STUDIES ON THE ANTI-CANCER POTENTIAL OF THE SESQUITERPENE LACTONE PARTHENOLIDE ZHANG SIYUAN (B Med Peking University, P.R.China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF COMMUNITY, OCCUPATIONAL AND FAMILY MEDICINE NATIONAL UNIVERSITY OF SINGPAORE 2005 ACKNOWLEGEMENTS I would like to gratefully acknowledge my supervisors, Prof Ong Choon-Nam and Dr Shen Han-Ming, for their consistent and enthusiastic professional guidance throughout my study They have brought me into this exciting biological world and, more importantly, they provided me many valuable approaches in doing research: Prof Ong who consistently emphasizes and reminds me of the fundamental theories and overall strategy of my study; Dr Shen who is outspoken in his insightful comments and suggestions with great inspirations I am also grateful for their patience and kindness throughout my study All of these are invaluable to me in my whole life It was also a great pleasure for me to work in the big family of Department of Community, Occupational, and Family Medicine in the last four years I was surrounded by a group of friendly people who have helped me carry out my study smoothly I would like to thank Prof David Koh for his general guidance and support during my study in COFM A special thank goes to our laboratory staffs: Mr Ong Her Yam who have provided me with an excellent working environment in COFM and Mr Ong Yeong Bing who had provided me with dedicated assistance in the animal study I am also grateful to my bench mates Dr Peter Colin Rose, Mr Won Yen Kim, Mr Shi Ran Xin, Ms Huang Qing for their useful comments and suggestions on my study I would also like to thank the staff in Clinical Research Center, NUS, for their technical assistance on flow cytometry and confocal microscopy A deep appreciation goes out to my wife, Zhao Min, whose dedicated love, understanding and support made this thesis possible ii TABLE OF CONTENTS Acknowledgements ii Table of Contents iii Summary x List of Figures xii Abbreviations xv List of Publications xix CHAPTER INTRODUCTION 1.1 Parthenolide 1.1.1 Introduction: feverfew and Parthenolide 1.1.2 Chemical structure, metabolism and bioactivities of parthenolide 1.1.2.1 Chemistry: sesquiterpene lactones and parthenolide 1.1.2.2 Transportation in cell system and bioavailability 1.1.2.3 Bioactivities of parthenolide 1.1.3 The molecular mechanisms involved in the bioactivities of parthenolide 1.1.3.1 Effects on NF-κB signaling 1.1.3.2 Effects on inflammatory-related molecules 1.1.3.3 Effects on Mitogen-activated protein kinase (MAPK) pathway 12 1.1.3.4 Effects on Janus Kinase (JAK)-Signal Transducers and Activators of Transcription (STAT) pathway and cytokine signaling 1.1.3.5 Effects on cell proliferation and induction of apoptosis 13 1.1.3.6 Effects on cell cycle regulation 15 14 1.1.4 in vivo study of parthenolide 16 iii 1.1.5 Toxicity and adverse side effects 1.2 17 17 Oxidative stress, biothiols and intracellular redox balance 1.2.1 Reactive Oxygen Species 17 1.2.1.1 Definition 17 1.2.1.2 Sources of ROS 18 1.2.2 Biothiols 19 1.2.2.1 Definition 19 1.2.2.2 Biological properties and metabolism 20 1.2.3 Anti-oxidant defense system 21 1.2.4 Redox balance 22 1.2.5 Biological consequences of redox imbalance 23 1.2.5.1 Lipid peroxidation 1.2.5.2 DNA damage 24 1.2.5.3 Signal transduction 24 1.2.5.4 Apoptosis 1.3 23 25 25 Apoptosis and Cancer 1.3.1 Introduction 25 1.3.2 Cell death receptors 27 1.3.3 Caspases 28 1.3.4 Bcl-2 protein family and mitochondria 30 1.3.5 Other important regulators in apoptosis 34 1.3.5.1 Thiols and intracellular redox balance in apoptosis 34 1.3.5.2 Endoplasmic reticulum (ER) stress and Ca2+ in apoptosis 35 1.3.5.3 MAPK in apoptosis 36 iv 1.3.6 Dysregulated apoptosis in cancer 1.4 37 39 TNF and NF-κB activation 1.4.1 TNF superfamily and TNF-induced apoptosis 40 1.4.2 TNFα-induced NF-κB activation and suppression of apoptosis by NF-κB activation 1.5 Cyclooxygenase, prostaglandin and cancer 41 44 1.5.1 Cyclooxygenase and prostaglandins metabolism 1.5.2 Cyclooxygenase-2: an important cancer promoter 46 1.5.2.1 Epidemiological and experimental evidence 46 1.5.2.2 Mechanisms of the carcinogenic property of cyclooxygenase-2 1.6 44 47 49 Objectives of the study CHAPTER THE CRITICAL ROLE OF INTRACELLULAR THIOLS AND Ca2+ IN PARTHENOLIDE-INDUCED CELL DEATH 2.1 Introduction 52 2.2 Materials and Methods 53 2.2.1 Reagents 53 2.2.2 Cell culture and treatments 54 2.2.3 Determination of intracellular GSH and GSSG content 54 2.2.4 Measurement of intracellular protein thiols 55 2.2.5 Measurement of intracellular ROS formation and Calcium release 56 2.2.6 Western blot 57 2.2.7 DNA content assay 58 2.2.8 TUNEL assay 58 v 2.2.9 Statistical analysis 2.3 59 59 Results 2.3.1 Parthenolide-induced intracellular thiols depletion 2.3.2 Effects of NAC and BSO on parthenolide-induced intracellular thiols depletion 2.3.3 Effects of parthenolide on overall intracellular ROS level 60 2.3.4 Effects of parthenolide on cytosolic calcium level 64 2.3.5 Effects of cellular redox status on parthenolide-induced apoptosis 2.4 59 68 64 75 Discussion CHAPTER INVOLVEMENT OF PROAPOPTOTIC BCL-2 FAMILY MEMBERS IN PARTHENOLIDE-INDUCED MITOCHONDRIAL DYSFUNCTION AND APOPTOSIS 3.1 Introduction 81 3.2 Materials and methods 83 3.2.1 Chemicals and reagents 83 3.2.2 Cell culture and treatment 83 3.2.3 Detection of Apoptosis 84 3.2.4 Western blot 84 3.2.5 Transfection and Immunostaining 85 3.2.6 Measurement of mitochondrial membrane potential (MMP) 86 3.2.7 Cell subfractionation and detection of release of mitochondrial proteins 86 3.2.8 Protein cross-linking 87 3.2.9 In vitro assay for caspase 3-like activity 88 3.2.10 Statistical analysis 88 vi 3.3 88 Results 3.3.1 Activation of caspase cascade by parthenolide in COLO205 cells 3.3.2 Parthenolide-induced Bid cleavage following caspase activation 91 3.3.3 Bax conformational changes and mitochondrial translocation in parthenolide-treated cells 3.3.4 Enhanced Bak protein level and Bak oligomerization in parthenolidetreated cells 3.3.5 Loss of MMP and release of mitochondrial proteins 3.4 88 92 97 97 102 Discussion CHAPTER SUPPRESSED NF-κB AND SUSTAINED JNK ACTIVATION CONTRIBUTE TO THE SENSITIZATION EFFECT OF PARTHENOLIDE TO TNFα-INDUCED APOPTOSIS IN HUMAN CANCER CELLS 4.1 Introduction 108 4.2 Materials and methods 110 4.2.1 Chemicals, reagents, and plasmids 110 4.2.2 Cell culture and treatments 111 4.2.3 Cell viability test and detection of apoptosis 111 4.2.4 Preparation of cytosolic and nuclear extracts 113 4.2.5 Electrophoretic mobility shift assay (EMSA) 113 4.2.6 Transient transfections and luciferase reporter gene assay 114 4.2.7 IKK and JNK in vitro kinase assay 115 4.2.8 Co-immunoprecipitation and western blot (WB) 116 4.2.9 Statistics 117 vii 4.3 117 Results 4.3.1 Parthenolide sensitizes cancer cells to TNFα-mediated apoptosis 117 4.3.2 Parthenolide inhibits NF-κB activation 120 4.3.3 Parthenolide prevents recruitment of the IKK complex to TNF receptor 121 4.3.4 Pretreatment of parthenolide leads to a sustained JNK activation in TNFαtreated cells 4.3.5 Sustained JNK activation plays an important role in the sensitization effect of parthenolide to TNFα-mediated apoptosis 4.4 Discussion 127 132 133 CHAPTER PARTHENOLIDE SUPPRESSES THE GROWTH OF COLORECTAL CANCER XENOGRAFTS BY INDUCING APOPTOSIS AND TARGETING CYCLOOXYGENASE-2 5.1 Introduction 139 5.2 Materials and methods 140 5.2.1 Chemicals and reagents 5.2.2 Cell culture and treatment 141 5.2.3 Cell growth inhibition and induction of apoptosis 141 5.2.4 Determination of COX-2 protein level and PGE2 level in vitro 142 5.2.5 In vivo nude mice implantation and treatment 142 5.2.6 Evaluation of BrdU incorporation in HCA-7 xenografts 143 5.2.7 Evaluation of apoptotic cell death in HCA-7 xenografts 144 5.2.8 Evaluation of COX-2 expression in HCA-7 xenografts 145 5.2.9 Evaluation of PGE2 level in vivo 146 5.2.10 Statistics 5.3 140 147 Results 147 viii 5.3.1 Cells with higher expression level of COX-2 are more susceptible to parthenolide-induced cytotoxicity 5.3.2 Direct effects of parthenolide on COX-2 expression and PGE2 production 5.3.3 Parthenolide inhibits HCA-7 cell growth in vivo 153 5.3.4 Parthenolide induces apoptotic cell death in HCA-7 xenografts 159 5.3.5 Parthenolide inhibits COX-2 expression in HCA7 xenografts 159 5.3.6 Effects of parthenolide feeding on PGE2 level in vivo 5.4 147 163 152 163 Discussion CHAPTER GENERAL DISCUSSION AND CONCLUSION Anti-cancer potential of parthenolide – thiol-depletion induced disruption of cellular homeostasis 6.1.1 Parthenolide depletes cellular thiol and induces oxidative stress 170 6.1.2 Parthenolide-induced thiol depletion is associated with ER stress and calcium burst 6.2 Anti-cancer potential of parthenolide – induction of apoptosis 173 6.2.1 Proapoptotic approach: direct induction of apoptosis by parthenolide 177 6.2.2 Apoptosis-permissive approach: potentializing cancer cells in response to apoptosis induced by other chemotherapeutic agents 6.3 Anti-cancer property parthenolide – an in vivo nude mice xenografts model 6.4 Limitations in current study and further directions 180 6.5 188 6.1 Conclusions 171 175 184 187 CHAPTER REFERENCES ix SUMMARY Parthenolide is the major sesquiterpene lactone responsible for the bioactivities of Feverfew (Tanacetum parthenium), a traditional herbal medicine which has been used in treatment of fever, migraine and arthritis for centuries This compound is known to have potent anti-inflammatory properties, which is executed by inhibiting major inflammationresponsive pathways, such as nuclear factor kappa-B (NF-κB) pathway, mitogenactivated protein kinase (MAPK) signaling and signal transducers and activators of transcription (STAT) signaling pathway, blocking the expression of pro-inflammatory cytokines However, its anti-cancer properties are less studied Thus, the main objective of this study is to systematically investigate the anti-cancer properties of parthenolide The following investigations have been conducted: (i) the effects of parthenolide on intracellular redox balance and the biological consequences of parthenolide-induced thiol-depletion; (ii) the molecular mechanisms involved in parthenolide-induced apoptosis; (iii) the anti-cancer potential of parthenolide by investigating its sensitization ability to cancer cells in response to death receptor ligands induced apoptosis; (iv) the anti-cancer property of parthenolide using an in vivo nude mice xenografts model Firstly, parthenolide induced a rapid depletion of biothiols and a concomitant increase of ROS level which resulted in the disruption of intracellular redox balance As a consequence of unbalanced redox status, a severe endoplasmic reticulum (ER) stress was observed, as evidenced by an increased expression of ER stress marker protein GRP78 and cellular calcium burst All these changes led to a typical apoptotic cell death To further elucidate the mechanisms of parthenolide-induced apoptosis, a series of experiments were conducted by focusing on the changes of mitochondria and Bcl-2 protein family members It was demonstrated that parthenolide triggered the activation of x the caspase cascade The changes of pro-apoptotic Bcl-2 family members including Bid cleavage, Bax translocation and Bak dimerization were also found to play a role in promoting parthenolide-induced apoptosis In addition to the direct induction of apoptosis, parthenolide also significantly sensitized various cancer cells in response to TNFα-mediated apoptosis The inhibition of NF-κB activation and induction of a sustained JNK activation were proved to be the major mechanisms contributing to parthenolide’s sensitization effect To further validate the anti-cancer property of parthenolide, an in vivo nude mice xenograft study was conducted It was observed that parthenolide-feeding significantly reduced the tumor formation by inhibiting the cancer cell proliferation and inducing apoptosis Parthenolide also significantly suppressed cyclooxygenase-2 (COX-2) expression and COX-2-derived prostaglandin synthesis, suggesting COX-2 may be an important molecular target of parthenolide In conclusion, the present study provides experimental evidence from both in vitro cell culture and in vivo animal model demonstrating the anti-cancer properties of parthenolide These novel findings provide a new insight of the parthenoldie’s bioactivity which may help to develop it into a potential anti-cancer drug in the near future xi LIST OF FIGURES Figure 1.1 Feverfew and chemical structure of parthenolide Figure 1.2 Formation of parthenolide-thiol adducts Figure 1.3 Fenton and metal catalyzed Haber-Weiss reaction 18 Figure 1.4 Thiol and disulfides 20 Figure 1.5 Cycling of biothiols 21 Figure 1.6 Glutathione (GSH) synthesis 22 Figure 1.7 Mitochondria and Bcl-2 family: the central point of apoptosis signaling 29 Figure 1.8 TNFα-induced apoptosis and NF-κB activation 43 Figure 1.9 Cyclooxygenase and prostaglandin synthesis 46 Figure 1.10 Cyclooxgenase-2 promotes cancer formation 48 Figure 2.1 Effects of parthenolide on intracellular GSH concentration 61 Figure 2.2 Effects of parthenolide on intracellular protein thiols 62 Figure 2.3 Effects of NAC and BSO on intracellular GSH of parthenolide treated COLO205 cells 63 Figure 2.4 Effects of NAC and BSO on intracellular protein thiols of parthenolide treated COLO205 cells 63 Figure 2.5 Effects of parthenolide on overall intracellular ROS level detected by carboxy-H2DCFDA 65 Figure 2.6 Effects of NAC and BSO on parthenolide-induced overall intracellular ROS level 66 Figure 2.7 Effects of parthenolide on intracellular calcium level detected by Fluo-3 AM 67 Figure 2.8 Effects of NAC and BSO on parthenolide induced intracellular calcium release detected by Fluo-3 AM 69 xii Figure 2.9 Parthenolide-induced expression of ER stress protein GRP78 detected by western blot 70 Figure 2.10 Parthenolide-induced apoptotic cell death detected by sub-G1 assay 71 Figure 2.11 Parthenolide-induced apoptotic cell death detected by TUNEL assay 72 Figure 2.12 Different effects of NAC and BSO on parthenolide-induced apoptotic cell death detected by sub-G1 and TUNEL assay 73 Figure 2.13 Different effects of pro-treatment (pre) or co-treatment (co) of NAC/BSO on parthenolide-induced apoptotic cell death detected by sub-G1 assay 74 Figure 3.1 Parthenolide-induced initiator caspase activation 89 Figure 3.2 Parthenolide-induced effector caspase activation 90 Figure 3.3 Parthenolide-induced PARP cleavage and apoptotic cell death 93 Figure 3.4 Prevention of parthenolide-induced apoptotic cell death by caspase inhibitors 94 Figure 3.5 Prevention of parthenolide-induced apoptotic cell death by caspase inhibitors (continued) 95 Figure 3.6 Parthenolide-induced Bid cleavage 96 Figure 3.7 Bax conformational changes and mitochondrial translocation 98 Figure 3.8 Parthenolide-induced Bak overexpression and oligomerization 99 Figure 3.9 Parthenolide-induced changes of mitochondrial membrane potential (MMP) 100 Figure 3.10 Parthenolide-induced release of mitochondrial proapoptotic proteins 101 Figure 4.1 Parthenolide sensitizes cancer cells to TNFα-mediated apoptosis 118 Figure 4.2 TNFα-induced apoptotic cell death detected by TUNEL assay 119 Figure 4.3 Quantification of apoptotic cell death measured by DAPI staining in different human cancer cell lines 122 Figure 4.4 Parthenolide inhibits transcriptional activity of NF-κB determined by luciferase reporter gene assay 123 xiii Figure 4.5 Parthenolide inhibits p65 nuclear translocation and DNA binding 124 Figure 4.6 Parthenolide inhibits TNFα-induced IKK activation and IκB degradation 125 Figure 4.7 Parthenolide interrupts the recruitment of IKKs to TNFR1 and TRAF2 126 Figure 4.8 Parthenolide induces a sustained JNK activation after TNFα stimulation 128 Figure 4.9 JNK inhibitor SP600125 prevents the sensitization effects of parthenolide to TNFα-mediated apoptosis 129 Figure 4.10 Overexpression of DN-JNK1 and DN-JNK2 as well as CrmA suppresses parthenolide’s sensitization effects to TNFαmediated apoptosis 130 Figure 4.11 Overexpression of DN-JNK1 and DN-JNK2 suppresses parthenolide’s sensitization effects to TNFα-mediated apoptosis (continued) 131 Figure 5.1 Differential expression of cyclooxygenase and different PGE2 synthesis levels of two colorectal cancer cell lines HCA-7 and HCT116 148 Figure 5.2 Different sensitivity of HCA-7 and HCT-116 to parthenolideinduced cytotoxicity 149 Figure 5.3 Different sensitivity of HCA-7 and HCT-116 to parthenolideinduced apoptosis 150 Figure 5.4 Inhibition of COX-2 expression by parthenolide-treatment in HCA-7 cells 151 Figure 5.5 Inhibition of PGE2 synthesis by parthenolide treatment in HCA-7 cells 154 Figure 5.6 Effect of dietary feeding of the parthenolide on HCA-7 xenograft tumor growth in athymic female nude mice 155-156 Figure 5.7 Effect of parthenolide on cell proliferation within HCA-7 xenograft tumors examined by BrdU incorporation 157 Figure 5.8 Parthenolide-induced apoptosis within HCA-7 xenograft tumors examined by TUNEL immunohistochemistry staining 158 Figure 5.9 Effects of parthenolide on COX-2 expression in HCA-7 xenograft tumors examined by COX-2 immunohistochemistry staining Figure 5.10 Effects of parthenolide feeding on PGE2 level in vivo Figure 6.1 Mechanisms involved in parthenolide (PN)-induced apoptosis 160-161 162 185 xiv ABBREVIATIONS 5-HT 5-Hydroxytryptamine 8-OHdG 8-hydroxy-2’- deoxyguanosine Act D actinomycin D AICD activation-induced-cell-death AIF apoptosis-inducing factor ANT adenylate translocator Apaf-1 apoptosis-activating factor ASK1 apoptosis signal-regulating kinase ATP adenosine triphosphate Bak Bcl-2 homologous antagonist Bax Bcl-2 associated X protein BH3 Bcl-2 homology domain Bid BH3-interacting domain death agonist BrdU 5-Bromo-2'-deoxy-uridine BSA bovine serum albumin BSO buthionine sulfoximine CAM cell adhesion molecule CARD caspase activation and recruitment domain c-FLIP cellular FLICE inhibitory protein CHX cycloheximide COX cyclooxygenase CsA cyclosporin A Cyto c cytochrome c DCFH-DA 2',7'-dichlorodihydrofluorescein diacetate DED death effector domain DEVD-CHO Asp-Glu-Val-Asp-CHO DISC death-inducing signaling complex DMSO dimethyl sulfoxide DR death receptor DSS disuccinimidyl subernate xv DTNB 5,5-dithiobis-2-nitrobenzonic acid DTT dithiothreitol EDTA ethylene diamine-tetra-acetic acid EIA enzyme immunoassay ER endoplasmic reticulum ERK extracellular regulated protein kinase ETC electron transport chain FADD Fas-associated death domain protein FBS Fetal bovine serum FILP FLICE inhibitory protein G3PDH glyceraldehydes-3-phosphate dehydrogenase GRase glutathione reductase GSH reduced glutathione GSSG oxidized glutathione/ glutathione disulphide IAPs inhibitors of apoptosis ICAM-1 intracellular cell adhesion molecule-1 IKC IKK complex IKK IκB kinase IL interleukin INFγ interferon-γ iNOS inducible isoform of nitric oxide synthase IκB NF-κB inhibitory protein JAK Janus kinase JNK c-Jun N-terminal kinase LPS lipopolysaccharide MAPK mitogen-activated protein kinase MDA malondialdehyde MEKK1 mitogen-activated protein kinase MEKK3 mitogen-activated protein kinase MKK MAPK kinase MMP mitochondrial membrane potential MPT mitochondrial permeability transition MRP multidrug resistance transporter P-glycoprotein xvi MTD maximal tolerated dose MTT NAC 3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide N-acetylcysteine NADH nicotinamide-adenine dinucleotide (reduced) NADPH nicotinamide-adenine dinucleotide phosphate (reduced) NEM N-ethylmaleimide NF-κB nuclear factor-kappaB NLS nuclear localization sequence NO nitric oxide NSAIDS non-steroidal anti-inflammatory drugs OPT o-phthalaldehyde PARP poly(ADP-ribose) polymerase PG prostaglandin PI propidium iodide PMSF phenylmethylsulfonyl fluoride PT permeability transition PTPC membrane permeability transition pore complex PUFA polyunsaturated fatty acid Rh-123 rhodamine 123 ROS reactive oxygen species RT-PCR reverse transcription polymerase chain reaction SDS sodium dodecyl sulfate SH sulphydryl Smac second mitochondrial activator of caspases SOD superoxide dismutase STAT signal transducers and activators of transcription tBid truncated Bid TNFR1 TNF receptor TNFα tumor necrosis factor α TPA 12-o-tetradecanoylphorbol-13-acetate TRADD TNF-R1-associated death domain protein TRAF2 TNF receptor associated factor TRAIL TNF-related apoptosis-inducing ligand xvii TUNEL TdT-mediated dUTP nick end labeling UV ultraviolet light VCAM-1 vascular cell adhesion molecule-1 VDAC voltage-dependent anion channel XIAP X-linked inhibitor of apoptosis protein Z-IETD-FMK benzyloxycarbonyl-Ile-Glu-Thr-Asp-(OMe) fluoromethyl ketone Z-VAD-FMK benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone γ-GCS γ-glutamyl cysteine synthestase Δψ m mitochondrial membrane potential xviii LIST OF PUBLICATIONS Zhang,S., Ong,C.N., and Shen,H.M (2004) Critical roles of intracellular thiols and calcium in parthenolide-induced apoptosis in human colorectal cancer cells Cancer Lett 208, 143-153 Zhang,S., Ong,C.N., and Shen,H.M (2004) Involvement of proapoptotic Bcl-2 family members in parthenolide-induced mitochondrial dysfunction and apoptosis Cancer Lett 211, 175-188 Zhang,S., Lin,Z.N., Yang,C.F., Shi,X., Ong,C.N., and Shen,H.M (2004) Suppressed NF-κB and sustained JNK activation contribute to the sensitization effect of parthenolide to TNFα-induced apoptosis in human cancer cells Carcinogenesis.25, 2191-2199 Zhang,S., Won, Y.K., Ong,C.N., and Shen,H.M (2004) Anti-cancer properties of sesquiterpene lactones, Curr Med Chem (In press) Zhang,S., Ong,C.N., and Shen,H.M (2004) Parthenolide suppresses the growth of colorectal cancer xenografts by targeting cyclooxygenase-2 and inducing apoptosis (manuscript in preparation) Abstracts: Zhang,S., Ong,C.N., and Shen,H.M.(2003) Mitochondrial dysfunction mediates parthenolide-induced apoptosis in human colorectal cells Proceedings of the 94th Annual Meeting of American Association for Cancer Research Zhang,S., Lin,Z.N., Yang,C.F., Shi,X., Ong,C.N., and Shen,H.M (2004) Suppressed NF-κB and sustained JNK activation contribute to the sensitization effect of parthenolide to TNFα-induced apoptosis in human cancer cells Proceedings of first ShangHai Symposium on Signal Transduction and Cancer xix ... Abbreviations xv List of Publications xix CHAPTER INTRODUCTION 1. 1 Parthenolide 1. 1 .1 Introduction: feverfew and Parthenolide 1. 1.2 Chemical structure, metabolism and bioactivities of parthenolide 1. 1.2 .1. .. balance 1. 2 .1 Reactive Oxygen Species 17 1. 2 .1. 1 Definition 17 1. 2 .1. 2 Sources of ROS 18 1. 2.2 Biothiols 19 1. 2.2 .1 Definition 19 1. 2.2.2 Biological properties and metabolism 20 1. 2.3 Anti- oxidant... Limitations in current study and further directions 18 0 6.5 18 8 6 .1 Conclusions 17 1 17 5 18 4 18 7 CHAPTER REFERENCES ix SUMMARY Parthenolide is the major sesquiterpene lactone responsible for the bioactivities