SCREENING AND EVALUATION OF THE ANTICANCER POTENTIAL OF SCORPION VENOMS AND SNAKE VENOM L-AMINO ACID OXIDASE IN GASTRIC CANCER DING JIAN NATIONAL UNIVERSITY OF SINGAPORE 2014 SCREENING AND EVALUATION OF THE ANTICANCER POTENTI AL OF SCORPION VENOMS AND SNAKE VENOM L-AMINO ACID OXIDASE IN GASTRIC CANCER DING JIAN (B.S.c) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY VENOM AND TOXIN RESEARCH PROGRAMME DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2014 ACKNOWLEDGEMENTS I would like to take this opportunity to express my sincere gratitude to those who give me help during my pursuit of PhD degree for the last four years. It is obvious that this thesis could not be finished in time and with good quality without their support. First, I would thank my supervisor Prof Gopalakrishnakone, P, who introduced this interesting project to me. As a supervisor, Prof Gopal helped me design experiments, guided me to learn the knowledge and skills in toxicology and cancer research, and more importantly encouraged me to be confident and move forward when the project was not going smoothly. His attitudes towards work and life also impressed me and let me know the importance of the balance between these two factors. Second, I would deliver my deep appreciation to my co-supervisor Prof Bay Boon-Huat, Head of Anatomy department, NUS. Prof Bay interviewed me and enrolled me from Zhejiang University, China, to NUS. Furthermore, Prof Bay took me in as a member of team Anatomy and as part of his research group. I have benefited so much from the friendly and supportive environment in his group. Prof Bay also guided me and supported me with detailed instructions during the whole processes of my PhD project. He has discussed with me for most experimental problems and revised my drafts of publications, proposals and thesis as well. ii Next, I would like to thank Dr Wu Ya Jun, Ms Chan Yee Gek for their help in sample processing and viewing of TEM and SEM, respectively. I appreciate the support on SILAC work from our collaborator Dr Jayantha Gunaratne`s team, Quantitative Proteomics Group, Institute of Molecular & Cell Biology, Singapore. The appreciation also goes to Ms Ng Geok Lan, Ms Yong Eng Siang, Mr Poon Zhung Wei, Mr Gobalakrishnakone, Ms Pan Feng and Dr Cao Qiong for their efforts in the lab management and the technique assistance to my bench work. Similarly, the support from Ms Carolyne Ang, Ms Diljit Kour and Ms Violet Teo for administrative issues should not be ignored. I am also deeply grateful for the guidance and help from my seniors, Dr Feng Luo, Dr VGM Naidu, Dr M M Thwin, Dr Yu Ying Nan, Dr Alice Zen Mar Lwin, Dr Chua Pei Jou and Dr Jasmine Li Jia En. I would appreciate the partnership and friendship of my colleagues from Anatomy department, Ms Guo Tian Tian, Ms Oliva Jane Sculy, Ms Eng Cheng Teng, Mr Denish Babu, Mr Ashwini Kumar, Ms Cynthia Wong, Ms Shao Fei, Dr Xiang Ping, Ms Ooi Yin Yin, Dr Parakarlane R, Mr Lum Yick Liang, and all the staff and students in Department of Anatomy. The research wouldn`t be done in smooth and the life wouldn`t be joyful without their company. The last but not the least, I would especially thank my parents who raise me up, support my education, teach me good behaviours and always encourage, care and love me. The love from my parents and my elder sister is the ever motivation to make progress. iii TABLE OF CONTENTS DECLARATION ......................................................................................................... i ACKNOWLEDGEMENTS ....................................................................................... ii TABLE OF CONTENTS .......................................................................................... iv SUMMARY ................................................................................................................. x LIST OF FIGURES ................................................................................................. xiii LIST OF TABLES .................................................................................................. xvii ABBREVIATIONS ................................................................................................ xviii PUBLICATIONS .................................................................................................... xxii CHAPTER 1 ................................................................................................................ 1 INTRODUCTION....................................................................................................... 1 1.1 Gastric cancer .......................................................................................... 2 1.1.1 Epidemiology ............................................................................................... 2 1.1.2 Risk Factors ................................................................................................. 4 1.1.3 Classification ............................................................................................... 6 1.1.3.1 Types of gastric cancer ......................................................................... 6 1.1.3.2 Histological classification of gastric cancer .......................................... 6 1.1.4 Early screening, diagnosis and prognosis .................................................... 8 1.1.5 Molecular changes in gastric cancer: genetic and epigenetic alterations 10 Microsatellite instability: ................................................................................ 10 Involvement of p53: ........................................................................................ 11 HER-2:.............................................................................................................. 12 E-cadherin: ...................................................................................................... 12 1.1.6 Treatment of gastric cancer: conventional and targeted therapies ......... 14 1.2 Scorpion venoms and toxins and their effects on cancer ........................... 18 1.2.1 Animal venoms and toxins, an introduction ............................................. 18 1.2.2 Scorpion biology ........................................................................................ 20 1.2.3 Scorpion venoms and toxins ..................................................................... 23 1.2.3.1 Sodium channel toxins (NaScTxs) from scorpion venoms ................. 24 1.2.3.2 Potassium channel toxins (KTxs) from scorpion venoms ................... 25 1.2.3.3 Calcium and Chloride channel toxins from scorpion venoms ........... 26 iv 1.2.3.4 Scorpion venom peptides with no disulfide bridges .......................... 27 1.2.3.5 High molecular weight enzymes ........................................................ 28 Hyaluronidase: ............................................................................................ 29 Phospholipase A2 (PLA2): ............................................................................ 29 Proteases: ................................................................................................... 30 1.2.3.6 L-amino acid oxidases (LAAOs) from scorpion and snake venoms ... 31 1.2.4 The anticancer potential of scorpion venoms and toxins ......................... 32 1.3 Scope of study ......................................................................................... 37 CHAPTER 2 .............................................................................................................. 39 MATERIALS AND METHODS ............................................................................. 39 2.1 Scorpion venom preparation, purification and characterization ............... 40 2.1.1 Scorpion venom preparation .................................................................... 40 2.1.2 Protein concentration measurement ....................................................... 40 2.1.3 Sodium dodecyle sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) ............................................................................................................................ 41 2.1.4 Size exclusive gel filtration ........................................................................ 42 2.1.5 Cation exchange chromatography ............................................................ 43 2.1.6 MALDI-TOF Mass spectrometry ................................................................ 43 2.2 Cell culture .............................................................................................. 44 2.3 Functional studies to evaluate the anticancer effects of scorpion venom and LAAO in vitro .................................................................................................. 46 2.3.1 Cell proliferation/viability assay ................................................................ 46 2.3.2 Cytotoxixity determination by Lactate Dehydrogenase (LDH) assay ........ 46 2.3.3 Cell cycle analysis ...................................................................................... 47 2.3.4 Cell apoptosis detection by Annexin V & PI staining ................................ 48 2.3.5 Transmission electron microscopy (TEM) ................................................. 49 2.3.6 Scanning electron microscopy (SEM) ........................................................ 49 2.3.7 Cell migration and invasion assay ............................................................. 50 2.3.8 Evaluation of Caspase-3 activity ..............................................................