A STUDY INTO THE DIFFERENT MECHANISMS OF ANTICANCER EFFECT OF DOCOSAHEXANOIC ACID IN COLON CANCER CELLS MANAV DEPARTMENT OF COMMUNITY, OCCUPATIOBNAL AND FAMILY MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2004 A STUDY INTO THE DIFFERENT MECHANISMS OF ANTICANCER EFFECT OF DOCOSAHEXANOIC ACID IN COLON CANCER CELLS MANAV (B MED, GSMU, INDIA) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF COMMUNITY, OCCUPATIOBNAL AND FAMILY MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2004 ACKNOWLEDGEMENTS I would like to express my respect and gratitude to Professor Ong Choon Nam and Professor Lee Hin Peng As my supervisors, they ensured that I remained focused on achieving my goal Their observations and guidance helped me to establish the overall direction of the research and to move forward with investigation in depth What I learned from them, especially their approach to a scientific question, is an invaluable lesson for me not only in the academic perspective but also in my personal life I would also like to express my sincere thanks to: Prof David Koh, Head of the Department, for his support during the course of the study; Dr Shen Han Ming, for his advice and stimulating discussions; Mr Ong Her Yam, Madam Lee Bee Lan, Madam Jin Su, Madam New Ai Li and Mr Ong Yeong Bing and Madam Zhao Min, for their guidance and help in the laboratory work.; My seniors and colleagues Zhang Siyuan, Won Yen Kim, Shi Ranxin, Huang Qing and Lu Guodong ; they were responsible for the fine-tuning of my techniques and ideas; National University of Singapore, for providing me a research scholarship for this study Finally, I thank my family for their never-ending love and support I dedicate this thesis to them i TABLE OF CONTENTS Acknowledgements i Table of contents ii Abbreviations vii List of publications ix Summary x CHAPTER I: INTRODUCTION 1.1 Polyunsaturated Fatty Acids (PUFAs) 1.1.1 Structure of PUFAs 1.1.2 Synthesis of n-3 and n-6 PUFAs 1.1.3 Dietary sources 1.1.4 Intestinal absorption, metabolism 1.1.5 Functions of PUFAs 1.1.6 Competition between n-3 and n-6 PUFAs 1.2 PUFAs and colorectal cancer 1.2.1 Epidemiological Studies 1.2.2 Animal studies 1.2.3 Intervention studies in humans 10 1.2.4 In vitro studies 10 1.3 Apoptosis 11 ii 1.3.1 Apoptosis - A brief introduction 11 1.3.2 PUFAs and apoptosis 13 1.4 Oxidative Stress 14 1.4.1 Reactive Oxygen Species (ROS) – Definition and source 14 1.4.2 Role of oxidative stress in carcinogenesis and apoptosis 15 1.4.3 PUFAs and oxidative stress 16 1.4.3.1 PUFAs and lipid peroxidation 16 1.4.3.2 PUFAs and ROS 17 1.5 Mitogen Activated Protein Kinases (MAPKs) 18 1.5.1 MAPK signaling pathways- Introduction 18 1.5.2 MAPKs and ROS 19 1.5.3 PUFAs and MAPKs 20 1.6 Peroxisome Proliferator-Activated Receptors (PPARs) 1.6.1 Functions of PPARs 21 1.6.2 PPARγ and colon cancer 22 1.6.3 PUFAs and PPARs 23 1.7 Objectives of the Study 24 CHAPTER II: MATERIALS AND METHODS 25 2.1 Cell lines and chemicals 26 2.2 Cell culture and fatty acid supplementation 27 2.3 Determination of cell viability - MTT assay 27 2.4 Evaluation of cell morphological alterations 28 iii 2.5 Determination of sub-G1 population 28 2.6 Measurement of cellular caspase-3 activity 29 2.7 Western Blotting 29 2.8 JNK in vitro kinase assay 30 2.9 Transient transfections 31 2.10 Luciferase assay 31 2.11 Measurement of ROS 32 CHAPTER III: EFFECT OF DHA ON VIABILITY OF COLON CANCER CELLS 3.1 Introduction 34 3.2 Results 35 3.2.1 Cytotoxic effects of PUFAs 35 3.2.1.1 Effect of different fatty acids on cell viability of HT-29 cells 35 3.2.1.2 Effect of DHA on cell viability of different colon cancer cell lines 35 3.2.1.3 Effect of metabolic pathway inhibitors on DHA-induced cell death 35 3.2.2 DHA induced cell death is apoptotic 36 3.2.3 DHA induced apoptosis is caspase mediated 37 3.2.3.1 Determination of caspase-3 activation 37 3.2.3.2 Effect of caspase-3 inhibitor on DHA-induced cell death 37 3.3 Discussion 37 CHAPTER IV: EFFECT OF DHA ON ROS GENERATION AND MAP KINASE ACTIVATION iv 4.1 Introduction 50 4.2 Results 50 4.2.1 Effect of DHA on MAPK signaling pathways 50 4.2.1.1 DHA induces MAPK activation 50 4.2.1.2 Effect of MAPK inhibitors on DHA-induced cell death 51 4.2.1.3 Effect of JNK mutants on DHA-induced cell death 52 4.2.2 Role of ROS in DHA induced cell death 53 4.2.2.1 Generation of ROS by DHA 53 4.2.2.2 Effect of antioxidants and H202 on DHA-induced MAPK activation 53 4.2.2.3 Effect of antioxidants on DHA-induced cell death 4.3 Discussion 54 54 CHAPTER V: DHA INHIBITS TNF-α INDUCED COX-2 EXPRESSION AND NF-ΚB TRANSCRIPTION THROUGH THE PPARγ PATHWAY 5.1 Introduction 66 5.2 Results 67 5.2.1 DHA inhibits TNF-α induced COX-2 and NF-κB activation 67 5.2.2 DHA induces transcriptional activition of PPARγ 68 5.2.3 DHA regulates COX-2 expression and NF-κB activation through PPARγ-dependent pathway 69 5.2.4 DHA induces apoptosis in colon cancer cells by a PPARγ-independent mechanism 5.3 Discussion 69 70 v CHAPTER VI: DISCUSSION AND CONCLUSIONS 6.1 Chemotherapeutic actions of DHA 81 6.2 Chemopreventive role of DHA 85 6.3 Comparison of results from in vitro and in vivo studies 87 6.4 Conclusions 88 REFERENCES 90 vi ABBREVIATIONS AA Arachidonic acid ALA Alpha-linolenic acid APC Adenomatous polyposis coli BADGE Bis-phenol A diglycidyl ether BSA Bovine serum albumin COX Cyclooxygenase DAPI 4’, 6-diamidino-2-phenylindole DHA Docosahexanoic Acid DMEM Dulbecco’s Modified Eagle’s Medium ERK Extracellular regulated kinase FA Fatty acid JNK c-Jun N-terminal kinase LA Linoleic acid LOX Lipoxygenase MAPK Mitogen-activated protein kinase MTT 3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide NAC N-acetyl cysteine NF-κB Nuclear factor- κB PBS Phosphate buffered saline PARP Polyadenosine diphosphate-ribose polymerase PPAR Peroxisome Proliferator-Activated Receptor vii PPRE Peroxisome Proliferator-activated receptor response element PUFA Polyunsaturated fatty acid ROS Reactive oxygen species TNF-α Tumor necrosis factor-alpha viii malondialdehyde DNA adducts in human colorectal mucosa: relationship with diet and the presence of adenomas Cancer Epidemiol Biomarkers Prev 11, 267-273 Li,D., Ng,A., Mann,N.J., and Sinclair,A.J (1998) Contribution of meat fat to dietary arachidonic acid Lipids 33, 437-440 Li,L., Feng,Z., and Porter,A.G (2004) JNK-dependent phosphorylation of c-Jun on serine 63 mediates nitric oxide-induced apoptosis of neuroblastoma cells J Biol Chem 279, 4058-4065 Liu,H., Nishitoh,H., Ichijo,H., and Kyriakis,J.M (2000) Activation of apoptosis signalregulating kinase (ASK1) by tumor necrosis factor receptor-associated factor requires prior dissociation of the ASK1 inhibitor thioredoxin Mol Cell Biol 20, 2198-2208 Lockshin,R.A and Zakeri,Z (2002) Caspase-independent cell deaths Curr Opin Cell Biol 14, 727-733 Lofberg,R (2003) Review article: medical treatment of mild to moderately active Crohn's disease Aliment Pharmacol Ther 17 Suppl 2, 18-22 Louis,E., Peeters,M., Franchimont,D., Seidel,L., Fontaine,F., Demolin,G., Croes,F., Dupont,P., Davin,L., Omri,S., Rutgeerts,P., and Belaiche,J (2000) Tumour necrosis factor (TNF) gene polymorphism in Crohn's disease (CD): influence on disease behaviour? 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and the caspase inhibitor z-VAD-fmk was able to block the DHA-induced apoptosis The effect of DHA on the mitogen-activated signaling kinases (MAPKs) was also