Anti-cancer Effects of Ganoderma Lucidum Triterpenoids. Guo-Sheng Wu. Doctor of Philosophy in Biomedical Sciences

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1 Anti-cancer Effects of Ganoderma Lucidum Triterpenoids by Guo-Sheng Wu Doctor of Philosophy in Biomedical Sciences 2013 Institute of Chinese Medical Sciences University of Macau

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3 Anti-cancer Effects of Ganoderma Lucidum Triterpenoids by Guo-Sheng Wu SUPERVISOR: Yi-Tao Wang Doctor of Philosophy in Biomedical Sciences 2013 Institute of Chinese Medical Sciences University of Macau

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5 Author s right 2013 by Wu, Guosheng

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7 Acknowledgements Many people, institutions and public organizations deserve my thanks and have contributed to this study. I would like to express my gratitude to my supervisor Prof. Yi-Tao Wang for giving me opportunities to study in Institute of Chinese Medical Sciences (ICMS) and participate in research projects. I appreciate Dr. Jin-Jian Lu, whose unfailing encouragement, guidance, feedback, and friendship helped improve my work in countless ways. Thanks to Prof. Simon Ming-Yuen Lee and Dr. Xiu-Ping Chen for valuable suggestions and kind help during the past four years. Thanks to Prof. Shao-Ping Li and Dr. Qing-Wen Zhang for providing crude drug (G. lucidum) and extracted compounds, respectively. I also would like to thank Dr. Ying Zheng, Dr. Ru Yan, Dr. Mei-Wan Chen for your encouragement. A special appreciation goes to Jia-Jie Guo, who showed enthusiasm of resolving scientific problems and assisted me quite a lot in the past three years. Other colleagues who supported me throughout this work are greatly appreciated and include Dr. Yue-Lin Song, Guo-Zhen Cui, Ying-Bo Li, Zheng-Ming Qian, Zai-Jun Zhang, Wen Tan, Zhang-Feng Zhong, Shang Li, Zeng-Tao Xu, Yuan-Ye Dang, De- Jun Hu, Sheng-Peng Wang, Jing Xie. I also express my thanks to Dr. Guang Hu, Xiao-Jia Chen, Gang Du, Jia Guan, Kun Feng, Jing Li, Lan-Zhen Meng, Wang-Hui 1

8 Jing, Zhi-Qiang Dong, De-Qiang Li, Wei-Hua Huang, Guang-Ping Lv, Lan-Ying Wang, Wen-Jin Wu for your encouragement. I am grateful to Li-Ting Huang, Jiao-Lin Bao, Shuai Yuan, Ren-Bo Ding for sharing a good memory and enjoying board role-playing games together. It has been a great experience and I have been very fortunate to get to know so many people in ICMS, including Xiao-Jing Zhang, Wen-Wen Zhao, Jin-Ming Zhang, Hai-Tao Li, Hai-Jing Zhong, Li-Na Ma, Jian Gong, Xiu-Jiang Cai, Wei Lin, Yi-Ming Niu, Wen-Shan Xu, Zhe-Rui Zhang, Chao Bi, Ping Li, Mei-Jun AoLi, Hao-Ye Li, Ya-Ping Li, Qian Ding, Rui-E Chen, Yang-Yang Hu, Na Ni, Yan-Juan Huang, Kan Zhu, Yang Yang, Ting Li, Feng-Qian Chen, Li-Juan Liu, Sheng-Hui Chen, Xiu-Dan Zhan, Yao-Jun Ju. Thanks to the two individuals who shaped my identity and character as well as my interest, I dedicate this thesis to my parents. Although they do not understand English and have no idea about scientific work, I will interpret this thesis word by word and tell them what I did in this sleepless and beautiful city, Macao. Of the institutions that have supported me, none has been more important than ICMS: our ICMS department, our Ph.D. program, and our technical support. Beyond ICMS, my thanks go to University of Macau (UM), which supplied the financial support to me; and to UM library, a peace and quiet place for thinking and considering, where I completed this thesis eventually. The current study was supported by the grant (029/2007/A2, 077/2011/A3, 074/2012/A3) from Science and Technology Development Fund of Macau Special Administrative Region and research fund of University of Macau (UL016/09Y3/CMS/WYT01/ICMS, SRG026-ICMS13-LJJ, MYRG208(Y3-L4)- ICMS11-WYT, and UL016/09Y4/CMS/WYT01/ICMS). 2

9 Abstract Ganoderma lucidum triterpenoids (GLTs), structurally a class of highly oxidized lanostanes, have been demonstrated to exhibit a board spectrum of anti-cancer properties, which include anti-proliferative, anti-metastatic, anti-oxidative, and antiinflammatory effects, etc. Recently, some purified compounds from these triterpenoids were tested for anti-cancer effect in vitro and in vivo, and several possible targets involved in this process were identified. However, the specific signal pathways and precise molecular mechanisms responsible for their anti-cancer properties remain unclear. In this study, three forms of G. luciudm triterpenoids, an ethanol-soluble acidic component (ESAC) from G. lucidum, several pure ganoderic acids (GA-DM, GA-A, GA-F) and a mixture mainly contain ganoderiol A and its analogues (GAEE), were used to systemically analyze their anti-cancer effects in vitro and in zebrafish model. So far the acquired achievements are summarized and listed as follows: (1) The anti-proliferative effects of ESAC and GA-DM against human breast cancer cells are confirmed. DNA damage, G1 cell cycle arrest and apoptosis are closely related to the growth inhibition. (2) To clarify the anti-angiogenic effects of G. lucidum triterpenoids, transgenic Tg (fli1: EGFP) zebrafish model is used to screen potential compounds. G. lucidum triterpenoids treatment does not inhibit angiogenesis in zebrafish, and ESAC exposure shows a slight promoter on angiogenesis. Moreover, ESAC truly protect blood vessel from zebrafish vascular damage, which suggests a vascular protective role of G. lucidum triterpenoids. (3) GAEE is shown to inhibit adhesion and migration abilities of highly metastatic breast cancer MDA-MB-231 cells. However, GAEE treatment does not alter the expression and enzyme activities of matrix metalloproteinase (MMP) and 3

10 urokinase plaminogen activator (upa), and has no effect on transactivation of nuclear factor-kappa B (NF-κB), which is mainly responsible for cell invasion. The underlying mechanism revealed that GAEE decreases the expression of focal adhesion kinase (FAK) and attenuates the interaction of FAK and SRC, hence blocks paxillin activation, and results in inactivation of RhoA, CDC42, which are responsible for cell adhesion and migration. These results suggest that GAEE may be a potential anti-cancer agent targeting FAK-SRC-paxillin signaling. Based on the scientific work we carried out and the achievements mentioned above, we conclude that the anti-cancer profile of GLTs is promising and further studies are needed to potentiate these triterpenoids as anti-cancer agents. 4

11 Declaration I declare that the thesis here submitted is original except for the source materials explicitly acknowledged and that this thesis as a whole, or any part of this thesis has not been previously submitted for the same degree or for a different degree. I also acknowledge that I have read and understood the Rules on Handling Student Academic Dishonesty and the Regulations of the Student Discipline of the University of Macau. 5

12 Table of Contents Acknowledgements... 1 Abstract... 3 Declaration... 5 List of Tables and Figures... 9 List of Abbreviations Chapter 1 Introduction General Background (Cancer Treatment Strategies) Targeting cancer stem cells Targeting tumour blood vessel formation Targeting cancer metastasis Conclusions Specific Background (Anti-cancer Properties of G. lucidum Triterpenoids) G. lucidum triterpenoids G. lucidum extracts containing triterpenoids G. lucidum triterpenoid-enriched extracts Purified compounds from G. lucidum Ganoderic Acid Ganoderiol Other triterpenoids Conclusions Research Goals and Objectives Research Methodology and Design Potential Contributions Organization of the Thesis Statement of Originality Chapter 2 The Anti-proliferative Effect of GLTs Introduction Materials and Methods Cell culture and Materials Preparation and chemical analysis of ESAC Preparation and chemical analysis of GAEE Observation of morphologic changes Cell proliferation assay Colony formation assay Flow cytometry analysis of DNA content DNA fragmentation assay Mitochondrial membrane potential assay Western blot analysis Comet assay Immunocytochemical labeling Statistical analysis Results Chemical characterization of ESAC

13 Chemical characterization of GAEE Screening GLTs by MTT assay ESAC decreases the cell viability in breast cancer cells ESAC mediates G1 cell cycle arrest in breast cancer MCF-7 cells ESAC induces DNA damage in MCF-7 breast cancer cells ESAC induces apoptosis in MCF-7 breast cancer cells GA-DM decreases the cell viability in breast cancer cells GA-DM mediates G1 cell cycle arrest in MCF-7 cancer cells GA-DM induces apoptosis in MCF-7 cancer cells GA-DM induces DNA damage in MCF-7 cancer cells Conclusions Chapter 3 Effects of GLTs on Blood Vessels Formation in Zebrafish Model Introduction Materials and Methods Ethics statement Chemicals and reagents Zebrafish and embryo Zebrafish embryo collection, drug treatment for anti-angiogenic assay Zebrafish embryo collection, drug treatment for pro-angiogenic assay Results GLTs do not inhibit blood vessel formation in zebrafish model ESAC promotes angiogenesis in zebrafish ESAC rescues VRI-induced blood vessel loss in zebrafish Conclusions Chapter 4 Effect of GLTs on Breast Cancer Cell Metastasis Introduction Materials and Methods Materials Cell culture Observation of morphologic changes Hoechst staining MTT assay Flow cytometry analysis of DNA content Transwell assay Wound healing assay Cell adhesion assay Preparation of cytoplasmic and nuclear lysates Western blot analysis Gelatin zymography Casein-plasminogen zymography Immunofluorescence Immunoprecipitations Statistical analysis Results Screening GLTs by wound healing assay in MDA-MB-231 cells

14 Cytotoxicity of GAEE in MDA-MB-231 cells GAEE did not cause cell cycle arrest and apoptosis in MDA-MB-231 cells GAEE inhibited migration, adhesion, but slightly affected invasion in MDA-MB-231 cells Effect of GAEE on NF-κB/MMPs/uPA signaling Effect of GAEE on FAK signaling GAEE inhibited phosphorylation of paxillin and blocked the interaction between FAK and paxillin Effect of GAEE on the expression of Rho GTPases and actin assembly Conclusions Chapter 5 Conclusions Conclusions The summary of current study The summary of GLTs targets on anti-cancer effect Limitations of Current Study Perspectives for Future Work Future work for current study Future work for the anti-cancer research of GLTs References Curriculum Vitae

15 List of Tables and Figures Table 1.1. The anti-proliferative properties of GLETs in vitro Table 1.2. The anti-cancer properties of some typical triterpenoids compounds isolated from G. lucidum Fig Schematic depiction of cancer metastasis Fig The chemical structures of several triterpenoids Fig The schematic diagram of the anti-cancer effects of GLTs Fig The research design of this thesis Fig The flow chart of extraction and chemical characteristics of ESAC Fig Chemical assay of GAEE Fig MTT assay of GLTs in human breast cancer MCF-7 cells Fig ESAC decreases the cell viability in breast cancer cells Fig ESAC mediates G1 cell cycle arrest in MCF-7 cells Fig ESAC induces DNA damage in MCF-7 cells Fig ESAC induces apoptosis in MCF-7 cells Fig GA-DM decreases the cell viability in breast cancer cells Fig GA-DM mediates G1 cell cycle arrest in MCF-7 cancer cells Fig GA-DM induces apoptosis in MCF-7 cancer cells Fig GA-DM induces DNA damage in MCF-7 cancer cells Fig Toxic assay of GLTs on zebrafish Fig The effects of GLTs on blood vessel formation in ISVs of Tg (fli1: EGFP) zebrafish embryos Fig The effects of GLTs on blood vessel formation in SIVs of Tg (fli1: EGFP) transgenic zebrafish Fig The proangiogenic effects of GLTs on VRI-induced damaged blood vessels in Tg (fli1: EGFP) zebrafish embryos Fig Screening GLTs by wound healing assay Fig Effects of GAEE on cell viability in MDA-MB-231 cells Fig Effects of GAEE on cell cycle progression and apoptosis Fig Effects of GAEE on cell migration, adhesion and invasion Fig Effects of GAEE on NF-κB/MMPs/uPA signaling in MDA-MB-231 cancer cells Fig GAEE suppressed FAK signaling in MDA-MB-231 cells Fig GAEE inhibited activation of paxillin in MDA-MB-231 cells Fig Effects of GAEE on Rho GTPases expression and actin assembly Fig The summary of potential compounds in these study Fig The new findings of this study

16 List of Abbreviations AKT AP-1 AR protein kinase B activator protein-1 androgen receptor Arp2/3 actin-related protein 2 and 3 CDK CSCs CTCs DLAV DMSO ECM EGFP EMT cyclin-dependent kinase cancer stem cells circulation tumour cells dorsal longitudinal anastomotic vessel dimethyl sulfoxide extracellular matrix enhanced green fluorescent protein epithelial mesenchymal transition ERK1/2 extracellular signal regulated kinase 1/2 FAK FPT GAs GAEE GLTs GLCTs GLETs HMG-CoA hpf HUVEC IL ISV focal adhesion kinase farnesyl protein transferase ganoderic acids ganoderiol A-enriched extract G. lucidum triterpenoids G. lucidum extracts containing triterpenoids G. lucidum triterpenoid-enriched extracts 3-hydroxy-3-methylglutaryl-coenzyme A hours post fertilization human umbilical vein endothelial cell interleukin intersegmental vessel 10

17 JC-1 JNK LA LLC LNCaP MMP MTT NF-κB NO N-WASP PBS PMA ROS SIV SOD TGF TRAIL VEGF VEGFR VRI 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide Jun N-terminal kinase lucidenic acid Lewis lung carcinoma lymph-node carcinoma of the prostate matrix metalloproteinase 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide nuclear factor-kappa B nitric oxide neural Wiskott-Aldrich syndrome protein phosphate buffered saline phorbol-12-myristate-13-acetate reactive oxygen species subintestinal vessel superoxide dismutase transforming growth factor tumour-necrosis-factor-related apoptosis-inducing ligand vascular endothelial growth factor vascular endothelial growth factor receptor vascular endothelial growth factor receptor inhibitor 11