Studies on the anti-tumour activities of banlangen alkaloids on murine neuroblastoma cells.

Yip, Hon Yan Kelvin.Thesis (M.Phil.)--Chinese University of Hong Kong, 2010.Includes bibliographical references (leaves 218-242).Abstracts in English and Chinese.ACKNOWLEDGEMENTS --- p.iABBREVIATIONS --- p.iiABSTRACT --- p.viiCHINESE ABSTRACT (摘要） --- p.xiPUBLICATIONS --- p.xivTABLE OF CONTENTS --- p.xvChapter CHAPTER 1: --- GENERAL INTRODUCTION --- p.1Chapter 1.1 --- Neuroblastoma --- p.2Chapter 1.1.1 --- An overview of neuroblastoma --- p.2Chapter 1.1.2 --- Epidemiology of neuroblastoma --- p.3Chapter 1.1.3 --- Clinical presentations of neuroblastoma --- p.5Chapter 1.1.4 --- Diagnosis and clinical assessment of neuroblastoma --- p.8Chapter 1.1.5 --- Staging of neuroblastoma --- p.10Chapter 1.1.6 --- Genetic aberrations of neuroblastoma --- p.12Chapter 1.1.7 --- Therapies of neuroblastoma --- p.15Chapter 1.2 --- Banlangen alkaloids --- p.20Chapter 1.2.1 --- An overview of Banlangen alkaloids --- p.20Chapter 1.2.2 --- "Pharmacokinetics of indirubin, tryptanthrin and their derivatives" --- p.24Chapter 1.2.2.1 --- Bioavailability of indirubin and its derivatives --- p.24Chapter 1.2.2.2 --- Toxicity of indirubin and its derivatives --- p.25Chapter 1.2.2.3 --- Bioavailability of tryptanthrin --- p.26Chapter 1.2.2.4 --- Toxicity of tryptanthrin --- p.27Chapter 1.2.3 --- "Pharmacological effects of indirubin, tryptanthrin and their derivatives" --- p.28Chapter 1.2.3.1 --- Selective inhibitor on kinases --- p.29Chapter 1.2.3.2 --- Anti-inflammatory activities --- p.31Chapter 1.2.3.3 --- Anti-tumour activities --- p.32Chapter 1.2.3.3.1 --- Anti-leukemic activity --- p.32Chapter 1.2.3.3.2 --- Apoptosis-inducing activity --- p.34Chapter 1.2.3.4 --- Anti-viral properties --- p.37Chapter 1.2.3.5 --- Anti-microbial properties --- p.37Chapter 1.3 --- Aims and Scope of This Study --- p.39Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.41Chapter 2.1 --- Materials --- p.42Chapter 2.1.1 --- Cell lines --- p.42Chapter 2.1.2 --- "Cell culture media, reagents and buffers" --- p.43Chapter 2.1.3 --- General staining solutions --- p.46Chapter 2.1.4 --- Drugs and chemicals --- p.47Chapter 2.1.5 --- Reagent for primary cultures preparation --- p.48Chapter 2.1.6 --- Reagents for cell proliferation assay --- p.48Chapter 2.1.7 --- Reagents for DNA extraction --- p.50Chapter 2.1.8 --- Reagents for gel electrophoresis of nucleic acids --- p.51Chapter 2.1.9 --- Reagents and buffers for flow cytometry --- p.53Chapter 2.1.10 --- Reagents and buffers for measuring caspase activity --- p.54Chapter 2.1.11 --- "Reagents, buffers and materials for Western blot analysis" --- p.58Chapter 2.1.12 --- Reagent for Hoechst staining --- p.68Chapter 2.2 --- Methods --- p.69Chapter 2.2.1 --- Culture of cell lines --- p.69Chapter 2.2.2 --- Determination of cell viability --- p.70Chapter 2.2.3 --- Determination of cell proliferation by tritiated thymidine ([ 3H]-TdR) incorporation assay --- p.72Chapter 2.2.4 --- "Isolation, culture and cytotoxicity test of murine peritoneal macrophages" --- p.73Chapter 2.2.5 --- "Isolation, culture and cytotoxicity test of murine bone marrow cells" --- p.74Chapter 2.2.6 --- Cytotoxicity test of primary cortical neurons from SD rats --- p.75Chapter 2.2.7 --- Determination of colony forming ability --- p.75Chapter 2.2.8 --- Analysis of cell cycle profile /DNA content by flow cytometry --- p.76Chapter 2.2.9 --- Detection of DNA fragmentation by agarose gel electrophoresis --- p.77Chapter 2.2.10 --- Quantitative detection of DNA fragmentation by Cell Death ELISAplus kit --- p.78Chapter 2.2.11 --- Detection of intracellular reactive oxygen species (ROS) generation --- p.80Chapter 2.2.12 --- Measurement of caspase activity --- p.81Chapter 2.2.13 --- Hoechst 33342 staining --- p.83Chapter 2.2.14 --- Cell morphological study --- p.83Chapter 2.2.15 --- Analysis of morphological changes by flow cytometry --- p.84Chapter 2.2.16 --- Assay for acetylcholine esterase (AChE) activity --- p.85Chapter 2.2.17 --- Protein expression study --- p.86Chapter 2.2.18 --- Statistical analysis --- p.89Chapter CHAPTER 3: --- IN VITRO STUDIES ON THE ANTI PROLIFERATIVE EFFECT OF INDIRUBIN-3'-OXIME AND TRYPTANTHRIN ON NEUROBLASTOMA CELLS --- p.90Chapter 3.1 --- Introduction --- p.91Chapter 3.2 --- Results --- p.95Chapter 3.2.1 --- Effects of indirubin-3'-oxime and tryptanthrin on the proliferation of human and the murine neuroblastoma cells --- p.95Chapter 3.2.2 --- Kinetic and reversibility studies of the anti-proliferative effect of indirubin-3'-oxime and tryptanthrin on the murine neuroblastoma Neuro-2a BU-1 cells --- p.107Chapter 3.2.3 --- Cytotoxic effect of indirubin-3'-oxime and tryptanthrin on the murine neuroblastoma Neuro-2a BU-1 cells --- p.115Chapter 3.2.4 --- Effects of indirubin-3'-oxime and tryptanthrin on the clonogenicity of the murine neuroblastoma Neuro-2a BU-1 cells --- p.120Chapter 3.2.5 --- Cytotoxicity of indirubin-3'-oxime and tryptanthrin on primary cells --- p.123Chapter 3.2.6 --- Effects of tryptanthrin on the cell cycle profile and expression of cyclins and cyclin-dependent kinases (CDKs) in the murine neuroblastoma Neuro-2a BU-1 cells --- p.132Chapter 3.2.7 --- Effect of indirubin-3'-oxime on the cell cycle profile in the murine neuroblastoma Neuro-2a BU-1 cells --- p.133Chapter 3.3 --- Discussion --- p.142Chapter CHAPTER 4: --- IN VITRO STUDIES ON THE APOPTOSIS-INDUCING EFFECT OF INDIRUBIN-3'-OXIME ON NEUROBLASTOMA CELLS --- p.150Chapter 4.1 --- Introduction --- p.151Chapter 4.2 --- Results --- p.156Chapter 4.2.1 --- Induction of DNA fragmentation in the indirubin-3'-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.156Chapter 4.2.2 --- Induction of chromatin condensation in the indirubin-3 '-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.160Chapter 4.2.3 --- Induction of caspase activities in the indirubin-3 '-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.162Chapter 4.2.4 --- Induction of Reactive Oxygen Species (ROS) in the indirubin-3' -oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.169Chapter 4.2.5 --- Expression of pro-apoptotic and anti-apoptotic proteins in the indirubin-3 '-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.173Chapter 4.3 --- Discussion --- p.177Chapter CHAPTER 5: --- STUDIES ON THE DIFFERENTIATION-INDUCING ACTIVITY OF TRYPTANTHRIN ON NEUROBLASTOMA CELLS --- p.188Chapter 5.1 --- Introduction --- p.189Chapter 5.2 --- Results --- p.193Chapter 5.2.1 --- Effects of tryptanthrin on the cell size and cellular complexity of the murine neuroblastoma Neuro-2a BU-1 cells --- p.193Chapter 5.2.2 --- Morphological studies on tryptanthrin-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.196Chapter 5.2.3 --- Effect of tryptanthrin on the acetylcholine esterase (AChE) activity in the murine neuroblastoma Neuro-2a BU-1 cells --- p.198Chapter 5.2.4 --- Effects of tryptanthrin on the expression of tau protein and other mediators involved in the differentiation pathway --- p.200Chapter 5.3 --- Discussion --- p.204Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.209REFERENCES --- p.21