EVALUATION OF THE CHEMOTHERAPEUTIC AND CHEMOPREVENTIVE POTENTIAL OF TRITERPENOIDS FROM PORIA COCOS LING HUI (MBBS, FIRST MILITARY MEDICAL UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS First of all, I would like to express my sincere gratitude to my supervisors, Assoc Prof. Lawrence Ng Ka-Yun and Dr. Chew Eng Hui. Deep appreciation goes to Prof. Ng for setting direction for this PhD project, and for his guidance throughout this project. Especially, I thank him for his trust and confidence in me, as well as his continual support and encouragement after he left this university. Heartfelt thanks go to Dr. Chew, who guided me through the toughest final year as my main supervisor. I truly thank her for her hands-on tutoring on molecular biology-related techniques, and most importantly, for her enthusiastic and inspiring discussion on cancer research. I could not imagine a more patient, helpful and friendlier supervisor than her. Special thanks to Dr. Leslie Gapter, who has been my initial mentor on this PhD project. Her great sense of responsibility, generosity and kindness is greatly appreciated. Thanks to Dr. Lin Haishu for his unconditional help on this project. I am grateful to all my fellow students working in the same lab, who had accompanied me throughout the four years of PhD study. Sincere thanks to Mr. Zhang Yaochun and Mr. Surajit Das for their friendship and all their emotional support. Deep appreciation goes to Dr. Huang Meng for his friendship, encouragement and confidence in me. My most heartfelt thanks also go to other fellow students Yang Hong, Wang Zhe and Chun Xia for their friendship and help. Last but not least, I would like to give my greatest gratitude to my wife, Ms Zhang Huiwen. Her love, encouragement and confidence in me have been the main i force driving me forward. Finally, I extend my thanks to my parents for their constant support and care. ii TABLE OF CONTENTS TITLE PAGE ACKNOWLEDGEMENTS ………………………………………………………i TABLE OF CONTENTS ……………………………………………………… .iii ABBREVIATIONS …………………………………………………………… .vii SUMMARY………………………………………………………………………ix LIST OF TABLES ……………………………………………………………….xii LIST OF FIGURES …………………………………………………………… .xiii PUBLICATIONS ……………………………………………………………… xvii Chapter 1: Introduction ………………………………………………………… 1.1 Natural products as source of anticancer agents ……………………4 1.2 Triterpenoids from Poria cocos …………………………………….9 1.2.1 Triterpenoids ………………………………………………….9 1.2.2 Triterpenoids from Poria cocos………………………………16 1.3 Apoptosis ………………………………………………………… .24 1.3.1 Overview of apoptosis…………………………………….… 24 1.3.2 Characteristics of apoptosis………………………………… .25 1.3.2.1 Morphological features……………………………… .26 1.3.2.2 Biochemical features ………………………………….28 1.3.3 Pathways of apoptosis ………………………………………34 1.3.3.1 Extrinsic pathway ……………………………………35 iii 1.3.3.2 Intrinsic pathway …………………………………….38 1.3.4 Promoting apoptosis as strategy against cancer………….….42 1.4 Prostaglandins in cancer development …………………………… 50 1.4.1 Overview of prostaglandins …………………………………50 1.4.1.1 PLA2 family enzymes ………………………………51 1.4.1.2 COX enzymes ………………………………………53 1.4.2 Prostaglandins and cancer……………………………………54 1.4.2.1 PLA2 enzymes and cancer ………………………….56 1.4.2.2 COX enzymes and cancer ……… ……………… .59 1.4.3 Prostaglandins as target for cancer prevention………………60 1.4.3.1 Inhibition of arachidonic acid release …………………….61 1.4.3.2 Inhibition of arachidonic acid conversion to prostaglandins.62 1.5 Matrix metalloproteinases and tumor invasion ……………….…….64 1.5.1 Overview of tumor invasion …………………………….… 64 1.5.2 Matrix metalloproteinases …………………….………….….65 1.5.2.1 The MMP family……………………………….…65 1.5.2.2 Regulation of MMP activity …………………… .66 1.5.3 MMP-2 and MMP-9 ………………………………….…… .71 1.5.3.1 The role of MMP-2 and MMP-9 in tumor invasion …… .71 1.5.3.2 Regulation of MMP-2 and MMP-9……………………….73 1.5.3.3 MMP-2 and MMP-9 as target for control of tumor invasion.76 1.6 Aims of study ……………………………………………………… 79 iv Chapter 2: Isolation and identification of triterpenoids from P. cocos………… 81 2.1 Introduction ……………………………………………………….81 2.2 Materials and methods ………………………………………… 83 2.3 Results …………………………………………………………….87 2.3.1 Separation of alcoholic extracts into four fractions …… .87 2.3.2 Isolation of pure compounds …………………………… 88 2.3.3 Identification of purified compounds …………………….90 2.3.4 Cytotoxicity test ……………………………………….…95 2.4 Discussion ………………………………………………….……97 Chapter 3: Polyporenic acid C induces caspase-8-mediated apoptosis in human lung cancer A549 cells ……………………………………………………………….100 3.1 Introduction …………………………………………………… 101 3.2 Materials and methods ………………………………………….102 3.3 Results ………………………………………………………… 109 3.4 Discussion …………………………………………………… 123 Chapter 4: Pachymic acid inhibits A549 cells growth and modulates arachidonic acid metabolism…………………………………………………………………… .129 4.1 Introduction ………………………………………………… .130 4.2 Materials and methods ……………………………………… 132 4.3 Results ……………………………………………………… .140 4.4 Discussion…………………………………………………… 158 v Chapter 5: Investigation of lanostane-type triterpenoids against breast cancer cell invasion ……………………………………………………………………… 162 5.1 Introduction ………………………………………………… 163 5.2 Materials and methods …………………………………………165 5.3 Results………………………………………………………….169 5.4 Discussion…………………………………………………… .184 Chapter 6: General discussion, conclusion and future work…………………….188 6.1 General discussion ……………………… 188 6.2 Conclusion …………………………… 193 6.3 Future work ………………………… .195 REFERENCES ………………………… .198 APPENDICES ……………………… 237 vi ABBREVIATIONS AIF AP-1 BCA CAD CCK-8 CDDO CDDO-Me c-FLIP COX DEDA DISC DMBA ERK FADD FBS GAPDH IAP ICAD/DFF45 IKK IκBα JNK LDH MAC MAPK MMPs MP MS MT-MMPs mTOR MTT NF-κB NMR NSAIDs NSCLC PA PAK2 PARP apoptosis inducing factor activator protein-1 bicinchoninic acid caspase-activated DNase cell counting kit-8 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate FADD-like IL-1β-converting enzyme (FLICE)-inhibitory protein cyclooxygenase 7,7-dimethyl-5,8-eicosadienoic acid death-inducing signaling complex 7, 12-dimethylbenz[a]anthracene extracellular signal-regulated kinase Fas-associated death domain fetal bovine serum glyceraldehyde-3-phosphate dehydrogenase inhibitors of apoptosis proteins inhibitor of caspase activated DNase or DNA fragmentation factor 45 IκB kinase inhibitor of kappaBα c-Jun NH2-terminal kinase Lactate dehydrogenase mitochondrial apoptosis-induced channel mitogen-activated protein kinase Matrix metalloproteinases melting point mass spectrometry membrane-type MMPs mammalian target of rapamycin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide nuclear factor kappa B nuclear magnetic resonance non-steroidal anti-inflammatory drugs non-small cell lung cancer pachymic acid p21-activated kinase poly-ADP ribose polymerase PGE2 PI PI3K PKC prostaglandin E2 propidium iodide phosphatidylinositol 3-kinase protein kinase C vii PLA2 cPLA2 iPLA2 sPLA2 PPAC PS RIP-1 RT-PCR SCLC SP-1 STATs TIMPs TLC TNF TPA TRADD TRAF2 TRAIL uPA phospholipase A2 calcium-dependent cytosolic PLA2 calcium-independent PLA2 secretary PLA2 polyporenic acid C phosphatidylserine receptor interacting protein reverse transcription - PCR small cell lung cancer stimulatory protein-1 signal transducers and activators of transcription tissue inhibitors of metalloproteinases thin layer chromatography tumor necrosis factor 12-O-tetradecanoylphorbol-13-acetate TNF receptor-associated death domain tumor necrosis factor receptor associated factor TNF-related apoptosis inducing ligand urokinase plasminogen activator viii SUMMARY Poria cocos (also known as Fuling) is one of the most famous herbs used in Traditional Chinese Medicine for its diuretic, sedative and tonic effects. The aim of this PhD project is to examine the efficacies of triterpenoids from Poria cocos against human cancers. This project was initiated with separation and isolation of triterpenoids contained in alcoholic extracts of Poria cocos using flash column chromatography. In total, eight compounds were obtained and identified as (1) pachymic acid, (2) dehydropachymic acid, (3) 3-acetyloxy-16α-hydroxytrametenolic acid, (4) polyporenic acid C, (5) 3-epi-dehydropachymic acid, (6) 3-epi-dehydrotumulosic acid, (7) tumulosic acid, and (8) 29-hydroxypolyporenic acid. The antiproliferative activity of these triterpenoids was examined using a cell proliferation assay. Due to its relatively stronger antiproliferative activity, polyporenic acid C (PPAC) was subjected to further evaluation for its apoptosis-inducing effect. 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Cancer Res 64(20): 7570-8. 236 Appendices Appendix I 13C NMR spectrum of Compound (pachymic acid) 237 Appendices Appendix II 13C NMR spectrum of Compound (dehydropachymic acid) 238 Appendices Appendix III acid) 13 C NMR spectrum of Compound (3-acetyloxy-16α-hydroxytrametenolic 239 Appendices Appendix IV 13C NMR spectrum of Compound (polyporenic acid C) 240 Appendices Appendix V 13C NMR spectrum of Compound (3-epi-dehydropachymic acid) 241 Appendices Appendix VI 13C NMR spectrum of Compound (3-epi-dehydrotumulosic acid) 242 Appendices Appendix VII 13C NMR spectrum of Compound (tumulosic acid) 243 Appendices Appendix VIII 13C NMR spectrum of Compound (29-hydroxypolyporenic acid) 244 [...]... isothiocyanates from cruciferous vegetables have been shown to possess preventive and/ or therapeutic effects against various cancer types in vitro and 7 Chapter 1 in vivo (Beliveau and Gingras 2007) These findings further reinforce the importance of natural products as sources of agents with anticancer potential 8 Chapter 1 1.2 Triterpenoids from Poria cocos 1.2.1 Triterpenoids The triterpenoids are a large and. .. to be through inhibition of topoisomerase II and they are clinically used in the treatment of neoplasia such as lymphomas and bronchial and testicular cancers (Hande 1998) Derivatives of camptothecin constitute the third class of anticancer agents in clinical use Camptothecin was first extracted from the Chinese ornamental tree, Camptotheca acuminata, also known as the “tree of joy” Despite its remarkable... However, due to limited research on Poria cocos- originated triterpenoids, the cancer-preventive and –therapeutic potential of these lanostane-type triterpenoids remains largely unknown The subsequent sections provide (1) an overview of natural products-derived anticancer agents, (2) a review on anticancer triterpenoids, and (3) a literature review on triterpenoids from Poria cocos In addition, three cancer-related... Together, the findings presented in this PhD study had expanded on the current understanding of the anticancer potential of triterpenoids from Poria cocos xi LIST OF TABLES Table 1.1 Differences between apoptosis and necrosis ……………………… 25 Table 2.1 13C-NMR spectral data for compound 1-8 (75 MHz in C5D5N; δ in ppm).95 Table 2.2 IC50 values of compounds 1-8 against A549 cell growth ……………… 97 xii LIST OF. .. progression, these findings had indicated the chemopreventive potential of PA against lung carcinogenesis The anticancer potential of Poria cocos- originated triterpenoids was further explored by examining their efficacy against phorbol ester-stimulated matrix metalloproteinase secretion and breast cancer cell invasion PPAC, PA and dehydropachymic acid were found to reduce the gelatinolytic activity of matrix... Medicine, Poria cocos is one of the most commonly used components in complex herbal prescriptions Scientific research has shown that 16 Chapter 1 alcoholic extracts of Poria cocos possess multiple pharmacological activities that benefit human beings Triterpenoids are the main constituents in the alcoholic extracts of Poria cocos and are therefore believed to be the active components responsible for the biological... activities of this plant (Akihisa et al 2007) Most of the identified triterpenoids contained in Poria cocos belong to the lanostane group These lanostane-type triterpenoids are observed to exhibit a wide range of activities including anti-rejection, anti-emetic, anti-inflammatory and anticancer effects For instance, ethanol extract of Poria cocos has been reported to relieve the acute rejection of heart... unfavorable responses Apart from the studies mentioned above, the majority of research interests have been focused on investigating the anti-inflammatory activities of Poria cocos- originated triterpenoids markedly inhibit the The methanol extracts of Poria cocos are found to inflammatory activity provoked 12-O-tetradecanoylphorbol-13-acetate (TPA) in mice (Yasukawa et al 1995) another study, triterpenoids including... of the currently used anticancer agents are developed in one way or another from natural products (Gordaliza 2007) These anticancer drug candidates include vinblastine, vincristine, the camptothecin derivatives, topotecan and irinotecan, etoposide, and paclitaxel (Cragg and Newman 2003) One class of natural products that have attracted interest as potential anticancer candidate in recent years is the. .. stage, the cell damages are expanded, and eventually lead to the appearance of benign tumors Finally, during the progression phase, new clones of tumor cells with increased proliferative capacity, invasiveness, and metastatic potential are produced To combat cancer, a variety of strategies have been proposed and developed The conventional treatments of cancer include surgery, chemotherapy, radiotherapy . EVALUATION OF THE CHEMOTHERAPEUTIC AND CHEMOPREVENTIVE POTENTIAL OF TRITERPENOIDS FROM PORIA COCOS LING HUI (MBBS, FIRST MILITARY MEDICAL UNIVERSITY) A THESIS SUBMITTED. MMP-9 expression. Together, the findings presented in this PhD study had expanded on the current understanding of the anticancer potential of triterpenoids from Poria cocos. . progression, these findings had indicated the chemopreventive potential of PA against lung carcinogenesis. The anticancer potential of Poria cocos- originated triterpenoids was further explored