells is an important aspect that need to be seriously considered.

PUFA-Activated Macrophages Possess Tumoricidal Action In addition to direct toxicity of PUFAs on tumor cells as discussed above, it is noteworthy that these fatty acids may also have the ability to activate macrophages and regulate T-cell signaling (see Figs. 3.1, 3.2, and 3.3). Schlager et al. [424–430] showed that the fatty acids play an important in the resistance or sensitivity of tumor cells to killing by antibody and complement. They reported that mouse peritoneal macrophages treated with lymphokine (LK, cytokine) induced changes in the lipid composition of peritoneal macrophages: cellular content of cholesterol and PUFA

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content of cellular lipids (especially 18:3, GLA or ALA) increased two- to threefold after 8 hr. when the cells showed maximal tumoricidal activity, and all these changes reverted to normal by 24 hr. when the peritoneal macrophages are no longer tumoricidal. Peritoneal macrophages enriched in linolenic acid (18:3, GLA or ALA) were markedly tumoricidal and showed an increase in superoxide release, suggesting that endogenous levels of 18:3 regulate tumor cytotoxic activity of peritoneal macrophages solely by their ability to metabolize or mobilize 18:3 fatty acid without any changes in their protein synthesis, oxidative metabolism, or augmented capacity for tumor target binding. These results are in tune with our observation that GLA has potent tumoricidal action [252, 254–258, 279, 280, 339, 358, 404, 405, 431–433]. Possible relationship between immunocytes (that include macrophages, leukocytes, T cells), cytokines, ROS, and how they could be related to PUFAs has been outlined in Figs.  3.1, 3.2, and 3.4. It is evident from this data that PUFAs are capable of interacting with immunocytes in many ways that ultimately will have an impact on cancer and its relationship to cancer.

PUFAs Are Involved in Various Mitochondrial Processes Dietary or supplemented PUFAs after absorption from the gut, are distributed to cells and enrich various cellular membranes. This incorporation of PUFAs in cell membranes not only influences various cell metabolic processes and their survival but also has the ability to modulate almost all mitochondrial processes [358, 434, 435]. Our studies revealed that RIN5F (rat insulinoma cells) when exposed to alloxan- and streptozotocin-induced cytotoxic action can be prevented by various PUFAs [147, 390, 391]. Of all the fatty acids tested, AA was found to be the most potent in preventing chemical-induced cytotoxicity to RIN5F cells both in vitro and in vivo [147, 390, 391]. It is interesting to note that oral administration of AA is as potent as that of intraperitoneally administered AA suggesting that oral AA is very rapidly absorbed and effective. In addition, it was noted that both alloxan and streptozotocin interfere with AA formation possibly by blocking the action of desaturases and hence, experimental animals administered diabetogenic chemicals (alloxan and streptozotocin) have decreased AA content in their plasma phospholipid fraction [147, 436]. Similarly, patients with type 2 diabetes mellitus also have low plasma levels of AA in their phospholipid fraction [436]. This decrease in AA may be responsible for low plasma levels of LXA4 seen in type 2 diabetes mellitus [437]. Thus, in diabetes mellitus there is a deficiency of both AA and its metabolite LXA4. LXA4 also has anti-diabetic actions [147, 390, 391, 436]. These results pertaining to AA and LXA4 in the prevention of diabetes mellitus and their cytoprotective action against chemical-induced apoptosis of pancreatic beta cells are interesting since,both AA and LXA4 have growth inhibitory actions on tumor cells [252, 404, 416, 420–423, 431, 432]. This dichotomy in the actions of AA (and other PUFAs) and LXA4 on normal cells (protecting RIN5F and other normal cells from the cytotoxic action of various chemicals) vs tumor cells

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(inducing apoptosis) is rather surprising. These results suggest that the way PUFAs are handled by normal and tumor cells is different [358] and is discussed in some detail below. In addition, n-3 PUFAs have significant cytoprotective action especially of the myocardium [361, 438]. Studies revealed that n-3 fatty acids protect myocardium against oxidative-induced damage due to their ability to modulate mitochondrial reactive oxygen species (ROS) production [358, 435, 439]. These results are supported by the studies performed using fat-1 transgenic mice that synthesize n-3 fatty acids at the cost of AA. Fat-1 mice showed a decrease in ROS production from ETC complex I. Thus, fatty acid changes in the liver (and other tissues) mitochondria modulate oxidative stress [440] by altering ROS production from ETC complex I. This implies that the chemopreventive actions of fish oil may also be due to reduced oxidative stress in the mammary tissue when exposed to mammary carcinogen 7,12-dimethylbenz(α)anthracene (DMBA) [358, 441]. In contrast, tumor cells when exposed to PUFAs produced a significant increase in the generation of reactive oxygen species (ROS) that results in their apoptosis that is caspase dependent involving both intrinsic and extrinsic pathways [87, 358, 442–444]. These evidences [358, 435–437, 439–444] suggest that there is a differential metabolism of PUFAs by normal and tumor cells such that normal cells are protected from oxidative stress whereas tumor cells are exposed to increased oxidative stress [251–254, 358]. This differential action and metabolism of PUFAS by normal and tumor cells are welcome especially when PUFAs are employed in the management of cancer [147, 168, 246, 254, 339, 358, 390, 391, 396, 445–451]. This cytoprotective action of PUFAs is possibly mediated by their products: PGE1, lipoxins, resolvins, protectins, and maresins [358, 438, 446]. Thus, some of the beneficial actions of PUFAs can be ascribed to some of their products such as PGE1, PGI2, lipoxins, resolvins, protectins, and maresins and their ability to enhance NO and alter the expression of NF-kB, IkB, caspases, cytochrome C, Ras, Myc, Fos, Fas, p53, COX-2, and LOX.

 UFAs Augment Cytotoxic Action of Anti-cancer Drugs P and Reverse Drug Resistance In addition to the observation that PUFAs show differential action on normal and tumor cells as described above, it is noteworthy that PUFAs especially GLA, AA, EPA, and DHA enhance the tumoricidal action of anti-cancer drugs: vincristine, cis-platinum, and doxorubicin on various cancer cells in  vitro and in  vivo [452– 462]. The results of various studies suggested that PUFAs not only enhance the cytotoxicity of anti-cancer drugs to tumor cells but also reverse tumor cell drug resistance [358, 452–463]. Despite the fact that tumor cell drug resistance can be reversed in an in vitro system, it remains to be seen how these results could be translated to an in vivo system and especially to humans. One of the major issues to use various PUFAs to reverse tumor cell drug resistance to in vivo is the fact that these

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fatty acids bind rather tightly to albumin and other proteins which render them unavailable to tumor cells [464]. Since, in an in vivo situation, tumor mass is infiltrated by inflammatory cells such as leukocytes, macrophages, and monocytes (including T cells), there is certain degree of inflammation surrounding the tumor that leads to outpouring of albumin and other proteins that can bind to the PUFAs and prevent their uptake by tumor cells. Thus, despite the presence of PUFAs in the milieu surrounding the tumor mass, they may not be available and able to bring about their tumoricidal action. Hence, it is important that methods need to be devised wherein PUFAs can be directly delivered to the tumor cells. Alternatively, sufficient amounts of PUFAs need to be infused to patients with cancer so that they are able to saturate all the binding sites of the albumin and still available in adequate amounts to the tumor cells. This is a very difficult situation since it is not easy to saturate all the binding sites of albumin for PUFAs. One alternative is to infuse PUFAs intra-arterially close to the site of the tumor so that PUFAs are able to reach the tumor cells through their perusing artery(ies). This will be an invasive procedure that may not be suitable for all patients with cancer and is not without risks. The other possibility is to conjugate PUFAs to monoclonal antibodies to VEGF/EGF and other tumor receptors/antigens and infuse such a conjugate. Such an effort has never been tried. Hence, this remains conjectural.

Differential Metabolism of PUFAs by Normal and Tumor Cells The possibility that PUFAs have differential action on normal and tumor cells (e.g., AA protects pancreatic beta cells from the cytotoxic action of alloxan and streptozotocin, whereas AA can kill tumor cells) suggests that the metabolism of these fatty acids could be different in these cells. For instance, AA is metabolized to form the 5-lipoxygenase metabolite, 5-HETE (5-hydroxyeicosatetraenoic acid), by prostate cancer cells that enhance their growth, whereas inhibition of 5-lipoxygenase induced their apoptosis. Exogenous 5-HETE inhibited apoptosis of prostate cancer cells induced by 5-lipoxygenase inhibitors, confirming a critical role of 5-­lipoxygenase activity in the survival of these cells [358, 465]. These findings suggest that the way PUFAs are metabolized by tumor cells may have an impact on the survival, proliferation, and progression of cancer. It is likely that that free AA and GLA are tumoricidal, but when AA is converted to form 5-HETE by 5-­lipoxygenase, the tumor cells are stimulated to grow [358, 466, 467]. Cyclooxygenase-2 (COX-2) activity is upregulated in many cancers that is responsible for increased levels of PGE2 secreted by them and also enhanced PGE2 levels in the tumor milieu. This may explain as to why non-steroidal anti-­ inflammatory drugs (NSAIDs) that inhibit COX-2 prevent colon cancer and cause their apoptosis, though COX-2 inhibitors are not very effective in the prevention of cancer. One potential mechanism for tumor cell apoptosis on exposure to COX-2 inhibitors could be attributed to the accumulation of PGE2 substrate, namely, AA or diversion of AA to another pathway such as formation of LXA4, a known tumor cell

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growth inhibitor. Colon adenocarcinomas overexpress AA-utilizing enzyme, fatty acid-CoA ligase (FACL) 4, in addition to COX-2. It is likely that unesterified AA in the cells (more so in cancer cells) is a signal for induction of their apoptosis. Tumor cells engineered with inducible overexpression of COX-2 and FACL4 act as “sinks” for unesterified AA as evidenced by the observation that activation of the enzymatic sinks blocked apoptosis, and the reduction of cell death was inversely correlated with the cellular level of AA. Cell death caused by TNF-α is prevented by removal of unesterified AA. This suggests that AA and other PUFAs are the mediators of the tumoricidal action of TNF-α and imply that the cellular level of unesterified AA and other PUFAs could be a general mechanism by which apoptosis is regulated, whereas COX-2 and FACL4 promote carcinogenesis by lowering AA and other PUFA concentrations in the cytoplasm of the cell and its milieu [358, 409, 467– 470]. NSAIDs upregulate 15-LOX-1, and its (15-LOX-1) inhibition inhibits NSAID-induced apoptosis that could be restored by 13-S-HODE (13-S-hydroxyoctadecadienoic acid is the product of 15-LOX-1 protein; the other product of 15-LOX-1 is 15-S-HETE, but in this study 15-S-HETE formation was not noted) but not by its parent, LA.  These results suggest that NSAIDs induce apoptosis in colon cancer cells via upregulation of 15-LOX-1  in the absence of COX-2 [358, 471–473]. Hydroperoxides generated by 5-, 12-, or 15-lipoxygenases from LA, ALA, and AA induced apoptosis of erythroleukemia and neuroblastoma cells in a concentration- and time-dependent manner, while leukotrienes, prostaglandins, and thromboxanes were not cytotoxic [474]. Studies have also shown that disruption of the 5-LO signaling pathway mediates growth arrest and apoptosis in breast cancer cells by the induction of PPARs and activation of PPARs with shunted endoperoxides [358, 475, 476]. These results imply that delivery of free unsaturated fatty acids to the tumor cells and generation of hydroperoxides by 5-, 12-, or 15-lipoxygenases from PUFAs and simultaneous inhibition of COX-2 enzyme induce apoptosis of tumor cells. PUFAs also suppress fatty acid synthase enzyme to induce apoptosis of tumor cells [358, 460, 477–482]. Based on these results, it is suggested that measuring the expressions of desaturases, COX and various LOX enzymes, and the concentrations of the metabolites of PUFAs such as 13-S-HODE, 15-S-HETE, 5-HETE, lipoxins, resolvins, protectins and maresins, prostaglandins, leukotrienes and thromboxanes in tumor cells to know how they (tumor cells) are handling PUFAs and what products are being formed and to which metabolites of PUFAs they (tumor cells) will be sensitive to undergo apoptosis will be known. In this context, it is interesting to note that our recent studies revealed that prostaglandins and leukotrienes do not have much effect on the growth of tumor and normal cells (except PGE1 and PGE2 which inhibited the growth of tumor cells). In contrast to this, lipoxins, resolvins, and protectins showed significant growth inhibitory action on tumor cells with little or no action on normal cells [420]. In addition, Fig. 3.13  (continued)  and Teff) and macrophage (M1 and M2) homeostasis. It is also likely that lipoxins, resolvins, protectins enhanced actionrole of in anti-­ PUFAs, PGs, LTs, TXs,and lipoxins, resolvins, protectinsthe andgrowth maresinsinhibitory have a regulatory the cancer drug bleomycin did not normal cells.the In expression a similar PD-1, fashexpression of PD-1, PD-L1on andtumor PD-L2.cells It is but proposed thaton PUFAs suppress PD-L1 and PUFAs PD-L2, whereas PGs, and TXs enhance their expression. may account ion, even inhibited theLTs growth of may tumor but not normal cells.This This suggests for the immunosuppressive action of PGs, LTs and TXs and tumor suppressive actions of PUFAs, that both PUFAS and their anti-inflammatory metabolites, lipoxins, resolvins, and lipoxins resolvins, protectins and maresins. It is proposed that co-administration of immune check protectins, selectively inhibit the growth and potentiate the action of anti-cancer point inhibitors and PUFAs may be of benefit in the management of cancer

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Connecting the Plasma Membrane to the Nucleus

Normal cells

Tumor cells Apoptosis

Dietary EFAs LA and ALA

Dietary EFAs LA and ALA

(-) LP↑

(-)

Desaturases↓

ROS↑ Desaturases

(+) ROS↓

ROS

PUFAs GLA, DGLA, AA EPA and DHA

PUFAs↓ GLA, DGLA, AA EPA and DHA

LP

(+) COX-1~

COX-1 5-, 12-, 15-LOXs

Supplementation of PUFAs

(-)

5-, 12-, 15LOXs↓~

(+) (-)

COX-2

PGs and TXs of 2 and 3 series, LTs of 4 and 5 series

Vit E↑ LP↓

COX-2↑

PGs and TXs of 2 and 3 series, LTs of 4 and 5 series↑

(+) Lipoxins, Resolvins, protectins and maresins

Lipoxins, Resolvins, protectins and maresins↓

(-) Normal Homeostasis

Inflammation, angiogenesis, immunosuppression cell proliferation↑

Fig. 3.13  Scheme showing the metabolism of EFAs and PUFAs in normal and tumor cells and actions of supplemented PUFAs on tumor cells. Possible normal-tumor cell crosstalk is also depicted. For details see text. In general, tumor cells have low activity of desaturases. This results in a deficiency of their long-chain metabolites such as GLA, DGLA, AA from LA and EPA and DHA from ALA.  This deficiency of long-chain metabolites of LA and ALA, in turn, leads to decreased formation of lipid peroxides (LP) that have a role in the regulation of cell proliferation. Thus, low content of GLA, DGLA, AA, EPA and DHA and low concentrations of LP seem to be responsible for continued proliferation of tumor cells. This may explain as to why supplementation of GLA, DGLA, AA, EPA and DHA and consequent increase in the formation of LP induce cell growth arrest and apoptosis of tumor cells. Furthermore, GLA, DGLA, AA, EPA and DHA also can induce the generation of ROS in macrophages, T cells and leukocytes that are toxic to tumor cells. Paradoxically, tumor cells have increased COX-2 activity that leads to increased generation of prostaglandins, leukotrienes and thromboxanes, which are pro-inflammatory molecules. In contrast, tumor cells seem to produce decreased amounts of anti-inflammatory lipoxins, resolvins, protectins and maresins. These metabolites also have the ability to suppress tumor cell growth. The activity of 5-, 12- and 15-lipoxygenases in tumor cells are variable and most of the time they are low that also contributes to their low content of lipoxins, resolvins, protectins and maresins. Thus, there appears to be an imbalance between pro-inflammatory (PGs, LTs and TXs↑) and anti-­ inflammatory (lipoxins, resolvins, protectins and maresins↓) metabolites of PUFAs. Lipoxins, resolvins protectins and maresins enhance leukocyte and macrophage phagocytosis and help in the clearance of debris of inflammation and tumor cell milieu and regulate T cell (TH1/TH 2 and Treg

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Dietary LA

Cell Membrane Phospholipids

Dietary LA

Phospholipase A2 Desaturases↓ Desaturases

Arachidonic Acid

Normal cell

Tumor cell

COX-1 & COX-2↑; 12-LO & 5-LO↓

COX-1 & COX-2↑; 12-LO & 5-LO↑

15-LO & Acetylated COX-2↑

15-LO & Acetylated COX-2↓

PGG2, PGH2, 12-HPETE, 5-HPETE, 12HETE, 5-HETE↑

PGG2, PGH2, 12-HPETE, 5-HPETE, 12HETE, 5-HETE↓

15-HPETE, 15(R)-HETE, 15(S)HETE, 13(S)-HODE ↓

15-HPETE, 15(R)-HETE, 15(S) HETE↑

PGD2, PGE2, PGF2, PGI2, 12-HHT, LTA4, LTB4, LTC4, LTD4, LTE4↓

PGD2, PGE2, PGF2, PGI2, 12-HHT, LTA4, LTB4, LTC4, LTD4, LTE4↑

Lipoxin A4, Lipoxin B4, 15-epi-Lipoxin A4, 15-epi-Lipoxin B4, 17-HDHA and 17-HpDHA ↑

Lipoxin A4, Lipoxin B4, 15-epi-Lipoxin A4, 15-epi-Lipoxin B4, 17-HDHA and 17HpDHA ↓

Enhance proliferaon, metastasis, inflammaon and suscepbility to chemotherapy

(-)

(+)

Suppress proliferaon, metastasis, inflammaon and resistant to chemotherapeucs

Fig. 3.14  Scheme showing metabolism of PUFAs in normal and tumor cells. Similar metabolism of EPA and DHA may exist in normal and tumor cells. (+) Indicates normal or excess of AA; (−) Indicates deficiency of AA. The handling of AA (and other PUFAs such as GLA, EPA and DHA) by normal cells and tumor cells is expected to be different that may explain its (AA) differential action. AA and other PUFAs protect normal cells from the cytotoxic action of alloxan, streptozotocin, benzo(a)pyrene, doxorubicin and other mutagenic and carcinogenic agents. In contrast, AA and other PUFAs are cytotoxic to tumor cells and enhance the anti-cancer action of chemotherapeutic agents and radiation. It is likely that to produce their differential action on normal and tumor cells, these fatty acids are metabolized differentially. It is suggested that when normal cells are exposed to AA and other PUFAs, cytoprotective metabolites are synthesized whereas tumor cells produce cytotoxic metabolites. It is also evident from various studies that normal cells do not accumulate toxic lipid peroxides while tumor cells do so as shown in Fig. 3.8. Tumor cells produce more prostaglandins, leukotrienes, thromboxanes, HETEs and HHTs compared to normal cells. In contrast, normal cells generate more lipoxins, resolvins, protectins and maresins that are cytoprotective compared to tumor cells. Similar to the metabolism of AA shown here, EPA and DHA may also be metabolized in a similar fashion. It has not yet been verified but, it is likely that cytoprotective molecules (such as lipoxins, resolvins, protectins and maresins) produced by normal cells are secreted into the tumor milieu so that they can inhibit tumor cell growth. Thus, normal cells may

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drugs on tumor but not normal cells. These results are in support of the proposal that normal and tumor cells metabolize PUFAs differently. Despite this, we are yet to exploit this knowledge in the clinic (see Figs. 3.13 and 3.14 for potential differences in the metabolism of PUFAs in normal and tumor cells).

PUFAs and Telomerase Activity Telomerase, also called terminal transferase, is a ribonucleoprotein that adds a species-­dependent telomere repeat sequence to the 3′ end of telomeres, a region located at each end of eukaryotic chromosomes in most eukaryotes. Telomere

Fig. 3.14  (continued)  try to impinge on the growth of tumor cells. Lipoxins, resolvins, protectins and maresins have anti-­inflammatory actions by themselves and by inhibiting the production of prostaglandins, leukotrienes and thromboxanes by tumor cells and local tumor infiltrating immunocytes. Since cancer is an inflammatory condition, suppressing local inflammation could be yet another mechanism by which cytoprotective molecules produced by normal cells try to inhibit tumor cell growth. It was reported (Lipids Health Dis 2010; 9: 112) that higher amounts of cyclooxygenase enzyme derived 12-HHT and a decrease in 12-LOX, 15-LOX-2, 15-LOX-1 and PGE activities corresponded to higher apoptosis and lower mitosis (12-HHT concentrations were higher, PGE2, 12-HETE, 15-HETE and 13-HODE was lower and 13-HODE/12-HETE ratio was higher). 12(S)-HETE augments the invasiveness of prostate carcinoma cells via selective activation of PKC-α (J Natl Cancer Inst 1994; 86: 1145–51), both ERK and cellular protein tyrosine kinase activation are involved in 12(S)-HETE-induced pancreatic cancer cell proliferation. Colorectal cancer is associated with decreased 15S-HETE and treatment of colon cancer cells with 15S-HETE inhibited cell proliferation and induced apoptosis in a PPAR γ-dependent pathway involving augmentation of TIEG (TGF-β-inducible early gene) and reduction of Bcl-2 expression (Int J Cancer 2003; 107: 837–43). Both AA and 15-L-(s)-hydroperoxyeicosatetraenoic acid (15-L-(s)-HPETE) showed cytotoxic action on MCF-7 (breast cancer) cells (Cancer Lett 1989; 46: 137–41. 13-S-hydroxyoctadecadienoic acid (13-S-HODE), a primary product of 15-LOX-1 metabolism of linoleic acid, is decreased in tumor cells. 13-S-HODE binds to PPAR-delta, decreases PPAR-delta activation, and down-­regulates PPAR-delta expression in colorectal cancer cells; induction of 15-LOX-1 expression is a critical step in NSAID (non-steroidal anti-inflammatory drugs such as aspirin) down-regulation of PPAR-delta and the resultant induction of apoptosis; and PPAR-delta is an important signaling receptor for 13-S-HODE-induced apoptosis (Proc Natl Acad Sci U S A 2003; 100: 9968–73). Addition of 13(S)-hydroxyeicosatetraenoic acid (HODE) decreased tumor cell proliferation. HCT-116 colorectal cells that were stably transfected with 15-LO-1 formed smaller tumors in nude mice. Thus, 13(S)-HODE induces changes in gene expression and has anti-tumorigenic effects (Prostaglandins Leukot Essent Fatty Acids 2004; 70: 7–15). Tumor cells form lipoxins (Res Commun Chem Pathol Pharmacol 1990; 68: 159–74) and deficient lipoxin production was paralleled by an attenuated conversion of AA to 12-HETE in cancer cells. In CML patients, blast crisis was associated with reduced lipoxins synthesis, while this capacity improved after retransformation into a second chronic phase (Blood 1991; 78: 2989–95). Lipoxins suppress the growth of tumor cells (Mol Cancer Ther 2010; 9: 2164–74) and resolvins, protectins and maresins may have similar action. Tumor cells that form substantial amounts of lipoxins, resolvins, protectins and maresins may be resistant to the chemotherapy and radiation. Tumor cells that form increased amounts of 17-HDHA and 17-HpDHA (formed from DHA and formation of similar compounds from AA and EPA) undergo apoptosis in response to chemotherapeutic drugs and radiation

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protects the end of the chromosome from DNA damage or from fusion with neighboring chromosomes. Telomerase is a reverse transcriptase enzyme that carries its own RNA molecule to elongate telomeres. Telomerase is active in normal stem cells and most cancer cells but is normally present at very low levels in somatic cells. High telomerase activity present in cancer cells can be inhibited by PUFAs due to their ability to downregulate human telomerase reverse transcriptase (hTERT) and c-myc expression via protein kinase C inhibition. Thus, PUFAs directly inhibit the enzymatic activity of telomerase and modulate the telomerase at the transcriptional level [358, 483, 484]. In addition, PUFAs modulate immune response (as discussed previously) and inhibit the adhesion of tumor cells to endothelium [485, 486], a property that will inhibit tumor metastasis and suppress tumor growth. As already discussed above, GLA and other PUFAs enhance tumor cell chemosensitivity [358, 452–463, 487, 488].

PUFAs Can Modulate G-Protein-Mediated Signals In addition to what is described above, PUFAs seem to have several other actions that account for their myriad actions. For instance, PUFAs modulate G-protein-­ mediated signal transduction [489]; mobilize Ca++ from intracellular stores [490], which may induce apoptosis [491]; activate PKC and enhance NADPH oxidase in macrophages [492] to enhance O2-. generation. AA and EPA (and possibly, DHA and other PUFAs including GLA) decreased Bcl-2 while increasing Bax in tumor cells [87, 493]. These actions are in addition to the action of PUFAs on p53 [87, 147, 390, 391]. In addition, DHA enhanced p27, inhibited cyclin-associated kinase, reduced pRb phosphorylation, and induced apoptosis of melanoma cells [358, 494, 495]. PUFAs can inhibit cell division by blocking translation initiation [496]. PUFAs enhance free radical generation in tumor cells that can directly activate heterodimeric Gi and G0 (small G proteins) [497], which are critical signaling molecules. Thus, PUFAs have several actions that enable them to induce apoptosis of tumor cells.

 ifferential Action Due to Differences in the Metabolism D of PUFAs Makes All the Difference Based on the preceding discussion, it is clear that PUFAs have differential action on normal and tumor cells and this could be due to their differential metabolism in these cells (see Figs. 3.10, 3.11, 3.12, 3.13, and 3.14). Such a differential metabolism may explain why unsaturated fatty acids are able to have differential action. As

References

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already discussed above, generation of hydroperoxides by 5-, 12-, or 15-­lipoxygenases from PUFAs is one pathway by which these fatty acids induce apoptosis of tumor cells. Lipoxins, resolvins, protectins (including neuroprotectin D1), and maresins possess cytoprotective actions by virtue of their potent anti-inflammatory actions. Thus, tumor cells generate toxic lipid peroxides when supplemented with PUFAs, whereas normal cells generate cytoprotective molecules: PGE1, lipoxins, resolvins, protectins, and maresins [34, 358, 498–509] (see Figs.  3.10, 3.11, 3.12, 3.13, and 3.14). It is evident from the preceding discussion that approaches are generic in nature but yet specific to tumor cells such that enhanced lipid peroxides are preferentially formed in the tumor cells, but not normal cells could form a novel therapeutic approach to selectively eliminate cancer cells by apoptosis. Thus, some of the salient features of involvement of various PUFAs and their metabolites in cancer can be summarized as follows: • Tumor cells have low activity of Δ6 and Δ5 desaturase enzymes. • This defect in desaturases leads to reduced concentrations of GLA, DGLA, AA, EPA, and DHA in tumor cells (tumor cells may have low concentrations of some of these PUFAs if not all). • Low PUFAs lead to low rates of lipid peroxidation in tumor cells. • Tumor cells may have relatively high concentrations of vitamin E. • Low concentrations of lipid peroxides may lead to high mitotic rate in tumor cells. • In view of these metabolic abnormalities, tumor cells are susceptible to the cytotoxic actions of lipid peroxides. • Several chemotherapeutic drugs, TNF, and radiation enhance free radical generation and formation of lipid peroxides in cancer cells. • Drug-resistant tumor cells have low amounts of PUFAs and hence generate low amounts of free radicals and form less amounts of lipid peroxides. • Enhancing the PUFAs content of tumor cells will induce apoptosis of tumor cells due to enhanced formation of toxic lipid peroxides. • PUFAs in combination with anti-cancer drugs and/or TNF enhance apoptosis of tumor cells and reverse drug resistance. • PUFAs are differentially metabolized by normal and tumor cells. • Cytoprotective molecules PGE1, lipoxins, resolvins, protectins, and maresins are formed in normal cells when supplemented with PUFAs, while their formation in tumor cells may render tumor cells drug resistant. • PUFAs and their metabolites have anti-angiogenic action. • Selective delivery of PUFAs may form a new approach to cancer in future.

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443. Giros A, Grzybowski M, Sohn VR, Pons E, Fernandez-Morales J, Xicola RM, Sethi P, Grzybowski J, Goel A, Boland CR, Gassull MA, Llor X. Regulation of colorectal cancer cell apoptosis by the n-3 polyunsaturated fatty acids Docosahexaenoic and Eicosapentaenoic. Cancer Prev Res (Phila). 2009;2:732–42. 444. Ponnala S, Rao KP, Chaudhury JR, Ahmed J, Rama Rao B, Kanjilal S, Hasan Q, Das UN.  Effect of polyunsaturated fatty acids on diphenyl hydantoin-induced genetic damage in vitro and in vivo. Prostaglandins Leukot Essent Fatty Acids. 2009;80:43–50. 445. Das UN, Rao KP. Effect of gamma-linolenic acid and prostaglandins E1 on gamma-­radiation and chemical-induced genetic damage to the bone marrow cells of mice. Prostaglandins Leukot Essent Fatty Acids. 2006;74:165–73. 446. Das UN, Ramadevi G, Rao KP, Rao MS.  Prostaglandins and their precursors can modify genetic damage-induced by gamma-radiation and benzo(a)pyrene. Prostaglandins. 1985;29:911–20. 447. Das UN. Tumoricidal and anti-angiogenic actions of gamma-linolenic acid and its derivatives. Curr Pharm Biotechnol. 2006;7:457–66. 448. Dhayal S, Morgan NG.  Pharmacological characterization of the cytoprotective effects of polyunsaturated fatty acids in insulin-secreting BRIN-BD11 cells. Br J Pharmacol. 2011;162:1340–50. 449. Suresh Y, Das UN. Long-chain polyunsaturated fatty acids and chemically induced diabetes mellitus: effect of omega-6 fatty acids. Nutrition. 2003;19:93–114. 450. Suresh Y, Das UN. Long-chain polyunsaturated fatty acids and chemically induced diabetes mellitus. Effect of omega-3 fatty acids. Nutrition. 2003;19:213–28. 451. Bazan NG. Omega-3 fatty acids, pro-inflammatory signaling and neuroprotection. Curr Opin Clin Nutr Metab Care. 2007;10:136–41. 452. Madhavi N, Das UN. Reversal of KB-3-1 and KB-Ch-8-5 tumor cell drug-resistance by cis-­ unsaturated fatty acids in vitro. Med Sci Res. 1994;22:689–92. 453. Madhavi N, Das UN. Effect of n-6 and n-3 fatty acids on the survival of vincristine sensitive and resistant human cervical carcinoma cells in vitro. Cancer Lett. 1994;84:31–41. 454. Das UN, Madhavi N, Sravan Kumar G, Padma M, Sangeetha P. Can tumour cell drug resistance be reversed by essential fatty acids and their metabolites? Prostaglandins Leukot Essent Fatty Acids. 1998;58:39–54. 455. Germain E, Chajès V, Cognault S, Lhuillery C, Bougnoux P. Enhancement of doxorubicin cytotoxicity by polyunsaturated fatty acids in the human breast tumor cell line MDA-MB-231: relationship to lipid peroxidation. Int J Cancer. 1998;75:578–83. 456. Mahéo K, Vibet S, Steghens JP, Dartigeas C, Lehman M, Bougnoux P, Goré J. Differential sensitization of cancer cells to doxorubicin by DHA: a role for lipoperoxidation. Free Radic Biol Med. 2005;39:742–51. 457. Ilc K, Ferrero JM, Fischel JL, Formento P, Bryce R, Etienne MC, Milano G. Cytotoxic effects of two gamma linoleic salts (lithium gammalinolenate or meglumine gammalinolenate) alone or associated with a nitrosourea: an experimental study on human glioblastoma cell lines. Anti-Cancer Drugs. 1999;10:413–7. 458. Menendez JA, Ropero S, Lupu R, Colomer R. Omega-6 polyunsaturated fatty acid gamma-­ linolenic acid (18:3n-6) enhances docetaxel (Taxotere) cytotoxicity in human breast carcinoma cells: relationship to lipid peroxidation and HER-2/neu expression. Oncol Rep. 2004;11:1241–52. 459. Menéndez JA, Ropero S, del Barbacid MM, Montero S, Solanas M, Escrich E, Cortés-Funes H, Colomer R.  Synergistic interaction between vinorelbine and gamma-linolenic acid in breast cancer cells. Breast Cancer Res Treat. 2002;72:203–19. 460. Menendez JA, Ropero S, Mehmi I, Atlas E, Colomer R, Lupu R.  Overexpression and hyperactivity of breast cancer-associated fatty acid synthase (oncogenic antigen-519) is insensitive to normal arachidonic fatty acid-induced suppression in lipogenic tissues but it is selectively inhibited by tumoricidal alpha-linolenic and gamma-linolenic fatty acids:

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Chapter 4

PUFAs and Their Metabolites in Carcinogenesis

Abstract  DNA damage due to endogenous and exogenous agents leads to activation of oncogenes. To prevent the activation of oncogenesis, there are many endogenous DNA repair mechanisms. But less well-known is the observation that essential fatty acids and their metabolites participate in mutagenesis and carcinogenesis. Our studies revealed that essential fatty acids and their metabolites such as prostaglandins have modulator influence on mutagenesis and DNA repair process. It appears that bioactive lipids (that include not only essential fatty acids and their long-chain metabolites such as GLA, DGLA AA, EPA, and DHA but also prostaglandins, leukotrienes, thromboxanes, lipoxins resolvins, protectins, and maresins) are able to act on the immune system to eliminate cells harboring DNA damage (this includes cells that contain micronucleus and bacterial and viral DNA and their genes). This interaction among bioactive lipids, immunocytes, and cytokines seems to be critical to prevent carcinogenesis and cancer. Alternatively, these bioactive lipids are able to eliminate cancer cells by their direct cytotoxic action on tumor cells. In addition, some, if not all, of the bioactive lipids are able to activate immunocytes, enhance the formation of toxic lipid peroxides in tumor cells, and mediate the cytotoxic action of various cytokines to induce apoptosis of tumor cells and eliminate them. Keywords  Bioactive lipids · Immunocytes · Polyunsaturated fatty acids · Lipid peroxides · DNA damage · Cytokines · NK cells · ROS · Cancer

Introduction In general, it is believed that cancer is the result of alterations in cellular DNA due to its interaction/reaction with free radicals, potentially resulting in mutations that can adversely affect the cell cycle and consequently leading to the transformation of normal cell to tumor cell. It has been suggested that when a normal cell is exposed to excess ROS and when the antioxidant defenses of the normal cell are inadequate, it will lead to DNA damage, oncogene activation, and conversion of normal to tumor cell. It is likely that these elevated levels of ROS will lead to the formation of © Springer Science+Business Media, LLC, part of Springer Nature 2020 U. N. Das, Molecular Biochemical Aspects of Cancer, https://doi.org/10.1007/978-1-0716-0741-1_4

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high amounts (that are toxic to cell organelles) of lipid peroxides that ultimately produce DNA damage and mutations sufficient enough to activate one or more oncogenes resulting in carcinogenesis. It is also possible that when a normal cell is exposed to excess of ROS and lipid peroxides, DNA damage is significant enough to lead to apoptosis of the cell. Thus, this balance between sufficient degree of DNA damage to activate oncogenes and significant amounts of DNA damage to produce apoptosis of cell is critical in the pathogenesis of carcinogenesis. It can be argued that if the DNA damage is not adequate to produce apoptosis but significant enough to produce activation of oncogenes, a normal cell would get converted into a cancer cell. This argument implies that when normal cell that harbors DNA damage sufficient enough to produce activation of oncogenes expresses newer antigens on its cell membrane that can be recognized by the immune system, which induce apoptosis. Thus, the survival of the cell with activated oncogenes is essential for it to proliferate and become recognizable cancer mass. At the same token, the cell that is harboring damaged DNA with activated oncogenes and expressing newer antigens on its cell membrane will be recognized as foreign by the immune system and mount an attack that is if adequate will lead to elimination of the abnormal cells. On the other hand, at times, the cell with newer antigens on its membrane is not recognized or the recognition is not strong enough to mount adequate immune response that will lead to proliferation of abnormal cells, leading to the onset of cancer. This suggests that for a cancer cell to survive and proliferate to form a recognizable cancer mass, there need to be DNA damage and activation of oncogenes and inadequate immune response to recognize and eliminate the cancer cell. This immune avoidance or failure of the immune surveillance may depend, partly, on the ability of cancer cell to elaborate certain immunosuppressive molecules such as PGE2. This implies that majority of mutagens and carcinogens are not only DNA-damaging compounds but are also capable of suppressing immune response (or immunosuppressors). In addition, the apparent tug-of-war between the cancer cell and the immune system of the body (cancer cell trying to survive even when the immune system is trying to eliminate it by mounting immune response and secreting molecules that have the potential to induce apoptosis of cancer cell) results in an inflammatory response in which leukocytes, macrophages, T cells, and platelets are participants. If the immune response to the merging cancer cell is strong, it will lead to the secretion of IL-6, TNF-α, and other pro-inflammatory and apoptosis-inducing molecules and secretion of cytotoxic granules by T cells, NK cells, and CTL cells. In the event tumor cells are able to evade these immune attacks by secreting immunosuppressive molecules (such as PGE2) due to the expression of weak antigens on the surface of cancer cells so that the immune recognition and immune attack are not strong enough to eliminate the cancer cell, it will lead to a weak pro-­inflammatory response at the site of cancer resulting in smoldering low-grade inflammation. Paradoxically, macrophages that initially show anti-cancer actions over a period of time become collaborators in cancer cell survival and proliferation. It is likely that tumor cells are able to influence and, thus, subvert macrophage function to help them survive and proliferate. This may, in part, be due to the production of certain humoral factors secreted by tumor cells that, in turn, modulate the function of

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macrophages. In view of these observations, it is prudent to prevent DNA damage by various mutagens and carcinogens in order to prevent the transformation process of normal to a tumor cell. Since humans are constantly exposed to several endogenous and exogenous mutagens and carcinogens and free radicals that have the ability to cause DNA damage and consequently development of cancer, it is important that efficient DNA repair mechanisms are needed to prevent cancer. This implies that inefficient DNA repair system could lead to the development of cancer. Hence, in order to prevent cancer, cells have evolved not only efficient DNA repair processes but also adequate apoptotic mechanisms so that in the event DNA repair is not possible, cells that harbor damaged DNA are eliminated from the body to prevent the development of cancer. For the elimination of cells that harbor damaged DNA (mutations and activated oncogenes), their recognition and mounting adequate immune response is needed. Thus, it stands to reason that cells that contain abnormal mutations and activation of oncogenes express newer antigens on their cell surface. These newer antigens need to be strong enough to be recognized by the immune system. But, in majority of the instances, these newer antigens are weak and so are not well recognized by the immune system to mount an efficient attack to eliminate them. This could be one of the reasons as to why many cancer cells escape detection by the immune system and survive to proliferate and form tumors.

Genomic Instability and Cancer Genomic stability or instability depends on the efficiency of DNA damage repair (DDR) system. In order to protect from the development of cancer and prevent carcinogenesis, unrepaired DNA damage causes cell cycle arrest and apoptosis. Failure to result in cell cycle arrest and apoptosis can lead to accumulation of genome mutations that ultimately results in development of cancer. Thus, DDR has a critical role in protection against human cancer since a defective DNA repair results in unchecked proliferation. Hence, methods designed to augment DNA repair process and/or prevent the action of proteins involved in DNA repair could be a promising strategy to prevent cancer since such a cell may become normal if the repair process is successful or undergo apoptosis due to unrepaired DNA damage. DDR serves as a guardian of genomic stability to prevent tumorigenesis. At the other end of the spectrum, DDR acts as a negative factor to resist chemo- and radiotherapy. Thus, DDR functions as “a double-edged sword” in cancer prevention and cancer therapy [1]. Developing strategies that can effectively exploit DDR may form a potential cancer therapy target. But it is noteworthy that dysfunction of DNA repair pathways may be compensated for by other potentially compensatory DDR pathway(s), and paradoxically such pathways may enhance tumor cell resistance to various therapies. This implies that DDR pathways could be manipulated to prevent or reverse drug resistance and may also be tweeted to kill cancer cells that depend on a compensatory DNA repair pathway for survival [2–4]. It is to be noted that multiple genomic alterations are involved in the development of cancers that include alterations in oncogenes, tumor suppressor genes,

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DNA mismatch repair, and excision repair genes. Currently, a molecular diagnostic approach to cancers is being developed that tries to identify these abnormalities and develop suitable therapeutic or intervention strategies. Some of the best characterized tumor-related genes that can be detected using a variety of test systems include K-ras, myc, and p53 [5] especially in colorectal, pancreas, and lung cancers. Various methods of their detection include plaque hybridization, dot blot hybridization, combined PCR and RFLP or SSCP, and sensitive PCR. In this context, it is noteworthy that detection of neoantigens expressed by the cancer cells can be used as possible targets in cancer therapy by generating specific antibodies that can be tagged to the conventional chemotherapeutic drugs. Such an approach highlights the concept that immune system can recognize and control tumor growth. In 1893, William Coley showed that live bacteria can be used as an immune stimulant to treat cancer. This has been dubbed as Coley’s toxin that was found to stimulate production of TNF-α by stimulated macrophages and other immunocytes. But such an approach showed only limited efficacy since tumor cells have the ability to avoid recognition and elimination by the immune system and, in fact, sometimes use TNF-α and other similar humoral factors as growth enhancers [6]. However, recent advances showed that immune system can be manipulated to eliminate cancer cells which has been discussed in detail in the previous chapter. Though immune checkpoint inhibitors (including employing blocking antibodies to cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) and by chimeric antigen receptor (CAR) T cells) seem to be of immense promise, current evidence suggests that they are not useful in not more ~20–30% of patients. Furthermore, their use seems to be associated with significant side effects. Hence, more studies are needed that can fine-tune the immune checkpoint inhibitors such that they are more specific and have much less side effects. Some of the mechanisms by which tumors evade the immune system include inefficient methods of antigen processing and presentation machinery; recruitment of suppressor immune cells including regulatory T cells, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages; production of soluble factors that suppress the immune system such as TGF-β and IL-10; and upregulation of ligands that suppress TIL (tumor-infiltrating lymphocytes) activity, such as programmed death ligand-1 (PD-L1). In addition, MDSCs seem to directly encourage tumor growth and metastasis. It is possible that tumor cells are able to reprogram myeloid cells to create an immunosuppressive environment and drive tumor progression directly by promoting cancer stemness, angiogenesis, epithelial-to-mesenchymal transition, and metastasis [7]. Another important aspect that has not been studied well is the possibility that tumor cells may acquire new DNA material from the surrounding cells, bacteria, and viruses (endogenous or exogenous) that may impart newer functions to tumor cells that may help them to survive and thwart the endogenous immune surveillance system. Immune checkpoint therapy that involves using antibodies against CTLA-4 and PD-1 has been shown to inhibit the growth of tumors in mice and humans especially in the treatment of melanoma [8–10]. The underlying cause for all these events to occur in cancer (mutations, genomic instability, DDR, oncogene activation,

PGE2, LXA4, and Treg and Teff Cells and Inflammation

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immunologic changes, etc.,) is due to DNA damage. Thus, if the mutations can be avoided/prevented, DNA damage can be repaired appropriately in time, and/or cells harboring DNA damage can be eliminated by an efficient immune system, there is little scope for cancer to emerge.

Bioactive Lipids and Mutagenesis/Carcinogenesis In a series of studies, we showed that certain specific bioactive lipids such as GLA, DGLA, PGE1, and PGI2 (gamma-linolenic acid, dihomo-GLA, prostaglandin E1, and prostacyclin, respectively) have potent antimutagenic actions. These studies showed that radiation, benzo(a)pyrene (BP), 4-alpha-phorbol, and diphenylhydantoin (DPH)-induced genetic damage to human lymphocytes and mouse bone marrow cells can be prevented by GLA, DGLA, PGE1, and PGI2 [11–26]. In these studies, it was noted that all the mutagenic/carcinogenic agents tested produced significant damage to DNA that could be prevented by GLA, DGLA, PGE1, and PGI2. In a human study, wherein patients with epilepsy who are on long-term DPH therapy, when supplemented with GLA (in the form of evening primrose oil capsules that is rich source of GLA and LA) showed significant decrease in the number of peripheral lymphocytes with DNA damage (measured as lymphocytes containing micronuclei). In these patients, GLA supplementation enhanced circulating levels of GLA. But very little change in the levels of AA, derived from GLA, was noted. This suggests that decrease in the number of circulating lymphocytes containing micronuclei is due to GLA but not as a result of its conversion to AA [26]. Further studies revealed that there is a strong possibility that GLA induced apoptosis of circulating lymphocytes that harbored micronuclei or damaged DNA. This implies that GLA is able to educate the immune system to recognize and induce apoptosis of abnormal human lymphocytes. If this is true, it is an exciting possibility suggesting that GLA has the unique ability to augment the immune response against abnormal cells and enhance the Teff cells. How exactly GLA is able to produce this specific action is not clear. But some speculations can be made.

PGE2, LXA4, and Treg and Teff Cells and Inflammation The transcription factor B-lymphocyte-induced maturation protein-1(Blimp1/ PRDI-BFI) plays a crucial role in the terminal differentiation of many cells [27, 28]. Blimp1 is expressed in many cells, including B and T lymphocytes and myeloid cells. It regulates differentiation of T follicular helper cells. Blimp1 regulates the expression of IL-10 expression in Foxp3+ 3,6 and Foxp3–2,7–11 T cells and CD8+ T cells and modulates effector/memory differentiation. Thus, Blimp1 has a role in the function of both effector (Teff) and regulatory (Treg) cells. Deletion of Blimp1 in T cells leads to the development of chronic intestinal inflammation in mice that is

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similar to inflammatory bowel disease (IBD) seen in humans. This suggests that Blimp1 has a crucial role in the regulation of T-cell homeostasis. Recent studies revealed that Blimp1 activates IL-10 gene. Studies with Foxp3+ Treg cell-specific deletion of Blimp1 mice showed that Foxp3+ Treg cell-intrinsic expression of Blimp1 is required to control Treg and Teff cell homeostasis but is dispensable to prevent development of severe spontaneous intestinal inflammation. Blimp1 controls Treg and Teff cell functions by differentially regulating gene expressions in these cells [29]. It is noteworthy that PGE2, derived from AA and a pro-inflammatory molecule, promotes CD4 Th1/Th17 differentiation. Regulatory T cells, including the IL-10-­ producing Tr1 cells, counterbalance the pro-inflammatory activity of effector Th1/ Th17 cells. Tr1 cell differentiation and function are induced by IL-27 that is dependent on c-Maf in addition to AhR and Blimp-1. PGE2 suppresses IL-27-induced differentiation and IL-10 production of CD4 + CD49b + LAG-3 + Foxp3-Tr1 cells. This action of PGE2 is mediated through EP4 receptors. Thus, PGE2 reduces the expression of c-Maf expression by enhancing cAMP levels. PGE2 reduces IL-21 production in differentiating Tr1 cells. PGE2 can inhibit Tr1 differentiation and c-Maf expression independent of IL-21. PGE2 reduced Egr-2/Blimp-1 expression without any significant effect on STAT1/3 activation and AhR expression. Furthermore, differentiation of CD4 + CD49b + LAG-3+ Tr1 by PGE2 need not be associated with either induction of Foxp3 or production of IL-17. PGE2 inhibits expression and production of IL-27 from activated conventional dendritic cells (cDC) and also reduces murine Tr1 differentiation and function by acting on IL-27-­ differentiating Tr1 cells. Tus, PGE2 inhibits IL-27 production by cDC by its direct inhibitory effect on Tr1 differentiation by reducing c-Maf expression [30–32]. Lipoxin A4 (LXA4), an anti-inflammatory metabolite of AA, suppresses the production and action of PGE2. It may do so by altering the expression of c-Maf, AhR, and Blimp1 and suppressing cAMP levels in the cells. Thus, bioactive lipids may be actively involved in the regulation of generation and action of Treg and Teff cells and modulating lymphopoiesis (see Fig. 4.1). In the present discussion, emphasis has been on the role of PGE2 and LXA4; it is pertinent to mention that other PGs such as PGE1, derived from DGLA, and GLA by itself may have the ability to regulate the formation and function of Treg and Teff cells. Similarly, other anti-­ inflammatory compounds such as resolvins, protectins, and maresins may also show actions similar to LXA4. To what extent EPA and DHA (eicosapentaenoic acid and docosahexaenoic acid) function similar to AA, DGLA, or GLA remains to be determined. It is noteworthy that in our studies, it is observed that GLA-supplemented subjects who were on long-term diphenylhydantoin therapy for epilepsy showed a smaller number of circulating lymphocytes with micronuclei. It is interesting to note that GLA alone induced an insignificant increase in the amount of genetic damage in  vitro. Paradoxically, GLA administration to those who are on long-term diphenylhydantoin (DPH) therapy for epilepsy resulted in the detection of reduced number of micronuclei containing lymphocytes in their peripheral blood; but the degree of DNA damage was found to be increased (as assessed by DNA ladder

PGE2, LXA4, and Treg and Teff Cells and Inflammation

165

Dietary LA Multipotential hematopoietic

GLA

stem cell

?

?

PGE1

DGLA

Lymphoblast AA

LXA4

? Prolymphocyte

PGE2

NK cell

cGMP cAMP

T cell

(-) IL-17

Teff cell

IL-10

(-) IL-21

Treg cell

(-)

(-)

(-) IL-27

c-Maf AhR

(-)

Blimp1

Fig. 4.1  Scheme showing possible role of PUFAs, PGE2, and LXA4 on Teff and Treg cells. PGE2 produces its actions on Treg cells by modulating cAMP activity and c-Maf, AhR, and Blimp1 and, thus, may bring about its pro-inflammatory actions. In contrast, LXA4 and AA are anti-­ inflammatory in nature, and it is likely that these bioactive lipids may have opposite action on cAMP, c-Maf, AhR, and Blimp1. In addition, LXA4 suppresses the production and action of PGE2, and, thus, these two bioactive lipids (PGE2 and LXA4) may regulate Teff and Treg functions and inflammation. Thus, it is likely that the intracellular metabolism of GLA/DGLA/AA in Treg and Teff cells may ultimately determine the initiation, progression, and resolution of inflammation. It is not known but possible that these bioactive lipids have a role in lymphopoiesis because of their ability to alter lymphoblast survival, proliferation, and differentiation and erythropoiesis by regulating stem cell survival, proliferation, and differentiation. In a similar fashion, PGE2 may enhance the expression of PD-1 and PD-L1, while GLA, DGLA, AA, LXA4, resolvins, protectins, and maresins suppress their expression. (−) Indicates negative or suppressive or inhibitory action (PGE2 may have both inhibitory and augmentative actions on Treg function depending on its concentration, half-life, duration of exposure, and the concentrations of other molecules such as TXA2, LTs, lipoxins, resolvins, protectins, maresins, GLA/AA/DGLA, etc.)

pattern). This paradoxical action of GLA suggests that the fatty acid induces apoptosis of cells that harbor significant DNA damage [26] that, in turn, may be due to the ability of GLA and/or its metabolites to modulate Treg and Teff function. In this context, it is interesting to note that lymphokine-activated macrophages that induce apoptosis of tumor cells release linolenic acid (most probably GLA) ( [33–39]; see Chap. 3 for further discussion). Our own studies revealed that GLA,

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4  PUFAs and Their Metabolites in Carcinogenesis

AA, EPA, and DHA can induce apoptosis of tumor cells [40–46]. Since cancer cells harbor altered DNA (in the form of mutations, expressions of various oncogenes, etc.), it is reasonable to propose that GLA has the unique ability to activate the immune system in such a way that it is geared to recognize cells that harbor abnormal DNA and eliminate these abnormal cells by apoptosis. This proposal can be extended to suggest that bioactive lipids could regulate the expression of PD-1 and PD-L1 and, thus, serve as endogenous anti-cancer molecules. Some of these concepts are both supported and disputed by evidences available to date. For example, PGE2 has been shown to be involved in the regulation of vertebrate hematopoietic stem cell homeostasis implying that PGE2 may have a role in lymphopoiesis and in the regulation and generation of Treg and Teff cells [47]. Furthermore, PGE2 and its G protein-coupled receptor EP4 seem to participate in the mobilization of hematopoietic stem cells and hematopoietic progenitor cells (HSCs/HPCs) from the bone marrow to the circulation [48, 49], a process that is needed for the generation of Treg and Teff cells in times of need. These results coupled with the observation that resolution of inflammation requires cyclooxygenase-­2 (COX-2) activity and blocking COX-2 and PGE2 production perpetuated inflammation instead of attenuating inflammation, suggesting that under some very specific conditions, PGE2 may behave as an anti-inflammatory molecule. Under these circumstances, repletion with PGE2 restored homeostasis by enhancing the production of the anti-inflammatory molecule LXA4, a lipoxygenase metabolite of AA [50]. These results provide an inexorable link between cyclooxygenase and lipoxygenase pathways in the resolution of inflammation and the role of PGE2 as a feedback inhibitor that is needed to limit chronic inflammation seen in certain conditions such as lupus and rheumatoid arthritis. These findings may imply that (as has been argued previously by me) that the concentrations of PGE2 need to attain a peak to trigger the generation of LXA4 by the activation of lipoxygenase enzymes to initiate resolution of inflammation. In other words, inflammation needs to reach a peak before resolution process can be triggered and formation of LXA4 is initiated. Thus, the precursor of PGE2 and LXA4, namely, AA, has a very critical role both in the initiation of inflammation and its resolution. Thus, it can be argued that unless and otherwise sufficient degree of inflammation is triggered by the generation of adequate amounts of PGE2, there would not occur activation of lipoxygenase enzymes and generation of resolution-inducing molecule LXA4. In a similar fashion, the balance between Treg and Teff cells could be regulated by the balance between COX-2 and lipoxygenase enzymes, local concentrations of PGE2/LXA4, and the timing of their activation and half-life of PGE2/LXA4. This suggests that PGE2 may have both stimulatory and suppressive actions on Treg cells (see Fig. 4.1). This is supported by the observation that in cancer, inducible (i) or adaptive Treg expand, accumulate in tissues and peripheral blood of patients, and represent a functionally prominent component of CD4 + T lymphocytes. Phenotypically and functionally, iTreg are distinct from natural (n) Treg. A subset of iTreg cells that express CD39 and CD73 hydrolyze ATP to 5’-AMP and adenosine (ADO) that mediates suppression of immune cells which express ADO receptors. It is interesting to note that iTreg produce PGE2. iTreg-mediated suppression involves binding of ADO and

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PGE2 produced by iTreg to their respective receptors expressed on Teff cells, leading to the upregulation of adenylate cyclase and cAMP activities in Teff and, hence, their functional inhibition. Thus, methods designed to regulate PGE2/LXA4 concentrations in these cells may relieve iTreg-mediated suppression in cancer [51]. This knowledge is also relevant to possible therapeutic strategies in autoimmune diseases such as lupus, rheumatoid arthritis, type 1 diabetes mellitus, sepsis, and radiation-induced damage to various tissues. In lupus and type 1 diabetes perhaps, enhancing the action of Treg cells is needed, whereas in sepsis and radiation-induced damage, recovery may depend on sequential and fine-tuned modulation of Treg and Teff cell function, and appropriate modulation of inflammatory and anti-­ inflammatory events is needed for recovery and restoration of homeostasis. The fact that PGE2 may have both pro- and anti-inflammatory actions is evident from the report that deletion of microsomal PGE synthase-1 (mPGES1) exacerbates nonimmune inflammatory arthritis in mice [52]. In this study, it was noted that collagen antibody-induced arthritis model in mice that is deficient in mPGES1 produced a significant increase in the severity of disease with a significant upregulation of neutrophil but not macrophage recruitment to the inflamed joints. A dramatic reduction in the concentrations of PGE2 was noted at the peak of arthritis implying that PGE2 may have anti-inflammatory action. These results led the authors to suggest that PGE2 is an important negative regulator of neutrophil-mediated inflammation. In this study, the authors did not measure LXA4 levels. There is a distinct possibility that LXA4 was produced in this animal model at the peak of arthritis that may have been responsible for the anti-inflammatory action seen in the mPGE1-­ deficient mice. It is noteworthy that animals with Lyme arthritis showed a cyclic change in PGE2 production and similar level during both the initiation and resolution phases of inflammation [53] indicating a dual role of PGE2 in the regulation of inflammation: serving as a mediator of inflammation in the initiation stage of inflammation and as a resolution-inducing agent at the stage of resolution of inflammation. It has been suggested that PGE2 can suppress neutrophil functions [54] and reprogram activated M1 macrophages to M2 macrophages [55], inhibit CCL5 expression in activated macrophages [56], suppress TNF-α production [57], and, possibly, trigger AA release and drive the released AA to the production of pro-­ resolution lipid mediators such as LXA4 [58, 59]. Furthermore, as is evident from the preceding discussion LXA4 synthesis is triggered only at the peak of PGE2 levels. It looks as though PGE2 levels need to reach a peak to trigger LXA4 synthesis and initiate resolution of inflammation. It is not yet known whether PGE2 can be converted to LXA4 under some very specific and special circumstances. This is a distinct and exciting possibility that needs to be investigated in the future. Further evidence in support of the concept that PGE2 has anti-inflammatory action is derived from the observation that in human systemic inflammatory disease (such as sepsis), PGE2 and its receptor EP4 are downregulated, whereas the activity of 15-PGDH (15-prostaglandin dehydrogenase, which mediates PGE2 degradation) is enhanced. Mice with reduced PGE2 synthesis develop systemic inflammation and translocation of gut microbiota and have low IL-22 production that could be restored to normal by EP4 agonists. It was also demonstrated that PGE2–EP4 signaling acts

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4  PUFAs and Their Metabolites in Carcinogenesis

directly on type 3 innate lymphoid cells (ILCs), promoting their homeostasis, and drives them to produce IL-22. These results suggest that PGE2 has anti-inflammatory actions that seem to be mediated through IL-22 [60]. In addition, it was reported that inhibition of 15-PGDH activity potentiates tissue regeneration [61]. Such a tissue regeneration stimulus is likely to be of benefit in several clinical conditions. Since PGE2 has the ability to promote the expansion of several types of stem cells [62], efforts made to enhance its half-life are much desired in instances where tissue regeneration is needed. 15-PGDH-deficient mice had a twofold increase in PGE2 levels in various tissues (especially in the bone marrow, colon, and liver). These animals showed increased hematopoiesis and higher capacity to generate erythroid and myeloid colonies and showed a heightened response to colon injury induced by dextran sulfate sodium as evidenced by a twofold increase in cell proliferation in colon crypts and responded to partial hepatectomy by accelerated hepatic regeneration [61]. In response to bone marrow transplantation, these 15-PGDH-deficient animals showed accelerated recovery of neutrophils, platelets, and erythrocytes that seemed to be due to a fourfold increase in the production of CXCL12 and stem cell factor (SCF). Despite these favorable results reported in terms of suppressing unwanted inflammation, expansion of stem cells, and better tissue regeneration, it is not without some significant side effects and controversy. It was reported by Sasaki et al. [63] that a thioglycollate-induced exudation of leukocytes into the peritoneal cavity and pain nociception were suppressed in mice with the genetic deletion of PGIS (prostacyclin synthase). Both of these reactions were suppressed more effectively in the PGIS/mPGES-1 DKO mice than in the PGIS KO mice. As expected, mPGES-1 deficiency led to suppression in azoxymethane-induced colon carcinogenesis. On the other hand, PGIS deficiency upregulated both aberrant crypt foci formation at the early stage of carcinogenesis and polyp formation at the late stage. These results led the authors to suggest that PGIS and mPGES-1 cooperate to exacerbate inflammatory reactions but have opposing effects on carcinogenesis. It is evident from this data that PGI2 has anticarcinogenic action [63]. These results are in tune with our previous findings wherein we observed that PGI2 has potent antimutagenic actions, whereas PGE2 did not show such beneficial action [19, 24]. These results indicate that a delicate balance needs to be maintained between PGE2 and PGI2 levels such that inflammation and carcinogenesis are kept in check. In addition, it is known that PGE2 receptors EP2 and EP4 are upregulated on virus-specific cytotoxic T lymphocytes especially in those who have HIV, hepatitis C and B, and chronic lymphocytic choriomeningitis infections and show suppressed CTL survival and function. A combined blockade of PGE2 and PD-1 signaling improved viral control and augmented the number of functional virus-specific CTLs [64]. But in this study, the direct effect of PGE2 on PD-1 and PD-L1 expression was not studied. In a surgical stress mice model, it was found that surgical stress reduced CD8+ T cell total numbers in the spleen and impaired CTL function and blockade of PD-1 with specific antibody restored CD8+ T cell numbers and function. A concomitant dramatic increase in blood PGE2 levels was noted following surgical stress induction in this model. As expected, anti-PD-1 antibody along with PGE2

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inhibition restored CTL dysfunction induced by surgery [65]. Though these results do not directly address the interaction between PGE2 and PD-1, it is tempting to propose that PGE2 probably enhances the expression of PD-1 and PD-L1. This may account for immunosuppressive actions of PGE2  in these two studies [64, 65]. Furthermore, PGE2 acting on its receptor EP4 on T cells and dendritic cells facilitates TH1 differentiation and amplifies IL-23-mediated TH17 cell expansion that accounts for immune inflammation seen in multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases, and allergic skin disorders [66]. These results [47–66] emphasize the complex nature of interactions among Treg, Teff, and CTLA4 cells; PGE2, PD-1, and PD-L1; and various cytokines and their role in inflammation and immunosuppression or immunostimulation and cancer development and progression. PGE2 seems to have both pro- and anti-inflammatory actions that may be independent of its action on immune response. The therapeutic potential of PGE2 can be exploited only after a thorough study of its actions that need to include a time– and dose–response studies on Treg, Teff, and CTLA4 cells and its influence on PD-1 and PD-L1 expression and secretion and actions of various cytokines and kinetics of PG synthase and PGDH enzymes under different conditions. In addition, studies need to be performed as to the relationship between AA and PGE2/LXA4/ PGI2/PGFα/TXA2/LTs formation at various time points and doses and in various tissues under different physiological and pathological conditions. The complex nature of actions of PGE2 suggests that perhaps, it is more practical to employ AA/GLA/DGLA administration to derive beneficial actions of PGE2 and LXA4 assuming that cells/tissues are wise and empowered to use these precursors in the most beneficial fashion. This is evident from the observation that AA administration to experimental animals and patients with inflammatory conditions synthesizes LXA4 but not PGE2 [66, 67] and is safe to be given to normal humans [68].

Bioactive Lipids in Mutagenesis and Carcinogenesis It is evident from the preceding discussion that once mutagens and carcinogens attack a normal cell, it leads to a series of actions/reactions that ultimately ends in the conversion of the normal cell into a cancer cell or its elimination if the DNA damage harboring cell is recognized by the immune system and able to mount adequate immune response and include its apoptosis. Our previous study [69] showed that mutagens/carcinogens (such as (DEN) diethylnitrosamine, diphenylhydantoin, 4-α-phorbol, radiation, benzo(a)pyrene, and anti-cancer drugs) suppress delta-5 desaturase activity (the rate-limiting enzyme in the conversion of dietary LA to GLA, the precursor of AA) and, possibly, delta-6 desaturase (which is needed for the conversion of DGLA to AA) that may explain why tumor cell membranes have low levels of long-chain fatty acids, especially AA, and an impaired capacity to undergo lipid peroxidation. These results are supported by several other studies that showed that tumor cells have low activity of desaturases that could be responsible for the low content of PUFAs in tumor cell

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4  PUFAs and Their Metabolites in Carcinogenesis

membranes [70–72]. As a result of this low activity of desaturases and consequently decreased content of PUFAs, the cell membrane architecture will be altered and may become rigid. This, in turn, can lead to: (i) A decrease in the tumoricidal action of macrophages, NK cells, and other immunocytes (ii) Defective Teff and Treg cell functions (iii) Increase in the expression of PD-1 and PD-L1 (iv) An increase in the generation and release of pro-inflammatory cytokines IL-6 and TNF-α, due to the lack of negative feedback regulatory control by bioactive lipids (v) A decreased formation and release of anti-inflammatory and tumor cell growth suppressing molecules such as lipoxins, resolvins, protectins, and maresins (vi) An increase in the number of genetic mutations (leading to activation of various oncogenes)in tumor cells due to lack or deficiency of antimutagenic compounds such as GLA, DGLA, PGE1, and PGI2 (vii) An increased production of pro-inflammatory eicosanoids such as PGE2 (from AA), TXA2 (from AA), and LTs (from AA and EPA) (viii) Fusion of tumor cells with tumor-infiltrating macrophages, leukocytes, and surrounding normal cells and acquisition of tissue-infiltrating and metastasizing properties due to alterations in the cell membrane architecture and function and acquisition of new genetic material. It is proposed that normal cells surrounding the tumor cells may release AA/GLA/ EPA/DHA/lipoxins/resolvins/protectins/maresins to suppress the growth of tumor cells. It is likely that there is some sort of an interaction between normal and tumor cells with one trying to outwit the other. It is perfectly possible that normal cells surrounding the tumor cells try to restrict and contain the growth of tumor cells physically and biochemically by producing growth inhibitory compounds (that may include PUFAs, lipoxins, resolvins, protectins, maresins, etc.) that are aided by the tumorinfiltrating NK cells, T cells, macrophages, and leukocytes. These cells can produce various cytokines, ROS, and bioactive lipids, which contain the growth of tumor cells and sometimes induce apoptosis of tumor cells. In contrast, tumor cells try to overcome the growth inhibitory actions of surrounding normal cells by producing various metalloproteases, collagenases, and other compounds such as IL-10 and TGF-β that can and immune response suppressors such as PGE2 that aid them to evade the immune system and infiltrate the surrounding tissue and metastasize to distant organs. In addition, tumor cells are able to convert M1 to M2 macrophages that enhance the growth of tumor cells (see Fig. 4.2). Furthermore, tumor cells are extremely sensitive to the actions of ROS. Thus, generation of ROS by tumor-­infiltrating immunocytes and surrounding normal cells and chemotherapeutic drugs may actually produce further DNA damage, leading to the activation of several oncogenes that aid tumor cells to evade the immune system by constantly changing their antigenicity and/or mask their antigenic nature (partly due to changes in the cell membrane composition) so that they are not easily recognized by the body’s immune system. Thus, there is a

Bioactive Lipids in Mutagenesis and Carcinogenesis

171

Mutagens & Carcinogens

Treg cells

PUFAs↓

Normal Cell

(-)

Desaturases↓

ROS↑

(+) PGE2↑

T-Helper Cells

Genetic Damage↑

(-)

LXA4↓

(+)

ROS, RNS, IFN-γ, TNF-α

Apoptosis

Tumor Cell

Killer T Cells

Proliferation & Metastasis

Tumor antigens

M1Ф

TNF-α NF-kB↑ PUFAs

IL-4, IL-10, TGF-β, VEGF, IL-8, MMP-9, GM-CSF, Arginase ↑

LXA4

PGE2

(-)

MCP-1

NO ↓

M2Ф

NF-kB↑

(-) Fibroblasts, monocytes, macrophages and vascular endothelial cells

Fig. 4.2  Scheme showing the interaction among mutagens and carcinogens, PUFA metabolism, immunocytes, cytokines, ROS, and microenvironment surrounding the tumor cells and proliferation and metastasis of tumor cells. It is known that mutagens and carcinogens inhibit the activity of desaturases. This leads to a decrease in the formation of GLA, DGLA, and AA from dietary LA and EPA and DHA from ALA. This deficiency of AA, EPA, and DHA could result in decreased formation of their anti-inflammatory metabolites: lipoxins, resolvins, protectins, and maresins. As a result of this (deficiency of GLA, DGLA, AA, EPA, and DHA and lipoxins, resolvins, protectins, and maresins: termed as bioactive lipids), there could occur an increase in the generation and release of pro-inflammatory cytokines, IL-6 and TNF-α, due to the lack of negative feedback regulatory control by bioactive lipids. GLA, DGLA, and PGE1 have antimutagenic actions. Thus, the normal cells exposed to various mutagens and carcinogens will have DNA damages, leading to various mutations and ultimately activation of various oncogenes and conversion into a tumor cell. Deficiency of bioactive lipids will also lead to generation of excess ROS and RNS (reactive nitrogen species) (again due to the absence of negative feedback control exerted by bioactive lipids) that contribute to DNA damage. Mutagens and carcinogens may also have actions on Treg, killer cells, and macrophages and activate NF-kB that leads to the formation and release of excess of proinflammatory cytokines, ROS, and RNS contributing to immunosuppression and DNA damage. It is interesting to note that deficiency of AA, EPA, and DHA is associated with increase in the generation of pro-inflammatory eicosanoids such as PGE2 (from AA), TXA2 (from AA), and LTs (from AA and EPA). These pro-inflammatory PGE2 and similar molecules may be released by tumor cells, tumor microenvironment, and macrophages. Thus, a normal cell exposed to mutagens and carcinogens gets converted into a cancer cell. When tumor cells are supplemented with AA/ EPA/DHA/GLA/DGLA, there will be increased production of anti-inflammatory LXA4, resolvins, protectins, and maresins and PGE1 that have growth inhibitory actions and suppress production of ROS, RNS, IL-6, and TNF-α. PUFAs (especially GLA/AA/EPA/DHA) themselves have growth

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4  PUFAs and Their Metabolites in Carcinogenesis

constant cross talk between normal and tumor cells, and their microenvironment and the balance between various factors discussed above determine the final outcome. Growth inhibitory lipid peroxides are not formed in sufficient amounts in tumor cells (due to their low PUFA content) that would have restrained tumor cell growth. Thus, tumor cells seem to have evolved a variety of clever strategies to overcome natural defenses and sustain their growth (see Figs. 4.2 and 4.3). In this context, it is interesting to note that dexamethasone enhances PD-1 expression during T-cell activation [73]. It is noteworthy that corticosteroids not only block the activity of phospholipase A2 and thus inhibit the release of AA, EPA, and DHA and other PUFAs from the cell membrane phospholipid fraction but are also capable of blocking ∆6 and ∆5 desaturases. As a result, replenishment of cell

Fig. 4.2  (continued) inhibitory action on tumor cells and cause apoptosis of tumor cells by enhancing the formation of ROS and accumulation of lipid peroxides. It is also possible that normal cells surrounding the tumor cells may release AA/GLA/EPA/DHA/lipoxins/resolvins/protectins/maresins that also have growth suppressive action on tumor cells. Furthermore, GLA/AA/ EPA/DHA may aid in the conversion of M1 to M2 (pro-inflammatory nature to anti-inflammatory type) and, thus, suppress tumor cell growth. Thus, there is a constant cross talk between normal and tumor cells, and their microenvironment and the balance between various factors discussed above that determine the final outcome. Thus, when normal cells are exposed to mutagens and carcinogens, increased production of ROS/RNS and pro-inflammatory cytokines results in DNA damage and activation of oncogenes, and this, in turn, would lead to the conversion of a normal cell to tumor cell. On the other hand, when tumor cells are exposed to AA/GLA/EPA/DHA, there will be generation of excess of ROS/RNS and accumulation of toxic lipid peroxides in the tumor cells that ultimately lead to their apoptosis. Thus, it is paradoxical that ROS/RNS will convert a normal cell into a tumor cell, and these very toxic molecules when generated in excess in the tumor cells will lead to their apoptosis due to accumulation of lipid peroxides. Thus, ROS/RNS have both harmful and beneficial actions depending on the circumstances/context and their target. Antioxidants are helpful to quench ROS and prevent normal cell DNA damage and, thus, aid in the prevention of cancer. But if the tumor cells are already lurking around, antioxidants could be harmful since they will be quenching ROS/lipid peroxides that are needed to induce apoptosis of tumor cells (see Fig. 4.4). This may explain paradoxical results obtained with antioxidant supplementation wherein it was observed that subjects who are at high risk of cancer (such as smokers) when were given beta-carotene/vitamin E had higher incidence of lung cancer and other cancers compared to control. It is likely that these study subjects already had tumor cells (undetected due to their fewer number), and on supplementation of antioxidants, the tumor growth was enhanced as explained above. It is also noteworthy that GLA and DGLA and possibly, lipoxins, resolvins, protectins, and maresins have antimutagenic action. Hence, regular supplementation of GLA/DGLA/ AA/EPA/DHA and methods designed to enhance the formation of lipoxins, resolvins, protectins, and maresins may both prevent the conversion of normal cells to cancer cells and growth and spread of cancer as well. Though in the figure only LXA4 is shown, it needs to be understood this represents other anti-­inflammatory metabolites of PUFAs such as resolvins, protectins, and maresins as well. Similarly, PGE2 is mentioned as a representative of all pro-inflammatory eicosanoids. PUFAs refer to GLA/DGLA/AA/EPA/DHA. It needs to be mentioned here that it is not necessary that all PUFAs have the same actions; sometimes they may have diametrically opposite actions depending on their concentrations, context, ratio among various fatty acids, products formed from them (pro- or anti-­inflammatory metabolites and the ratio between these pro- and anti-inflammatory metabolites). The scheme is only a representative of their known actions, and there could be some other actions that are yet to be discovered/described. In general, if methods can be developed such that PUFAs can be selectively delivered to normal cells, then it is possible to prevent the development of cancer. Alternatively, if methods can be developed to selectively deliver PUFAs to cancer cells, then they (cancer cells) could be eliminated by apoptosis and regress cancer

Bioactive Lipids in Mutagenesis and Carcinogenesis

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Mutagens & Carcinogens

∆6 and ∆5desaturases↓ DNA damage and activation of oncogenes

GLA, DGLA, AA, EPA and DHA↓ TNF-α, IL-6↑

PGE2↑

Alteration in cell membrane fluidity

Lipoxins, resolvins, protectins and maresins↓

Macrophages, NK cells, Treg and Teff cells PD-1 and PD-L1 ↑

M1 ↓ M2↑

NK cells↓

Treg ↓

Teff↑

Immunosuppression, cancer progression and metastasis

Fig. 4.3  Scheme showing consequences of action of mutagens and carcinogens on the activity of desaturases and its downstream events, leading to the development of cancer and its progression. For details, see text. All these events can be prevented by availability of adequate amounts of PUFAs to normal cells. In the event of availability of adequate amounts of PUFAs to tumor cells, it would lead to generation of ROS and formation of toxic amounts of lipid peroxides and their apoptosis

membrane AA, EPA, and DHA content will not occur. Hence, eventually, the cell membrane (this may include not only tumor cell membrane but also T cells, NK cells, and macrophages) will be depleted of their PUFA content. As a compensatory mechanism, cells produce excess PGE2 which is again an immunosuppressor. Thus, over a period of time, it leads to PUFA deficiency state with excess of PGE2 resulting in excess production of IL-6 and TNF-α (as a result of absence of negative feedback control exerted by AA, EPA, and DHA.  It is likely that AA, EPA, and

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4  PUFAs and Their Metabolites in Carcinogenesis

DHA are more potent than PGE2 in suppressing IL-6 and TNF-α production), M1 to M2 switch, NK cell, and Teff and Treg cell dysfunction and decreased ROS generation by macrophages – events that result in immunosuppression and proliferation and progression of cancer. In such a scenario, supplementation of GLA/DGLA/AA/EPA/DHA is expected to result in decreased expression of PD-1, PD-L1, and macrophage activation, decrease in Treg function, and increase in Teff cell function and activation and ultimately induction of apoptosis of tumor cells. In order to expedite tumor cell apoptosis and enhance the action of GLA/DGLA/AA/EPA/DHA, one can consider concomitant administration of vitamin C, a pro-oxidant and inducer of tumor cell apoptosis (see Figs. 4.4 and [74–85]). The administration of vitamin C and GLA/ Vitamin C

Dehydroascorbate GLA/DGLA/AA/ EPA/DHA

GSH/NADPH↓ Radiation and anti-cancer drugs

(+)

ROS↑

(+) (-) GADPH

Lipid peroxides

PARP↑

(-) Glycolysis↓

NAD+↓

Antioxidants

ATP↓

(+) Tumor cell Apoptosis

Tumor cell proliferation

(+) Fig. 4.4  Scheme showing collaboration/cooperation and synergistic action between vitamin C and PUFAs in inducing apoptosis of tumor cells. This pro-apoptotic action of vitamin C and PUFAs may be seen in any type of tumor cells and have relatively no action on normal cells. Both vitamin C and PUFAs augment the anti-cancer action of conventional chemotherapeutic drugs and radiation. Antioxidants quench ROS and decrease the formation of lipid peroxides, may decrease PARP activity/levels, and, thus, enhance tumor cell proliferation, actions that are opposite to that of excess ROS and lipid peroxides that enhance apoptosis of tumor cells. Glyceraldehyde 3-­phosphate dehydrogenase (GAPDH) catalyzes the sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules. In addition, GADPH is also involved in transcription activation and initiation of apoptosis. (−) Indicates inhibition of the process, synthesis, or block in their formation. (+) Indicates enhancement of process, synthesis, or action

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175

DGLA/AA/EPA/DHA can be in conjunction with radiation and current chemotherapeutic drugs. Preliminary results indicate that both vitamin C and PUFAs when administered along with chemotherapeutic drugs and radiation not only enhance tumor cell apoptosis but also are able to reduce toxicity of standard chemotherapy and radiation. Our in vitro studies suggested that even drug-resistant cancer cells are susceptible to the apoptotic action of PUFAs [86–88]. Thus, chemotherapeutic drugs, radiation, PUFAs, and vitamin C can be employed simultaneously in the treatment of any type of cancer. In such an instance, it is recommended that plasma levels of vitamin C need to be measured to ensure that therapeutic concentrations of the vitamin are achieved. In general, vitamin C needs to be administered in the range of 1 gm to 5gm/kg body weight to obtain its anti-cancer action. Similarly, high doses of various PUFAs need to be administered intravenously so that adequate amounts of fatty acids are available to achieve its anti-cancer action. It may be mentioned here that PUFAs bind to albumin rather vividly [89], and so high doses of PUFAs and their frequent administration are needed to derive its beneficial actions. Thus, in summary, it can be said that PUFAs have both antimutagenic and anti-­ cancer actions.

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Chapter 5

Molecular Mechanism of Anti-cancer Action of PUFAs with Particular Reference to GLA in Glioma

Abstract  The manifold actions of bioactive lipids especially in the regulation of inflammation, immune response, mutagenesis, and carcinogenesis and their unique ability to modulate the actions of various cytokines and cell membrane structure and function and generation of ROS (including nitric oxide, NO) suggest that they may have a crucial role in the pathobiology of cancer. This is especially true of their ability to induce apoptosis/ferroptosis/necrosis and other forms of cell death. Our own studies and those of others clearly demonstrated that bioactive lipids especially GLA, AA, EPA, and DHA can selectively eliminate tumor cells when given in appropriate doses. T derive such an anti-cancer action bioactive lipids need to be delivered to the cancer cells. In vitro, in vivo, and limited clinical studies revealed that GLA can regress human glioma without any side effects. This selective tumoricidal action of GLA and other polyunsaturated fatty acids seems to reside in their ability to augment free radical generation (ROS, NO, PUFA radical, etc.) and consequent formation and accumulation of toxic lipid peroxides only in tumor but not in normal cells. In an extension of these studies, our recent studies showed that co-­ administration of these bioactive lipids in conjunction with high doses of vitamin C and conventional anti-cancer drugs and immune checkpoint inhibitors can induce remission of other solid tumors in humans. Thus, it is possible that even drug-­ resistant cancers could be effectively treated using this bioactive lipid-based therapeutic approach. Keywords  Glioma · Gamma-linolenic acid · Apoptosis · Ferroptosis · Bioactive lipids · Lipid peroxides · Antioxidants

Introduction It is evident from the discussions presented in the preceding chapters that PUFAs such as GLA, AA, EPA, and DHA and their anti-inflammatory metabolites, lipoxins, resolvins, protectins, and maresins (protectin conjugates in tissue regeneration (PCTR)), suppress tumor cell growth and under some very specific conditions are © Springer Science+Business Media, LLC, part of Springer Nature 2020 U. N. Das, Molecular Biochemical Aspects of Cancer, https://doi.org/10.1007/978-1-0716-0741-1_5

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able to induce their apoptosis/ferroptosis or even necrosis. In this chapter, I will be discussing specifically the anti-cancer action of GLA and its possible mechanisms of action with specific reference to glioma. Previously, I and my colleagues showed that gamma-linolenic acid (GLA, 18:3 n-6) when infused into the glioma tumor bed selectively killed anaplastic cells without harming normal cells. GLA also showed anti-angiogenic action and prevented new blood vessel formation, which supply neoplastic tissue. GLA induced apoptosis of tumor cells by generating free radicals and lipid peroxides only in tumor cells but not in normal cells, and, thus, it behaves as an agonist of free radicals to kill tumor cells.

Metabolism of Essential Fatty Acids (EFAs) The dietary cis-linoleic acid (LA, 18:2 n-6) and alpha-linolenic acid (ALA, 18:3 n-3) are essential fatty acids (EFAs). These are called as EFAs since they cannot be synthesized in the body and, hence, have to be obtained from diet. Both LA and ALA need to be converted to their long-chain polyunsaturated fatty acids such as gamma-linolenic acid (GLA, 18:3 n-6), dihomo-GLA (DGLA, 20:3 n-6) and arachidonic acid (AA, 20:4 n-6) from LA, and eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) from ALA by the action of desaturases and elongases as shown in Fig. 5.1. DGLA is the precursor of one series of prostaglandins. AA is the precursor of two-series prostaglandins (PGs) and thromboxanes (TXs) and four-series leukotrienes (LTs). Similarly, EPA is the precursor of three-­ series PGs and thromboxanes and five-series LTs. Both delta-6 desaturase and delta-5 desaturase are considered as the rate-limiting steps in the metabolism of EFAs. Various PGs, LTs, and TXs have potent pro-inflammatory actions. But it needs to be noted that three-series PGs and TXs and five-series LTs are much less potent pro-inflammatory molecules compared to two-series PGs and TXs and four-­ series LTs. For all practical purposes, LA, GLA, DGLA, AA, ALA, EPA, and DHA are called as PUFAs, whereas LA and ALA are EFAs. It is noteworthy that some, if not all, of the actions of EFAs can also be brought about by GLA, DGLA, AA, EPA, and DHA. In view of this, GLA, DGLA, AA, EPA, and DHA are also considered as “conditional EFAs.” It may be mentioned here that several scientists call LA, GLA, DGLA, AA, ALA, EPA, and DHA as EFAs, which is not technically correct. In the present discussion, PUFAs refer to GLA, DGLA, AA, EPA, and DHA, whereas EFAs refer to LA and ALA. In addition to forming precursors to pro-inflammatory PGs, TXs, and LTs, certain potent anti-inflammatory molecules are also formed from AA, EPA, and DHA.  Thus, AA is the precursor of lipoxin A4 (LXA); EPA is the precursor of resolvins of E series, whereas DHA is the precursor of D series of resolvins, protectins, and maresins. Lipoxins, resolvins, protectins, and maresins are also potent anti-inflammatory compounds. A new class of peptide-conjugated specialized pro-­ resolving mediators that have tissue regenerative actions that belong to the protectin family called as protectin conjugates in tissue regeneration (PCTR) has also been

Metabolism of Essential Fatty Acids (EFAs)

183

n-3

n-6

Diet

Cis-LA, 18:2

α-ALA, 18:3 Δ6 Desaturase

GLA, 18:3 Insulin, Folic Acid, Vitamin B12, B6, Vitamin C,Zn2+ , Se, Mg2+, Ca2+

DGLA, 20:3

Δ5 Desaturase

PGs of 1 series AA, 20:4

EPA, 20:5

DHA, 22:6

VEGF, Endoglin, sFlt1, PlGF

PGs of 2 series, TXs, LTs of 4 series

PGs of 3 series, TXs, LTs of 5 series

Lipoxins, Resolvins, Protectins

eNO, PGI2, PGI3 Pro-inflammatory in Nature

ROS

Anti-inflammatory in Nature

Proinflammatory Cytokines IL-6, TNF-α, HMGB1

Anti-inflammatory Cytokines IL-4, IL-10, IL-12

Obesity, HTN, Type 2 DM, Metabolic syndrome, Pre-eclampsia, Autism

Normal

Fig. 5.1  Scheme showing effect of vitamins, minerals and trace elements on metabolism of essential fatty acids and their role in various diseases

described. It may be mentioned here that, for ease of reference, at various places in this book, only the names of lipoxins, resolvins, protectins, and maresins are mentioned. At some places, only the name of lipoxins is given. It is to be noted that at all these places, lipoxins are mentioned as a representative of all anti-inflammatory compounds formed from GLA, DGLA, AA, EPA, and DHA, and these include resolvins, protectins, maresins, and PCTRs.

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5  Molecular Mechanism of Anti-cancer Action of PUFAs with Particular Reference…

Fig. 5.2  Metabolism of AA, EPA, and DHA by COX and LOX enzymes leading to the formation of lipoxins, resolvins, protectins, and maresins

AA, EPA, and DHA are metabolized not only by COX and LOX enzymes but also by CYP-450 that leads to the formation of various metabolites including epoxy eicosatetraenoic acids (EETs), hydroxyeicosatetraenoic acids (HETEs), and hydroperoxyeicosatetraenoic acids (HPETEs). EETs, HETEs, and HPETEs also participate in inflammatory and other cellular processes. COX-2 metabolizes free AA to form 11R, and 15S-HPETE, which are reduced to 11R,15-HETE by peroxidase and is converted to 11- and 15-oxo-EET. EPA and DHA may also undergo similar metabolic fate by cytochrome P450 (see Figs. 5.1, 5.2, 5.3, 5.4, and 5.5 for metabolism of EFAs and AA, EPA, and DHA and various products formed from them). It is possible that LA, GLA, AA, and docosapentaenoic acid (DPA) may also undergo similar metabolic fate(s) as that of DGLA, AA, EPA, and DHA. It is also relevant to record here that when the term “bioactive lipids” is used, it refers to all those lipids that have biologically significant action(s) and more specifically refers to GLA, DGLA, AA, EPA, DHA, and DPA.

Anti-cancer Action of PUFAs Our own studies and those of others revealed that GLA, AA, EPA, and DHA are selectively toxic to tumor cells (similar actions are als shown by conjugated LA but are probably less effective compared to GLA, AA, EPA and DHA [1, 2]) with little or no action on normal cells [3–11]. This selective action of these fatty acids is

Anti-cancer Action of PUFAs

185 COOH arachidonic acid

COX-1&2 O O O HO

OH

O

OOH PGG2 reduction COOH

COOH

COOH OH

O

OH

OOH 5-LO [H] at C15 O

COOH 12-LO or 15-LO

LTA4

OH

COOH LTB4

LXA4 synthase HO OH

OH

Leukotrienes

COOH

COOH

OH

LTB4 synthase

OH

PGD2

Prostanoids

COOH

COOH

enzymatic epoxidation O

OH PGH2 hPGD2S

HO PGE2a

OOH

COOH

O

COOH PGE2

15-LO

5-LO

LXB4 synthase OH

COOH LXA4

COOH

HO OH LXB4

Lipoxins

COOH

O 15-deoxy-D12.14-PGJ

O

2

OH D

12.14

-PGJ2

Cyclopentenone Prostaglandins

Fig. 5.3  Formation of various products from AA and their structures

Membrane-phospholipids Phospholipase A2 COOH

Arachidonic acid (AA)

Cytochrome P450 enzymes

CH2OH 20-Hydroxyeicosatetraenoic acid (20-HETE) COOH

O 11,12-Epoxyeicosatrienoic acid (11,12-EET)

Hormones and growth factor via GPCR

COOH

Eicosapentaenoic acid (EPA)

COOH

Nutrition: (w-3)-/(w-6)-FA

COOH CH2OH 20-Hydroxyeicosapentaenoic acid (20-HETE) COOH

O 17,18-Epoxyeicosatetraenoic acid (17,18-EETeTr)

COOH Docosahexaenoic acid (DHA)

Disease conditions: NO, ROS, CO

COOH CH2OH 22-Hydroxyeicosahexaenoic acid (22-HDHE) COOH O 19,20-Epoxydocosapentaenoic acid (19,20-EDP)

Regulation of vascular, renal and cardiac function

Fig. 5.4  Metabolism of AA, EPA, and DHA by cytochrome P450 enzymes. Products formed from this pathway have actions on vascular, renal, and cardiac tissues

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Arachidonic Acid

Omega hydroxylation

16,17& 18-OH

Epoxygenase

Cyt P450/Allylic oxidation

19, 20-OH Diepoxide/Epoxy alcohols

Hepoxilin

Allene oxide

Prostagladins

Oxidation products of PGs, LTs, TXs Glutathione adducts

Trioxilins

Fig. 5.5  Metabolism of AA by cytochrome P450 enzyme system. Both EPA and DHA may also undergo similar metabolism by the cytochrome P450 enzyme system

supported by the observation that in mixed culture experiments, in which both normal and tumor cells were grown together, GLA showed more selective tumoricidal action compared to DGLA, AA, EPA, and DHA [12]. In addition, direct intratumoral injection of GLA regressed human gliomas without any significant side effects [13, 14]. This specific tumoricidal action of these fatty acids could be attributed to their ability to trigger free radical generation and formation of toxic lipid peroxides in the tumor but not in normal cells. In addition, it was noted that the colony formation ability of tumorigenic F4 cells was inhibited by GLA and EPA. In contrast, the non-tumorigenic revertants were not affected. Even retransformed tumorigenic variants when supplemented with GLA showed growth inhibition. Pro-oxidants such as ferrous chloride and copper salts enhanced the cytotoxicity of GLA in tumorigenic but not in non-tumorigenic variants. Based on these results, it can be suggested that tumorigenicity per se is characterized by a high sensitivity to PUFAs, especially to GLA, and that transformed cells are specifically sensitive to the cytotoxic action of GLA [15]. Tumor cells have low d-6-d activity, which is necessary for the conversion of LA and ALA to their respective long-chain metabolites. Hepatocarcinogens, diethylnitrosamine (DEN), and 2-acetylamino-fluorine (2-AAF) have been shown to suppress the activity of d-6-d and d-5-d resulting in low levels of GLA and AA, EPA, and DHA in the tumor cells [16–18]

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 OS and Lipid Peroxides as Mediators of the Anti-cancer R Action of PUFAs (Bioactive Lipids: BAL) It is well documented that the higher the intracellular concentrations of lipid peroxides, the lower the mitotic rate and vice versa [19]. This is in tune with the observation that, in general, tumor cells are resistant to lipid peroxidation compared to normal cells [20, 21]. This observation is in tune with the fact that cancer cells have lower microsomal phospholipid content and decreased content of unsaturated fatty acids [22]. This low content of PUFAs may explain the low rate of lipid peroxidation seen in tumor cells. Tumor cells have been found to have low PUFA content and cytochrome P450 and elevated levels of lipid-soluble antioxidant α-tocopherol. Previously, we showed that PUFAs, especially GLA, can enhance free radical generation (especially superoxide anion and hydrogen peroxide) and augment the formation and accumulation of lipid peroxides in tumor but not in normal cells, a phenomenon that occurred despite the fact that the uptake of fatty acids was at least two to three times higher in the normal compared to tumor cells [23, 24]. It is likely that the low content of PUFAs in the tumor cells is a result of decreased activity of delta-6 and delta-5 desaturases that are needed for the metabolism of dietary LA and ALA to form their more unsaturated and longchain metabolites such as GLA, DGLA, AA, EPA, and DHA. The elevated levels of PGs reported in many cancers could be attributed to a compensatory increase in the formation of various PGs, LTs, and TXs. It is tempting to suggest that tumor cells have increased COX-2 and lipoxygenase activities to form excess amounts of PGE2 that have immunosuppressive, cell proliferation-inducing, and metastatic-enhancing potential actions seen with tumor cells [25–35]. This led to the use of nonsteroidal anti-­inflammatory drugs for the prevention of colon and other cancers, though results have been unsatisfactory. It is known that tumor cells have relatively higher content of antioxidant α-tocopherol [36]. But tumor cells may have variable concentrations of low superoxide dismutase (SOD), glutathione peroxidase, and catalase enzymes. As a whole, it can be said that substrate (i.e., PUFAs) deficiency, a relatively high content of vitamin E, and enhanced levels of PGs are the reason for the low rate of lipid peroxidation seen in tumor cells [37, 38]. In addition, free radicals such as H2O2 (hydrogen peroxide) trigger p53-dependent apoptosis that can be correlated with p53 nuclear translocation and are cell cycle related. p53 suppresses the expression of MnSOD, and overexpression of p53 decreases the levels of SOD. Since tumor cells have impaired p53 and higher SOD activities, this could lead to higher antioxidant capacity in tumor cells. This is in tune with the suggestion that p53 has a prooxidant type of activity. Thus, when tumor cells are exposed to free radicals (that can be induced by supplementing tumor cells with PUFAs), they need to enhance its SOD activity in order to antagonize the cytotoxic action of ROS.  Such an oxidative stress can augment the expression of p53 that, in turn, could suppress SOD, tilting the balance more towards a pro-oxidant state which ultimately causes apoptosis of tumor cells. But our studies showed that PUFA-induced ROS generation fails to augment the antioxidant capacity of tumor cells, and, hence,

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they undergo apoptosis. Thus, tumor cells seem to be extremely sensitive to oxidant stress. In addition, free radicals and lipid peroxides can inactivate several enzymes, denature proteins, and deplete cellular ATP content that ultimately induce apoptosis. Free radicals cause ATP depletion in the cells by activating PARP (poly(ADP-­ribose) polymerase), the substrate of caspase-3. Thus, efforts made to enhance free radical generation in tumor cells especially by PUFAs induce apoptosis by several mechanisms. When tumor cells are exposed to PUFAs especially GLA, there is an increase in phosphorylation of Bcl-2 that also induces apoptosis. Our studies showed that GLA is more toxic to tumor cells with little or no effect on normal cells (GLA > AA > EPA > DHA) [39–46]. This selective tumoricidal action of GLA was not blocked by cyclooxygenase and lipoxygenase inhibitors, indicating that PGs, TXs, and LTs do not have any role in PUFA-induced apoptosis of tumor cells. GLA produced almost a two- to threefold increase in free radicals and lipid peroxidation products in tumor cells but not in normal cells. Thus, it can be said that the low rates of lipid peroxidation seen in tumor cells are at least, in part, due to PUFA deficiency and a relative increase in their antioxidant content. Schlager et al. demonstrated that mice peritoneal macrophages are cytotoxic to tumor cells upon exposure to lymphokine (LK, cytokine). These lymphokine-­ activated macrophages showed almost a two- to threefold increase in their linolenic acid (this could be GLA since macrophages do not synthesize ALA but can form GLA from LA) content when their tumoricidal action is at peak. This enhanced cellular lipid and fatty acid content returned to control when the macrophages lost their tumoricidal activity. These lymphokine-activated macrophages and those that were enriched in linolenic acid also showed markedly tumoricidal activity that correlated with a transient but significant increase in superoxide release. This indicates that endogenous levels of linolenic acid (presumably GLA) regulate the tumoricidal activity of macrophages. This tumoricidal action of activated macrophages was found to be not associated with any new protein synthesis, oxidative metabolism, or augmented capacity for tumor target binding [47]. These results are in tune with our observation that it is GLA but not ALA that has potent tumoricidal action [48–52]. It was also observed that GLA (and also AA, EPA, and DHA) can enhance the cytotoxic action of anti-cancer drugs [49, 50].

PUFAs and Immune System in Cancer One of the important functions of the immune system is to identify the specific antigens that are expressed by the tumor cells and mount an immune response and eliminate them. In general, the tumor cells express antigens that are unique to them that are not found on normal cells. The immune system recognizes these tumor-­ specific antigens as foreign which enables them to recognize these new antigens present on the tumor cells. Tumor antigens are presented on MHC class I molecules in a similar way to viral antigens, which allows killer T cells to recognize them as

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foreign and eliminate the tumor cells. In an occasional instance, antibodies that are generated against tumor cells use complement system to kill the tumor cells. Paradoxically, despite the presence of a robust immunological system in the body, it often fails to kill the tumor cells, and tumor cells evade the immune system by producing immunosuppressive molecules such as TGF-β, PGE2, and certain blocking antibodies. This makes the immune system ineffective in eliminating the tumor cells, and so they grow. Studies have also shown that sometimes macrophages may actually promote tumor growth by secreting some cytokines and growth factors that may enhance the growth of the tumor cells. Macrophages that encourage inflammation are called M1 macrophages, whereas those that decrease inflammation and encourage tissue repair are called M2 macrophages. This difference is reflected in their metabolism: macrophages that metabolize L-arginine to NO induce inflammation, while those convert L-arginine to ornithine show anti-inflammatory properties [53]. The M1 “killer” phenotype of macrophages is induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) that render macrophages to secrete high levels of IL-12 and low levels of IL-10. In contrast, the M2 “repair” phenotype macrophages secrete anti-inflammatory cytokine, IL-10. IL-4 cytokine can also encourage macrophages to become M2 type. M2 macrophages produce high levels of IL-10 and TGF-β and low levels of IL-12. Tumor-­ associated macrophages (TAM) are mainly of the M2 phenotype, and they promote tumor growth [54]. Though the exact mechanism by which M2 macrophages contribute to tumor growth and progression is not clear, it is opined that TNF-α secreted by these macrophages activate nuclear factor-kappa B (NF-kB) that, in turn, induces the production of monocyte chemotactic protein 1 (MCP1) by tumor cells. MCP1 is expressed mainly by tumor cells, fibroblasts, monocytes, macrophages, and vascular endothelial cells in the tumors. There is a close association between the expression of MCP1 and the accumulation of TAM. The expression of MCP1 also correlated significantly with the levels of VEGF, TNF-α, and IL-8. Increased expression of MCP1 and VEGF was found to be an early indicator of tumor progression and recurrence of tumor. Thus, MCP1 is an early indicator of tumor angiogenesis and growth, and, hence, it can be considered as a pro-tumorigenic protein [55]. TAM promote not only tumor cell proliferation and angiogenesis, but also their metastasis and NF-kB seem to be the master regulator of this macrophage polarization from M1 type to M2 type. NF-kB inhibition resulted in a significant decrease in the secretion of IL-10, a TH2 cytokine; a significant increase of TH1 cytokine production such as IL-12, TNF-α, and IL-6; suppression of VEGF production and matrix metalloproteinase-9 mRNA expression; a reduction of arginase mRNA expression; and an increase in NO production [56], factors that are needed for tumor growth. Recent studies have suggested that lipids could play a significant role in the generation of TH17–Treg cells, and yet other investigations showed that blocking PD-1, programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), a cell surface receptor that belongs to the immunoglobulin superfamily and expressed on T cells and pro-B cells, could be exploited to activate the immune system and treat cancer.

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PD-1, encoded by the PDCD1 gene, is expressed on T cells and pro-B cells. PD-1 binds two ligands, PD-L1and PD-L2. PD-1 and its ligands play a critical role in downregulating the immune system by preventing the activation of T cells. PD-1 promotes apoptosis of antigen-specific T cells and simultaneously reduces apoptosis of regulatory T cells (suppressor T cells), suggesting that drugs that block PD-1, the PD-1 inhibitors, may activate the immune system to attack tumors and are therefore useful in the treatment of cancer. Both PD-1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) are capable of actively modulating T-cell responses [57]. Both PD-1 and CTLA4 assist the tumor cells to escape from immunosurveillance, as they impair T-cell functions, often leading to exhaustion, decreasing secretion of IL-2, IFN-γ, and TNFα and dampening the proliferation of T cells, which would lead to their (T cells) reduced cytotoxic action. Hence, it stands to reason that blockade of PD-1 signaling and CTLA4 will enable the creation of an immunogenic tumor microenvironment that could lead to regression of tumors [58, 59] Immunostimulants activate naïve T cells to undergo proliferation and differentiate them into three distinct effector TH subsets based on their cytokine phenotypes. The three types of TH subtypes are TH1 cells that produce IFN-γ and protect against intracellular pathogens; TH2 cells that produce IL-4, IL-13, and IL-25 and help to clear extracellular pathogens; and TH17 cells that produce IL-17 and are meant to clear extracellular pathogens that are not effectively handled by either TH1 or TH2 cells. TH17 cells not only produce IL-17 but also secrete IL-21 and IL-22 and are capable of inducing the production of various pro-inflammatory cytokines, chemokines, and metalloproteinases from various tissues and cell types to help in the recruitment of neutrophils to tissues. Both IL-21 and IL-22 are not TH17-exclusive cytokines but are preferentially expressed in TH17 cells. PGD2, PGE2, PGF2α, PGI2, and thromboxane A2 derived from AA in response to stimuli are present in significant amounts at the sites of inflammation [60, 61]. Among them, PGE2 has immunomodulatory action and suppresses TH1 differentiation. Stimulatory action of PGE2 on TH17 differentiation or expansion in vitro is noted. PGE2 also has a role in the impairment of CTL function in coordination with PD-1. PGE2 has pro-inflammatory actions, and the immunosuppressive function of PGE2 may be responsible for the immunosuppression seen in cancer and its ability to limit the functions of NK cells, CD4, and CTLs [62, 63]. The increase in the production of PGE2 both by the tumor cells and monocytes/macrophages infiltrating the tumor has been held responsible for the defective cellular immune response, hypercalcemia, tumor cancer cell proliferation, tumor angiogenesis, and resistance of tumor to anti-VEGF therapy (see Fig. 5.6). In addition, PGE2 may have the ability to modulate NO generation. In contrast to the previous results, we also noted that both COX and LOX inhibitors (indomethacin and nordihydroguaiaretic acid when used as 60 and 20 mg/ml, respectively) enhanced the growth of IMR-32 cells. Though this growth-promoting action of indomethacin and NDGA could be attributed to their non-specific antioxidant actions, it is equally possible that some unknown products have been generated from PUFAs that have growth-promoting actions. In order to verify this possibility, we also studied the effect of LXA4,

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GLA

(-)

DGLA AA IL-17

NO

IL-10↑

p53

↓

PGE2 Treg cells↑

IL-6, TNF-α↓

Resistance to Anti-VEGF

CTL↓

Tumor cell proliferation, angiogenesis, metastasis↑

IFN-γ↓

CSC

↑↓

Fig. 5.6  Actions of GLA, AA, and PGE2 on various cytokines and immunocytes and its relationship to tumor cell behavior. CSC cancer stem cells, CTL cytotoxic T cells. PGE2 suppresses immune response and facilitates tumor growth. In contrast, GLA and AA enhance immune response and suppress tumor growth both by converting M2 to M1 macrophages and by direct toxicity to tumor cells by enhancing the formation of toxic lipid peroxides in tumor cells. GLA inhibits PGE2 synthesis and, thus, may abrogate PGE2-induced immunosuppression. AA may be converted to PGE2 by tumor cells and TAM and, thus, in an occasional instance may seem to encourage tumor growth and suppress immune response. Thus, the activity of COX-2 in the tumor cells determines how AA delivered to tumor cells (by plasma, surrounding normal cells and infiltrating macrophages, etc.) is processed by tumor cells. If the activity of COX-2 is high, it leads to PGE2 formation and increase in tumor growth. On the other hand, if COX-2 activity is low and TAM are able to deliver adequate amounts of ROS to tumor cells, it leads to the formation and accumulation of toxic lipid peroxides in tumor cell and induction of their apoptosis

resolvins, and protectins formed from AA, EPA, and DHA and noted that these antioxidant metabolites inhibited the growth of IMR-32 cells [49]. Thus, PGE1, PGE2 (may have both growth-promoting and growth-suppressive actions depending on the concentration of PG2 used), LTD4, LXA4, resolvins, and protectins seem to possess growth inhibitory action on IMR-32 cells. These results imply that the balance between various eicosanoids formed from their precursors and the cellular content of PUFAs in the surrounding milieu of tumor cells (i.e., normal cells surrounding the tumor cells) may determine whether tumor cells are induced to proliferate or suppress from further growth. This is so since we and others noted that GLA, DGLA, AA, EPA, and DHA have potent growth inhibitory action on several types of tumor cells both in  vitro and in vivo [39–46, 64–73]. In summary, these results showed that: (i) Though all PUFAs have tumoricidal action, among them, GLA is the most potent (GLA > AA > DGLA > EPA > DHA > LA = ALA). As the concentrations of AA, EPA, and DHA are increased, these fatty acids were also found

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to be toxic to normal cells, whereas GLA was not toxic to normal cells even at higher concentrations in which AA, EPA, and DHA were toxic to normal cells. Thus, GLA showed the most differential tumoricidal action [41]. (ii) GLA inhibited PGE2 synthesis, a compound that has pro-inflammatory action and enhances the production of IL-17 that is capable of enhancing the growth of tumor stem cells [74]. (iii) GLA inhibited angiogenesis [75]. (iv) Macrophages supplemented with GLA produced hydroxy GLA that is capable of killing tumor cells [47]. (v) GLA inhibited the production of IL-17. (vi) GLA enhanced the action of cytotoxic T cells. (vii) GLA blocks the action of programmed cell death protein (PD-1) so that immune response against tumor cells is enhanced. (viii) GLA has anti-inflammatory actions so that the inflammatory process involved in the progression of cancer cell growth is inhibited. (ix) GLA has anti-VEGF action and, thus, decreases angiogenic process to tumor that is needed for the cancer to grow. (x) GLA and other PUFAs can form toxic lipid peroxides in tumor cells that could lead to the apoptosis of tumor cells. But, unlike other PUFAs that form toxic lipid peroxides in significant amounts both in normal and tumor cells,

Fig. 5.7  (a) Effect of pretreatment with PUFAs on alloxan (i) and (ii) simultaneous treatment with PUFAs + STZ-induced changes in the concentrations of LXA4 in RIN cells in vitro. Concentrations of PUFAs used in this study are 15 μg. It is seen from the results shown in Fig. 5.2 that among the fatty acids tested, GLA induced the highest or at least as effective as that of other fatty acids in the generation of LXA4 from RIN cells in vitro. A alloxan, S streptozotocin, AA arachidonic acid, GLA gamma-linolenic acid, EPA eicosapentaenoic acid, DHA docosahexaenoic acid, C control

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GLA gives rise to toxic lipid peroxides in large amounts more specifically in the tumor cells compared to normal cells, and, thus, GLA is differentially toxic to tumor cells unlike other PUFAs such as AA, EPA, and DHA. (xi) Our studies showed that GLA can enhance while anti-cancer drugs such as streptozotocin (used in the treatment of malignant islet cell tumors) reduced the formation of lipoxin A4 (LXA4) (see Fig. 5.7). Other PUFAs also enhance the formation of LXA4, but GLA showed the highest. LXA4 inhibits the growth of tumor cells but is also cytoprotective to normal cells. Thus, GLA forms 15-, 12-, and 8-OH 20-carbon and 13-OH 18-carbon fatty acid derivatives which are conjugated dienes and/or hydroperoxyl groups of GLA peroxides that are toxic to tumor cells [76], and at the same time, GLA enhances the formation of LXA4 in normal cells so that normal cells are not killed by these lipid peroxides. On the other hand, AA, EPA, and DHA also form lipid peroxides in the tumor cells (GLA > AA > EPA > DHA) but form much less LXA4 in normal cells, and, hence, normal cells are not protected from the cytotoxic action of lipid peroxides that may explain the more specific and differential toxicity of GLA to tumor cells in comparison to AA, EPA, and DHA. Furthermore, GLA generated lipid peroxides in tumor cells within a short span of time, and these GLA-induced peroxides persist for longer time compared to lipid peroxides generated by the addition of AA, EPA, and DHA. (xii) GLA increased the expression of p53, which can induce apoptosis of tumor cells [75]. (xiii) GLA also decreased the expression of Bcl-2, an anti-apoptotic gene. (xiv) GLA also enhanced the anti-cancer action of radiation and other chemotherapeutic drug and, thus, can be used to augment the anti-cancer action of radiation and conventional anti-cancer drugs [50].

PUFAs (Especially GLA) Have Anti-angiogenic Action Malignant tumors need adequate blood supply to support their growth properties. This increased demand for blood supply needs the genesis of new blood vessels and is called as angiogenesis. Thus, malignant tumor is an angiogenesis-dependent condition, and they fail to survive if angiogenesis fails to occur at the most appropriate time. This tumor-associated angiogenesis is a complex and multistep process. Angiogenesis is necessary, but not sufficient, for tumor growth [77]. But angiogenesis is a common pathway for tumor growth and its progression. Despite this observation, anti-angiogenic agents failed to arrest tumor growth to a clinically significant degree in patients. Majority of these anti-angiogenic agents are proteins/peptides, and, hence, their long-term use led to the development of neutralizing antibodies, and so the tumors became resistant to their actions. In addition, many anti-angiogenic substances need to be given repeatedly and are relatively unstable and difficult to manufacture in large amounts. But for the production of angiogenic factors, which are produced mainly by the tumor cells [78], macrophages [79], leukocytes,

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platelets [80], and endothelial cells [81], a viable tumor mass or tumor cells are needed, which is the stimulus for the production of angiogenic factors. In other words, if the tumor cells are killed, then they (tumor cells) will not be able to produce angiogenic factors by themselves and incite the macrophages, endothelial cells, leukocytes, and platelets to produce VEGF and other angiogenic factors for the tumor cells to grow and progress. Thus, the most important strategy to eliminate tumor mass is to kill tumor cells directly with little or no action on surrounding normal cells. In this context, it is noteworthy that PUFAs and especially GLA have the potential to selectively reduce (i) the growth of and induce apoptosis of the tumor cells; (ii) inhibit the production of angiogenic factors including VEGF; (iii) block PGE2 production; (iv) prevent angiogenesis; (v) render macrophages infiltrating the tumor bed to produce more hydroxy fatty acids that are toxic to tumor cells; (vi) decrease IL-17 synthesis; (vii) enhance the action of cytotoxic T cells and decrease the expression of PD-1; (viii) suppress inflammation locally; (ix) enhance the expression of p53; (x) inhibit the expression of Bcl-2; and (xi) increase the production of LXA4 in the surrounding normal cells when GLA is injected into at least a portion of the neoplastic region, preferably glioma (especially, glioblastoma multiforme and other types of primary brain tumors and secondaries arising from other cancers elsewhere in the body that have metastasized to the brain). Thus, it is proposed that when a therapeutically effective amount of a solution of GLA is injected into the tumor bed, the tumor cells undergo apoptosis. It is suggested that GLA can be used in combination with tumor necrosis factor, anti-cancer drugs, lymphokines, specific polyclonal or monoclonal antibodies, and anti-cancer drugs such as vincristine, adriamycin, doxorubicin, cyclophosphamide, cis-platinum, L-asparaginase, procarbazine, camptothecin, taxol, 5-fluorouracil, or busulfan to suppress the growth of tumor cells. It is suggested that PUFAs including GLA may serve as inducers of free radicals such as superoxide anion, hydroxyl radical, nitric oxide (NO), and other free radicals. Under some well-defined conditions, PUFAs including GLA may themselves function as radicals or form radicals, and this can be dubbed as PUFA• and GLA• radicals. These PUFA• and GLA• radicals can ultimately produce apoptosis of cancer cells but will not produce apoptosis of normal cells since normal cells have relevant antioxidant defenses. Preliminary results showed that PUFAs/GLA could target tumor-derived stem cells in a very specific manner.

Mechanism of Action of PUFAs/GLA on Tumor Cells It is reported that the differences in the tumoricidal action of GLA with regard to other PUFAs such as DGLA and AA are the differences in the way these fatty acids are handled by the tumor cells and the type of lipid peroxides formed from these fatty acids. For instance, GLA is metabolized in the tumor cells to form 15-, 12-, and 8-OH 20-carbon and 13-OH 18-carbon fatty acid derivatives that seem to be responsible for the apoptosis of tumor cells. In contrast, DGLA undergoes an

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exclusive C-8 oxygenation pathway during COX-catalyzed lipid peroxidation. This is in addition to a C-15 oxygenation pathway shared by both DGLA and AA. This exclusive C-8 oxygenation leads to the production of distinct DGLA’s free radical derivatives that may be correlated with DGLA’s anti-proliferation activity. As shown below, DGLA undergoes a unique C-8 oxygenation reaction pathway, in addition to a C-15 oxygenation pathway shared by both DGLA and AA. COOH 9 C-8 oxygenation O 8 O 9 11

8 + 13

11

OOH 13

COOH

HO

15

COOH

8-OH-octanoic acid (8-HOA) *

COOH 15 DGLA

Heptanoic acid (HTA) *

COX, O2

O

9 8

C-15 oxygenation O 11

COOH 13

Pentane, in gasphase

15 OOH

OH

Hexanol (HEX) *

The C-8 oxygenation results in the formation of two exclusive DGLA-derived free radical metabolites, •C7H13O2 and •C8H15O3, while the C15 pathway produces two common radical metabolites, •C6H13O and •C5H11. On the other hand, HO COOHand called as 8-OH-octanoic acid (8-HOA) and OH

1-heaxanol (HEX), respectively, are expected to be formed from free radicals by abstracting an H• from the environment in the absence of the spin trapping agent. In a similar fashion, the carbon-centered radicals formed from COX-catalyzed AA peroxidation in vitro were showing an ESR (electron spin resonance)-active peaks and MS (mass spectra) ions of m/z 296, 448, and 548, all stemming from PGF2-type alkoxyl radicals. One of these was a novel radical centered on the carbon–carbon double bond nearest the PGF ring, caused by an unusual β-scission of PGF2-type alkoxyl radicals. similar to the The complementary non-radical product was 1-hexanol ( OH

one that is formed from DGLA as described above) product, instead of the more common aldehyde. Thus, it is evident that the peroxidation products formed from GLA, DGLA, and AA are totally different that may account for the differences in their tumoricidal action wherein GLA > DGA > AA with regard to their ability to induce apoptosis of tumor cells. Thus, it is evident that the lipid peroxidation end products formed from GLA, DGLA, and AA are different and so also the ability of these fatty acids to induce apoptosis of tumor cells explaining why GLA is more potent than DGLA and AA in killing the tumor cells. Thus, in summary, the formation of lipid peroxidation products and their relevant radical products formed from GLA, DGLA, and AA are as follows:

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GLA 15-OH-C12 FA• 12-OH-C20 FA• 8-OH-C20 FA• 13-OH-C18 FA•

DGLA •C5H11 •C6H13O •C7H13O2 •C8H15O3

AA •C5H11 •C6H13O •C14H21O4 •C20H34O5

It can be seen that the radicals formed from DGLA and AA as a result of lipid peroxidation process are very similar except for the last two products (namely, •C7H13O2 and •C8H15O3 from DGLA and •C14H21O4 and •C20H34O5 from AA), whereas the products formed from GLA seem to be entirely different as shown above. It is evident from these studies that both DGLA and AA when metabolized in tumor cells form similar lipid peroxides (such as •C5H11 and •C6H13O) and slightly different lipid peroxides (from DGLA, •C7H13O2 and •C8H15O3, and from AA, •C14H21O4 and •C20H34O5). The formation of at least two similar lipid peroxides from DGLA and AA can be ascribed to their common C20 chain length as both DGLA and AA are C20 molecules, whereas the two different lipid peroxides formed from DGLA and AA (from DGLA, •C7H13O2 and •C8H15O3, and from AA, •C14H21O4 and •C20H34O5) can be ascribed to the differences in the number of double bonds (DGLA has three double bonds, while AA has four double bonds). On the other hand, GLA is an 18-carbon chain length lipid containing 3 double bonds that may explain the formation of entirely different lipid peroxides compared to those formed from DGLA and AA. One distinct possibility as to why GLA is toxic to glioma cells (and other tumor cells) but not to normal neuronal cells (normal brain cells) can be related to the absence (or nearly absent) of GLA in normal brain cells (neuronal cells), while AA, EPA, and DHA (DHA > AA > EPA) are present in abundant amounts in them (the human brain is known to be rich in AA, EPA, and DHA) [82, 83]. Thus, glioma cells may be more sensitive to the toxic actions of GLA but not to that of AA, EPA, and DHA. On the other hand, normal neuronal cells of the brain contain not only large amounts of AA, EPA, and DHA but may also have the ability to convert GLA to AA due to the action of delta-5 desaturase so that AA formed is merged with the cell content of AA such that it is no longer toxic to normal neuronal cells. In contrast, glioma cells (and cancer cells) do not have this capacity to convert GLA to AA due to the low activity or almost absence of delta-5 desaturase in tumor cells, and so GLA will remain in free form that leads to its conversion to GLA-derived lipid peroxides that are toxic to tumor cells. This may explain as to why GLA is able to induce selective apoptosis of glioma cells while being nontoxic to normal neuronal cells. In this context, it is interesting to note that the anti-cancer actions of IFN and TNFα need the presence of substantial amounts of cellular content of GLA, AA, EPA, and DHA (~ GLA  ≥  AA > EPA > DHA). For instance, it was noted that when IFNsensitive tumor cells were exposed to IFN, accumulate lipid droplets in the cytoplasm surrounding the nucleus that coincided with the apoptosis of the tumor cells. On the other hand, when IFN-resistant cells were exposed to IFN (even very high doses of IFN), there was a significantly less amount of apoptosis of cancer cells, and no

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accumulation of lipid droplets were seen in these cells. In contrast to these results, when IFN-resistant cells were pre-treated with GLA/AA and then exposed to IFN, both induction of apoptosis and accumulation of lipid droplets in the tumor cells were seen. Further support to this is derived from the observation that IFN (α, β, and γ) induces the synthesis and activates phospholipase A2 (PLA2) [84] that, in turn, releases PUFAs (GLA, AA, EPA, and DHA) from the cell membrane lipid pool. It is these released GLA/AA/EPA/DHA/PUFAs that, in turn, induce apoptosis of tumor cells. This is confirmed by the fact that PLA2 inhibitors block the cytotoxic action of IFN on IFN-sensitive tumor cells. Similar observations were made with regard to the tumoricidal action of TNF-α as well. Thus, resistance of tumor cells to the cytotoxic action of IFN and TNF-α is due to a deficiency of their GLA/AA/EPA/DHA/PUFA content. This argument is supported by the fact that tumor cells are deficient in delta-6 and delta-5 deasturases, enzymes that are necessary to form GLA, DGLA, AA, EPA, and DHA from their precursor essential fatty acids: LA and ALA. Thus, GLA/AA/ EPA/DHA are needed for the elicitation of tumoricidal action of IFN and TNF-α. Other cytokines such as IL-2 and TGF-β also have actions similar to IFN and TNF-α on tumor cells. Both IFN and TNF-α induced macrophages and other immunocytes to release free radicals (including nitric oxide) that, in turn, induce apoptosis of tumor cells, and this was accompanied by increased formation of lipid peroxides and reduced concentration of antioxidants in the target cells [85–100]. These results are similar to those observed with the effect of PUFAs/GLA/AA/EPA/DHA on tumor cells, wherein it was noted that incubation of tumor cells with these fatty acids enhanced the formation of lipid peroxides in them (tumor cells), which coincided with the induction of apoptosis of tumor cells. This implies that a combination of IFN/TNF-α  +  PUFA/ GLA is likely to be more effective against tumor cells since IFN and TNF-α need PUFA/GLA to produce their cytotoxic action on tumor cells. But PUFA/GLA itself is sufficient to induce apoptosis of tumor cells since they are able to reverse resistance of tumor cells to IFN and TNF-α.

GLA for Glioma Management The GLA solution can be administered intratumorally (into the parenchyma of substance of the tumor) into the tumor bed even before surgery or after debulking surgery of the glioma mass, inserting a catheter into the tumor bed so that injections of the GLA solution could be performed on a daily basis after the surgical wound has healed and after confirming the position of the tip of the catheter by CT or MRI. It is also envisaged that in a situation where a patient has an inoperable glioma, GLA could be injected into the tumor mass to shrink its size due to apoptosis of the tumor cells such that the previously inoperable tumor mass now becomes operable and amenable to other treatment modalities including further injections of GLA or surgery or radiation or chemotherapy.

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Examples Based on the in vitro and in vivo studies [4–15, 23, 24, 42–50, 101–112] that showed that GLA can selectively kill tumor cells by enhancing free radical generation and formation of significant amounts of toxic lipid peroxides only in the tumor but not in normal cells and enhance the radiosensitivity of tumor cells and reverse tumor cell drug resistance to conventional chemotherapeutic drugs and intratumoral injection of GLA which induces apoptosis of glioma cells in animal glioma tumor models, we performed a limited open-label clinical study in patients with glioblastoma multiforme in 30 human patients (with stage 4 glioma: glioblastoma multiforme, anaplastic astrocytoma, and other types of brain tumors). These studies revealed that intratumoral delivery of GLA can induce apoptosis of glioma cells without any effects on normal neuronal cells.

Methods of Administration In this study, patients were admitted into the hospital. A complete clinical examination and biochemical assessment of the patient was done including CT/MRI scan of the brain and ultrasound examination of the abdomen to rule out any secondaries in the abdominal organs such as the liver, mesentery, etc., and an X-ray (radiograph) and/or CT and MRI scan of the chest was also performed to know whether there were any secondaries in the lungs. The purpose of CT and MRI scan of the brain was to find out the location, extent, and type of brain tumor or glioma. Carotid angiogram may be performed to know the blood supply to the tumor mass, the source of the origin of the blood supply to the glioma, and the extent of the blood supply (such as how many blood vessels are feeding the tumor, etc.). The CT and MRI scans with contrast medium were also done to know tumor mass and presence of any necrosis in the tumor mass and for subsequent comparison after the treatment. These patients underwent a debulking neurosurgical operation wherein the neurosurgeon removed a major portion of the tumor mass. During the surgery, after removing the tumor mass to the extent possible, the neurosurgeon instilled about 1–10 mg of GLA/PUFA into the tumor bed and closed the wound and the bony flap. Before closing the surgical site, the neurosurgeon also placed a catheter into the tumor bed, whose tip is in the tumor bed. The catheter is in turn connected to an Ommaya reservoir (as shown in Fig.  5.8) or a similar subcutaneous pump or is beneath the scalp or in any other place that is easily accessible for subsequent administration of GLA/PUFA solution. After 1 week to 10 days following the surgery and when the surgical wound has healed well, a CT or MRI scan of the brain is performed to know the residual glioma or brain tumor remaining after surgery, and then GLA/PUFA solution is injected/administered into the glioma tumor bed via the subcutaneous pump or Ommaya reservoir daily for the duration of the therapy that typically lasts anywhere from 7 days to 10 days or 15 days in one sitting.

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Fig. 5.8 Schematic representation of an implanted Ommaya reservoir or similar reservoir for infusion/ injection of PUFA/GLA solution into the tumor bed

Depending on the response of the glioma or brain tumor, the number of sittings can be repeated to the satisfaction of the treating physician or surgeon or till the tumor regresses to one’s satisfaction. The degree of regression of the tumor can be assessed by performing a CT or MRI scan with or without contrast medium after 7 to 10 or 15 days of administration of GLA/PUFA solution and comparing it to the preinjection or pre-administration of PUFA/GLA solution picture. In the event it is necessary, a repeat carotid angiogram can be performed to know whether the blood supply to the glioma or tumor mass has regressed in comparison to what was noted at the beginning of the study or prior to PUFA/GLA injection or administration. An angiography was performed and recorded during and immediately after the procedure (of injection or infusion of GLA/PUFA solution) and at periodic intervals thereafter. Using this methodology, 30 patients were treated by direct infusion or injection of GLA. The first six patients of glioma received GLA in doses varying from 0.25 mg/ day to 10 mg/day for 5–10 days. All these six patients showed significant reduction in the size of the tumor mass and survived for longer than expected. In another study, 15 patients were injected or infused with GLA in doses varying from 1 mg/day to 10 mg/ day for 7–10 days, 1 week after the neurosurgical procedure of debulking of the tumor mass. At the time of the surgery, these patients also received instillation of GLA 1 mg to 5 mg into the tumor bed at the end of surgery. All these patients showed 50–70% reduction in the size of the tumor mass as determined by repeat CT and MRI scans in which the CT and MRI scans performed 1 week after the neurosurgery (before the first injection of GLA after the surgery) were compared with CT or MRI scan performed 24 hours after the last injection (i.e., on the 8th or 11th day of GLA injection wherein GLA injection was given for 7  days or 10  days, respectively). Of the 15 patients treated in this fashion, 14 patients survived for more than 2 1/2 to 3 years after the completion of GLA therapy. This is 50–100% longer than what is expected since historical data indicates that patients with glioma (especially of glioblastoma multiforme) do not survive for more than 44 weeks (approximately 1 year after the diagnosis of glioma). In another study, nine patients, who had recurrence of glioma after

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Fig. 5.9  CT scan of the brain of a patient with glioma treated with intratumoral GLA. (a) CT scan of the brain with contrast of a 15-year-old patient with left temporal glioblastoma multiforme prior to the intratumoral injection of GLA. Note marked midline shift, suggesting raised intracranial tension and mass effect. (b) CT scan of the brain with contrast of the same patient taken 24 hours after seven intratumoral injections at the rate of 1 mg/day. CT scan demonstrates significant necrosis (black areas within the brain tumor mass) of the tumor and marked reduction in the midline shift, suggesting reduction in the intracranial tension and mass effect. There were no side effects due to the therapy

surgery, radiation therapy, and chemotherapy, who were given GLA at the rate of 1–5 mg/day for 7–10 days also showed regression of tumor mass and improved survival. Figure 5.9 shows a CT scan of a typical patient who has been treated with GLA recording the regression of tumor mass. In summary, this clinical study supports the contention that GLA not only has anti-cancer actions in vitro and in vivo but can be used as a potential drug for glioma and, possibly, other cancers [13, 14, 110, 112].

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86. Miyakawa Y, Kagaya K, Watanabe K, Fukazawa Y. Characteristics of macrophage activation by gamma interferon for tumor cytotoxicity in peritoneal macrophages and macrophage cell line J774.1. Microbiol. Immunol. 1989;33:1027–38. 87. Diamond RD, Lyman CA, Wysong DR.  Disparate effects of interferon-gamma and tumor necrosis factor-alpha on early neutrophil respiratory burst and fungicidal responses to Candida albicans hyphae in vitro. J Clin Invest. 1991;87:711–20. 88. Jiang H, Stewart CA, Leu RW. Tumor-derived factor synergizes with IFN-γ and LPS, IL-2 or TNF-α to promote macrophage synthesis of TNF-α and TNF receptors for autocrine induction of nitric oxide synthase and enhanced nitric oxide-mediated tumor cytotoxicity. Immunobiology. 1995;192:321–42. 89. Martin JH, Edwards SW. Interferon-gamma enhances monocyte cytotoxicity via enhanced reactive oxygen intermediate production. Absence of an effect on macrophage cytotoxicity is due to failure to enhance reactive nitrogen intermediate production. Immunology. 1994;81:592. 90. Sample AK, Czuprynski CJ. Priming and stimulation of bovine neutrophils by recombinant human interleukin1 alpha and tumor necrosis factor alpha. J Leukoc Biol. 1991;49:107–15. 91. Kharazmi A, Nielsen H, Bendtzen K. Modulation of human neutrophil and monocyte chemotaxis and superoxide responses by recombinant TNF-alpha and GM-CSF. Immunobiology. 1988;177:363–70. 92. Yoshikawa T, Takano H, Naito Y, Oyamada H, Ueda S, Kondo M.  Augmentative effects of tumor necrosis factor-alpha (human, natural type) on polymorphonuclear leukocyte-­ derived superoxide generation induced by various stimulants. Int J Immunopharmacol. 1992;14:1391–8. 93. Nunokawa Y, Tanaka S. Interferon-γ inhibits proliferation of rat vascular smooth muscle cells by nitric oxide generation. Biochem Biophys Res Commun. 1992;188:409–15. 94. Gao X, Zhang H, Belmadani S, Wu J, Xu X, Elford H, Zhang C. Role of TNF-α-induced reactive oxygen species in endothelial dysfunction during reperfusion injury. Am J Physiol Heart Circ Physiol. 2008;295:H2242–9. 95. Gauss KA, Nelson-Overton LK, Siemsen DW, Gao Y, DeLeo FR, Quinn MT. Role of NFκB in transcriptional regulation of the phagocyte NADPH oxidase by tumor necrosis factor α. J Leukoc Biol. 2007;82:729–41. 96. Neale ML, Fiera RA, Matthews N. Involvement of phospholipase A2 activation in tumour cell killing by tumour necrosis factor. Immunology. 1988;64:81. 97. Spriggs DR, Sherman ML, Imamura K, Mohri M, Rodriguez C, Robbins G, Kufe DW. Phospholipase A2 activation and autoinduction of tumor necrosis factor gene expression by tumor necrosis factor. Cancer Res. 1990;50:7101–7. 98. Seeds MC, Jones DF, Chilton FH, Bass DA.  Secretory and cytosolic phospholipases A2 are activated during TNF priming of human neutrophils. Biochim Biophys Acta. 1998;1389:273–84. 99. Latchoumycandane C, Marathe GK, Zhang R, McIntyre TM. Oxidatively truncated phospholipids are required agents of tumor necrosis factor α (TNFα)-induced apoptosis. J Biol Chem. 2012;287:17693–705. 100. Zhao L, Gandhi CR, Gao ZH. Involvement of cytosolic phospholipase A2 alpha signalling pathway in spontaneous and transforming growth factor beta induced activation of rat hepatic stellate cells. Liver Int. 2011;31:1565–73. 101. Dong M, Guda K, Nambiar PR, Rezaie A, Belinsky GS, Lambeau G, Rosenberg DW. Inverse association between phospholipase A 2 and COX-2 expression during mouse colon tumorigenesis. Carcinogenesis. 2003;24:307–15. 102. Leaver HA, Williams JR, Ironside JW, Miller EP, Gregor A, Su BH, Prescott RJ, Whittle IR.  Dynamics of reactive oxygen intermediate production in human glioma: n-6 essential fatty acid effects. Eur J Clin Investig. 1999;29:220–31.

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103. Bell HS, Wharton SB, Leaver HA, Whittle IR. Effects of N-6 essential fatty acids on glioma invasion and growth: experimental studies with glioma spheroids in collagen gels. J Neurosurg. 1999;91:989–96. 104. Vartak S, McCaw R, Davis CS, Robbins ME, Spector AA. Gamma-linolenic acid (GLA) is cytotoxic to 36B10 malignant rat astrocytoma cells but not to ‘normal’ rat astrocytes. Br J Cancer. 1998;77:1612–20. 105. Vartak S, Robbins ME, Spector AA.  The selective cytotoxicity of gamma-linolenic acid (GLA) is associated with increased oxidative stress. Adv Exp Med Biol. 1999;469:493–8. 106. Leaver HA, Wharton SB, Bell HS, Leaver-Yap IM, Whittle IR. Highly unsaturated fatty acid induced tumour regression in glioma pharmacodynamics and bioavailability of gamma linolenic acid in an implantation glioma model: effects on tumour biomass, apoptosis and neuronal tissue histology. Prostaglandins Leukot Essent Fatty Acids. 2002;67:283–92. 107. Antal O, Hackler L Jr, Shen J, Mán I, Hideghéty K, Kitajka K, Puskás LG. Combination of unsaturated fatty acids and ionizing radiation on human glioma cells: cellular, biochemical and gene expression analysis. Lipids Health Dis. 2014;13:142. 108. Leaver HA, Bell HS, Rizzo MT, Ironside JW, Gregor A, Wharton SB, Whittle IR. Antitumour and pro-apoptotic actions of highly unsaturated fatty acids in glioma. Prostaglandins Leukot Essent Fatty Acids. 2002;66:19–29. 109. Cai J, Jiang WG, Mansel RE. Inhibition of angiogenic factor- and tumour-induced angiogenesis by gamma linolenic acid. Prostaglandins Leukot Essent Fatty Acids. 1999;60:21–9. 110. Das UN.  From bench to the clinic: gamma-linolenic acid therapy of human gliomas. Prostaglandins Leukot Essent Fatty Acids. 2004;70:539–52. 111. Antal O, Péter M, Hackler L Jr, Mán I, Szebeni G, Ayaydin F, Hideghéty K, Vigh L, Kitajka K, Balogh G, Puskás LG.  Lipidomic analysis reveals a radiosensitizing role of gamma-­ linolenic acid in glioma cells. Biochim Biophys Acta. 1851;2015:1271–82. 112. Das UN. Gamma-linolenic acid therapy of human glioma-a review of in vitro, in vivo, and clinical studies. Med Sci Monit. 2007;13:RA119–RA31.

Chapter 6

Bioactive Lipid (BAL)-Based Therapeutic Approach to Cancer That Enhances Antitumor Action and Ameliorates Cytokine Release Syndrome of Immune Checkpoint Inhibitors Abstract  It is the aim of cancer therapy to selectively kill tumor cells with few or no side effects. But current therapeutic approaches such as surgery, radiotherapy, and chemotherapy including immune checkpoint inhibitors (ICI) are associated with mild to moderate to severe side effects that are considered to be responsible for significant morbidity and mortality to patients with cancer. The recently discovered and employed ICI therapy though produced significant remission in not more than 20–30% of the patients is capable of inducing severe side effects due to excess production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). It is paradoxical that appropriate amounts of IL-6 and TNF-α are needed to induce apoptosis of tumor cells. Hence, it is important to device newer methods of eliminating rather selectively tumor cells but at the same time prevent or eliminate or reduce cytokine storm-induced side effects. IL-6 and TNF-α induce the release of polyunsaturated fatty acids (PUFAs), especially GLA, DGLA, AA, EPA, and DHA, from the cell membrane phospholipids by activating phospholipases. Previously we and others showed that PUFAs induce apoptosis/ferroptosis/necrosis and other forms of tumor cell death and at the same time are capable of suppressing the release of excess IL-6 and TNF-α. NK cells, TILs (tumor-infiltrating cells), and γδ T cells release toxic granules (also called as cytolytic granules) that contain unsaturated fatty acids. Thus, PUFAs seem to be a universal component of cytolytic granules and are responsible for the cytotoxic action of NK cells, TILs, and γδ T cells especially against tumor cells. Hence, it is hypothesized that a combination of ICI/TILs/NK cells/γδ T cells and PUFAs is likely to form a novel, reliable, and robust method of inducing apoptosis of tumor cells with few side effects. Keywords  Checkpoint inhibitors · Polyunsaturated fatty acids · Arachidonic acid · Prostaglandins · NK cells · Indoleamine 2,3-dioxygenase · Cancer

© Springer Science+Business Media, LLC, part of Springer Nature 2020 U. N. Das, Molecular Biochemical Aspects of Cancer, https://doi.org/10.1007/978-1-0716-0741-1_6

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Introduction Cancer therapy is supposed/expected to eliminate tumor cells rather selectively with no or few actions on normal cells. But this goal is rarely achieved with the current methods of treatment including surgery, radiotherapy, and chemotherapy. In view of the significant morbidity and mortality associated with cancer therapy, some cancer patients try to avoid especially radiotherapy and chemotherapy and opt to take unconventional and unapproved forms of treatment. Recent studies showed that checkpoint inhibitors of PD-1 (programmed cell death 1 protein), PD-1 ligand (PD-­ L1), cytotoxic T-lymphocyte-associated protein 4 {CTLA-4 also called as CD152 (cluster of differentiation) 152}, and adoptive cell transfer (ACT) are of significant benefit in the management of cancer when used alone or in combination with existing chemotherapeutic drugs [1–7]. But these checkpoint inhibitors produce significant toxicity termed as immune-related adverse events (IRAEs) that include dermatologic, gastrointestinal, hepatic, endocrine, and other less common inflammatory events including cytokine release syndrome that can be lethal [8–10]. Furthermore, these checkpoint inhibitors are effective in not more than ~30–40% of the patients who receive the therapy [11, 12]. Previously, we and others showed that arachidonic acid (AA, 20:4 n-6) and other polyunsaturated fatty acids (such as gamma-linolenic acid, GLA; eicosapentaenoic acid, EPA; and docosahexaenoic acid, DHA) have selective tumoricidal action by augmenting free radical generation and formation of lipid peroxides in tumor cells with little or no effects on normal cells. In addition, AA and its metabolites have anti-inflammatory actions and suppress inappropriate cytokine release and action. This suggests that perhaps, supplementation of AA along with the conventional chemotherapeutic drugs and checkpoint inhibitors may prove to be more useful to eliminate cancer cells or inhibit their growth and circumvent their side effects.

Immune Checkpoint Inhibitors Immune checkpoint inhibitor  immunotherapy (or simply called as checkpoint inhibitors) therapy is a form of cancer immunotherapy. Tumor cells use these checkpoints to protect themselves from immune system attacks. The purpose of these immune checkpoint therapies is to block these inhibitory checkpoints to restore anti-cancer actions of the immune system [1]. The transmembrane programmed cell death 1 protein (PDCD1, PD-1; also known as CD279) and its ligand, PD-1 ligand 1 (PD-L1, CD274) serve as immune check point inhibitors. PD-L1 on the cell surface binds to PD1 on the T cells and other immune cells to suppress immune surveillance that enables tumor cells to survive [2, 3]. This upregulation of PD-L1 on the tumor cell surface is capable of inhibiting T-cell anti-cancer action. In view of this, use of antibodies against either PD-1 or PD-L1 will allow the T cells to attack the cancer cell(s) [4] (see Fig. 6.1).

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AA

COX

LXA4

PGE2 LXA4

PGE2 AA

COX/LOX AA

LP LP

LP AA and other PUFAs

Fig. 6.1  Scheme showing interaction among APC (antigen-presenting cell such macrophages/ dendritic cells) and T cells and the tumor cell. (Modified from “Immunotherapy in the precision medicine era: Melanoma and beyond”. December 13, 2016, https://doi.org/10.1371/journal. pmed.1002196). (-) indicates inhibition of production of PGE2 an immunoinhibitory and pro-­ inflammatory molecule produced from arachidonic acid (AA). PGE2 may be produced by the macrophages (or other APCs) and/or tumor cells or both. It is likely that PGE2 and other immunosuppressors may be produced by the microenvironment surrounding (tumor milieu) the tumor cells. As a result, CTLA-4 binding to the costimulatory B7 ligands found on antigen-­ presenting cells (APCs) prevents signaling through CD28. Antibodies targeting CTLA-4 release this checkpoint to allow T-cell activation (central immunoinhibition). Binding of the PD-L1 of the tumor cells to the PD-1 receptor on T cells results in peripheral immunoinhibition. Antibodies targeting either the ligand or its receptor release this checkpoint. Activated APCs (macrophages, T cells, and other immunocytes) may release AA and other PUFAs that are peroxidized by free radicals to form toxic lipid peroxides (LP) that act on tumor cells to induce their apoptosis. Under normal physiological conditions, there is a delicate balance maintained between PGE2 and LXA4 that are derived from AA. LXA4 has growth inhibitory actions on tumor cells, suppresses inappropriate inflammation, inhibits PGE2 production, and, thus, maintains normal homeostasis and probably keeps a check on the expression of PD, PD-L1, CTLA4, and IDO such that pathological immunosuppression does not occur as is seen in cancer. Tumor cells escape from the immunosurveillance by containing low amounts of AA (that has suppressive action on tumor cell growth and can induce their apoptosis); increased formation and release of PGE2 that has immunosuppressive actions, pro-inflammatory in nature and directly enhances tumor cell growth: actions that are diametrically opposite to LXA4. It is paradoxical that tumor cells that have low AA content (due to low activity of desaturases) produce high levels of PGE2 (due to increased COX-2 activity), whereas when AA is supplemented to these cells, they (tumor cells) undergo apoptosis (due to formation of toxic amounts of lipid peroxides: LP), form increased amounts of LXA4 (i.e., anti-­ inflammatory in nature), suppress PGE2 formation, and suppress the expression of PD, PD-L1, CTLA4, and IDO, events that ultimately lead to tumor cell elimination. Supplementation of EPA and DHA also may have actions similar to AA on tumor cells.

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CTLA-4 CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that, functioning as an immune checkpoint, downregulates immune responses [13–15]. The upregulation of CTLA-4 in activated T cells is needed to serve as a negative feedback control switch when bound to CD80 or CD86 on the surface of antigen-presenting cells to prevent excess stimulation of the immune system that could be harmful due to the release of pro-inflammatory cytokines. One of the antigen-presenting cells that could express CTLA-4 includes cancer cell. Hence, it is anticipated that anti-CTLA-4 antibodies could upregulate the T-cell function and eliminate tumor cells. This assumption is supported by the observation that anti-CTLA-4 therapy when used in combination with conventional chemotherapeutic drugs or other immune checkpoint inhibitors could successfully inhibit the growth of tumor cells in experimental animals [16]. It is noteworthy that patients who underwent therapy with immune checkpoint inhibitors especially with CTLA-4 antibodies or a combination of immune checkpoint inhibitors showed significant adverse events especially from excess release of pro-­ inflammatory cytokines also called as cytokine storm [17–19] (see Fig. 6.2).

Fig. 6.2 (a) Mechanism of action of immune checkpoint inhibitors. Notes: Treg depend on the activity of CTLA-4, PD-1, and PD-L1 to induce immunosuppression. Ipilimumab and tremelimumab are monoclonal antibodies that inhibit CTLA-4, while nivolumab, pembrolizumab, atezolizumab, and durvalumab inhibit PD-1 and PD-L1. These drugs reduce immune checkpoint activity on a Treg -rich microenvironment, thus diminishing tumor evasion. Treg regulatory T cells; TCR T-cell receptor, MHC major histocompatibility complex.

Fig. 6.2  (continued) (b). Crystal structure of CTLA4 as published in the Protein Data Bank (PDB: 1DQT). (c). Solution structure of the extracellular domain of human PD-1. (A) Best fit superposition of the protein backbone for the 35 converged structures obtained for hPD-1. (B) Ribbon representation of the backbone topology of the structure closest to the mean, in the same orientation. (C) Comparison of the NMR-based (red) and crystal (green, PDB accession number 3RRQ) structures of hPD-1. (D) Comparison of the structures of hPD-1, TCR & V-domain (PDB accession number 3OMZ), and CTLA-4 (PDB accession number 3OSK). (E) Comparison of the backbone topologies of human (red) and mouse (blue, PDB accession number 1NPU) PD-1. (F) Structure-based alignment of the sequences of human (Hu) and mouse (Mo) PD-1 (mature polypeptide numbering)

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Indoleamine-Pyrrole 2,3-Dioxygenase (IDO or INDO) Indoleamine-pyrrole 2,3-dioxygenase (IDO or INDO), the rate-limiting enzyme of tryptophan catabolism through the kynurenine pathway, induces the O2-dependent oxidation of L-tryptophan to N-formylkynurenine and has the ability to inhibit T-cell function. As a result, excess activity of IDO leads to tolerance of the immune system to tumor antigens by inhibiting T regulatory (Treg) and myeloid-derived suppressor cells. In addition, excess IDO activity is known to enhance tumor angiogenesis, one of the key events that is needed for the tumor cells to survive and grow [20, 21]. Thus, many tumor cells overexpress IDO to escape from the immune surveillance of the body. It is also noteworthy that tryptophan is needed for the proliferation of T cells. Hence, when the IDO activity is high in tumor cells, it leads to utilization of tryptophan selectively by the tumor cells, resulting in its (tryptophan) non-availability or low availability to T cells, resulting in their (T cells) decreased proliferation [22, 23]. This results in failure of the immune surveillance system and, thus, increases the survival and proliferation of tumor cells. Thus, IDO is considered as an immune checkpoint molecule, and IDO is produced by activated macrophages and other immunocytes. IDO suppresses the activation and action of T and NK cells, Treg cells, and myeloid-derived suppressor cells [21]. In mammals, tryptophan is essential for cell survival, and it cannot be synthesized de novo and, hence, is an essential amino acid. IDO regulates microbial growth by virtue of its ability to regulate tryptophan availability and accumulation of tryptophan catabolites in the inflammatory environment that have immunosuppressive actions [24, 25]. IDO is expressed by many normal cells and cancer cells [26, 27]. The presence of IDO in trophoblasts enables maternal tolerance to fetal allograft in view of its immunosuppressive actions. This implies that modulating IDO activity could be exploited in the management of autoimmune diseases [28–31]. Both depletion of tryptophan and tryptophan metabolites such as kynurenine, kynurenic acid, 3-hydroxy-kynurenine, and 3-hydroxy-anthranilic acid have the ability to suppress T-cell function due to their ability to induce T-cell apoptosis [32]. Aryl hydrocarbon receptor (AHR) is a direct target of kynurenine [32–35]. It is noteworthy that arachidonic acid (AA, 20:4 n-6) metabolites, bilirubin, cAMP, and tryptophan metabolites serve as ligands of the AHR. In addition, tryptophan can be utilized by gut microbiota to form indole derivatives, such as indole-3-acetic acid, indoxyl-3-sulfate, indole-3-propionic acid, and indole-3-aldehyde, which are also ligands for the aryl hydrocarbon receptor (AHR). It is known that activation of AHR of gut-resident T cells and innate lymphoid cells enhances production of IL-22, which protects against inflammation such as colitis. The susceptibility to colitis could be transferred to wild-type germ-free mice by transferring the microbiota (reviewed in 37). This two-way cross talk between microbes and the immune system may also be relevant to several autoimmune diseases including type 1 DM [36]. Tryptophan regulates the formation of neurotransmitter serotonin. Gut microbiota has a role in regulating host serotonin production. Spore-forming bacteria from

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the mouse and human microbiota enhance serotonin biosynthesis from colonic enterochromaffin cells (ECs), which supply serotonin to the mucosa, lumen, and circulating platelets [36–38]. Short-chain fatty acids acetate and butyrate produced by the gut microbiota influence the synthesis of serotonin by ECs, and, thus, it (serotonin) may have an important role in beta cell function and proliferation. For instance, during pregnancy, there is an expansion of the maternal population of pancreatic β cells. Serotonin stimulates β-cell proliferation. Inhibition of serotonin synthesis blocked β-cell expansion. Thus, an integrated signaling pathway linking β-cell mass to serotonin signaling pathway exists in the body [36, 39, 40]. This implies that tryptophan and consequently serotonin influence β-cell mass and development of type 1 DM. Of interest, the AHR is enriched in interleukin 17 (IL-17)-producing CD4+ T cells (TH17 cells) and controls the differentiation of naive CD4+ T cells [28, 41]. Its function in the context of T-helper-cell differentiation is ligand dependent; for example, FICZ promotes TH17 differentiation, whereas kynurenine results in the generation of regulatory T cells (Treg). AHR restricts autoimmunity by favoring the generation of Treg [27, 42, 43]. The immunosuppressive effects of kynurenine are mediated by the AHR and affect CD8+ T cells [28, 33]. In this context, it is noteworthy that there is a close interaction/cooperation between IL-22 and IL-17. Th17 cells also express IL-22, an IL-10 family member, at substantially higher amounts than T helper (Th)1 or Th2 cells. Like IL-17A, IL-22 expression was initiated by transforming growth factor β signaling in the context of IL-6 and other pro-­ inflammatory cytokines. The subsequent expansion of IL-22-producing cells was dependent on IL-23. It was demonstrated that IL-22 was coexpressed in vitro and in vivo with both IL-17A and IL-17F that synergize their actions to regulate genes associated with innate immunity [44]. These results imply that IL-22 and IL-17 have anti-inflammatory actions [36, 42–45] that are influenced by tryptophan metabolites through AHR. Recently, we noted that serotonin enhances the production of lipoxin A4 (LXA4), a potent anti-inflammatory metabolite of AA (unpublished data, 37). Thus, the ability of cancer cells to escape from immune surveillance system seems to, at least, partially depend on their ability to utilize tryptophan by upregulating an enzyme, tryptophan dioxygenase, to form kynurenine, an endogenous ligand for the aryl hydrocarbon receptor, as already discussed above, which mediates invasive tumor growth. Tryptophan conversion to kynurenine by the indoleamine 2,3-dioxygenase enzymes IDO and IDO2 and also by tryptophan dioxygenase (TDO) and the elevated levels of AHR in tumor to poor prognosis seen in patients highlight the significance of tryptophan metabolism in cancer and the ability of tumors to escape immune response. In this context, it is noteworthy that tryptophan is an activator of the biosynthesis of prostaglandins that are known to have immunosuppressive actions [46]. Thus, tryptophan metabolism is linked to AA–eicosanoid metabolism implying yet another mechanism by which it (tryptophan) brings about its immunosuppressive action.

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Immune Checkpoint Inhibitors in Cancer Realizing that currently available chemotherapeutic drugs and radiation and, of course, surgery have limitations, efforts have been and being done to develop what is called as “immune checkpoint inhibitors” that have the ability to upregulate immune system such that tumor cells could be eliminated in a selective fashion. Ipilimumab, an anti-CTLA-4 antibody; pembrolizumab and nivolumab that are monoclonal antibodies against PD-1; and combination BRAF and MEK inhibitors when used against tumors that have BRAF mutation resulted in significant antitumor action [47–50]. But it needs to be noted that these immune checkpoint inhibitors are not effective against many tumors, and still a significant number of patients remain unresponsive [11, 12]. This led to the suggestion that blocking IDO in combination with other immune checkpoint inhibitors could be of benefit to these unresponsive cancer patients. Preliminary studies did suggest that this could indeed be the case [51]. Initial studies with IDO inhibitors were encouraging. But a phase III clinical trial employing IDO inhibitor in combination with other checkpoint inhibitors gave a negative result, raising questions about the field in general [51]. Since only a small percentage of patients with cancer respond to immune checkpoint inhibitors [52], novel therapeutic approaches are needed to tackle cancer. In a recent study that evaluated the proportion of drugs with demonstrable benefit on survival or quality of life over available treatment options or placebo, both at time of approval and in the post-marketing period, showed that the EMA (European Medicines Agency) approved use of 48 oncology drugs (including immune checkpoint inhibitors and several monoclonal antibodies) in 68 indications, only 18 of the 68 (26%) were supported by a pivotal study powered to evaluate overall survival as the primary outcome [11]. It is surprising to note that among 68 cancer drug indications approved by the EMA in the period 2009–2013, with 5.4-year follow-up medication, only 35 (51%) were associated with a significant improvement in survival or quality of life over existing treatment options, placebo, or as add-on treatment. Based on this systemic evaluation of oncology approvals by EMA in 2009–2013, it was concluded that most drugs entered the market without evidence of benefit on survival or quality of life at a minimum of 3.3 years after market entry. These results imply that these drugs either extended or improved life for most cancer indications, and wherever there were survival gains over existing treatment options or placebo, they were often marginal. A meta-analysis of randomized trials that compared PD-1/PD-L1 inhibitor plus chemotherapy with chemotherapy in first line of treatment for advanced NSCLC (non-small cell lung cancer) (in which six trials involving 3144 patients) showed that the former treatment (PD-1/PD-L1 plus chemotherapy) significantly improved progression-free survival (PFS) (hazards ratio [HR], 0.62; 95% CI, 0.57–0.67; P < .001), overall survival (OS) (HR, 0.68; 95% CI, 0.53–0.87; P = .002), and ORR (objective response rate) (relative ratio [RR], 1.56; 95% CI, 1.29–1.89; P < 0.001), irrespective of PD-L1 expression level at the expense of increased treatment-related adverse events (AEs) [53].

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In another meta-analysis that included only randomized controlled studies (four trials with a total of 2174 patients), incorporating all available evidences to evaluate the efficacy and safety of anti-PD-1/PD-L1 antibody compared with chemotherapy in previously treated, progressive NSCLC patients showed a significant benefit to OS in the intention-to-treat population [combined hazard ratio (HR) 0.67; 95% CI, 0.61–0.75; P