Bioassay-guided isolation, characterization and mechanistic study of the bioactive components from Sophora flavescens for the anti-proliferative effect on human hepatoma cells.

by Tsang Kit Man.Thesis (M.Phil.)--Chinese University of Hong Kong, 2006.Includes bibliographical references (leaves 179-188).Abstracts in English and Chinese.ABSTRACT --- p.iABSTRACT IN CHINESE (摘要） --- p.iiiACKNOWLEDGEMENTS --- p.vCONTENTS --- p.viLIST OF FIGURES --- p.xiLIST OF TABLES --- p.xivABBREVIATIONS --- p.xviChapter CHAPTER ONE: --- INTRODUCTION --- p.1Chapter 1.1 --- Hepatocellular Carcinoma --- p.2Chapter 1.1.1 --- Incidence of Hepatocellular Carcinoma --- p.2Chapter 1.1.2 --- Therapies for Hepatocellular Carcinoma --- p.4Chapter 1.2 --- Multidrug Resistance of Tumor Cells --- p.8Chapter 1.3 --- Therapeutic Potential of Traditional Chinese Medicine on Human Hepatoma --- p.10Chapter 1.4 --- Sophora flavescens Ait --- p.13Chapter 1.5 --- Biological Activities of Sophorae Radix --- p.15Chapter 1.5.1 --- Antitumor Activities --- p.16Chapter 1.5.2 --- "Antibacterial, Antimalarial and Antiviral Activities" --- p.17Chapter 1.6 --- Objectives and Significance of Study --- p.19Chapter 1.6.1 --- Bioassay-guided Isolation of Active Compounds from Sophora flavescens --- p.19Chapter 1.6.2 --- Action Mechanisms of the Bioactive Compounds Isolated from Sophora flavescens --- p.20Chapter CHAPTER TWO: --- MATERIALS AND METHODS --- p.21Chapter 2.1 --- Cell Culture --- p.22Chapter 2.1.1 --- Cell Lines --- p.22Chapter 2.1.2 --- Cell Culture Media --- p.24Chapter 2.2 --- Isolation of Bioactive Compounds from Sophora flavescens --- p.25Chapter 2.3 --- MTT assay --- p.27Chapter 2.4 --- Cell Cycle Analysis --- p.28Chapter 2.5 --- Detection of Phosphatidylserine Externalization with Annexin V-FITC and PI --- p.29Chapter 2.6 --- DNA Fragmentation Assay --- p.30Chapter 2.7 --- Western Blot Analysis --- p.32Chapter 2.7.1 --- Extraction of Total Cellular Protein --- p.32Chapter 2.7.2 --- Extraction of Cytosolic Protein --- p.32Chapter 2.7.3 --- Determination of Protein Concentration --- p.33Chapter 2.7.4 --- Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.35Chapter 2.7.5 --- Electroblotting of Protein --- p.36Chapter 2.7.6 --- Probing of Proteins with Antibodies --- p.37Chapter 2.7.7 --- Enhanced Chemiluminescence (ECL) Assay --- p.39Chapter 2.8 --- Detection of Mitochondrial Membrane Potential by JC-1 Fluorescent dye --- p.39Chapter 2.9 --- cDNA Microarray Analysis --- p.40Chapter 2.9.1 --- Isolation of Total RNA --- p.40Chapter 2.9.2 --- Microarray Hybridization and Analysis --- p.41Chapter 2.9.3 --- Validation of Candidate Genes --- p.44Chapter 2.9.3.1 --- Determination of RNA Concentration --- p.44Chapter 2.9.3.2 --- First-Strand cDNA Synthesis --- p.44Chapter 2.9.3.3 --- Reverse-Transcription Polymerase Chain Reaction (RT-PCR) of Candidate Genes --- p.45Chapter 2.10 --- Two-Dimensional Polyacrylamide Gel Electrophoretic Analysis (2D-PAGE) --- p.47Chapter 2.10.1 --- Extraction of Total Cellular Protein for 2-D Gel Electrophoresis --- p.47Chapter 2.10.2 --- Determination of Protein Concentration --- p.47Chapter 2.10.3 --- First-Dimension Isoelectric Focusing (IEF) --- p.49Chapter 2.10.4 --- Second-Dimension SDS-PAGE --- p.49Chapter 2.10.5 --- Visualization of 2-D Gel by Silver Staining --- p.50Chapter 2.10.6 --- Identification of Differentially Expressed Proteins with Matrix Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) --- p.51Chapter 2.11 --- Statistical Analysis --- p.53Chapter CHAPTER THREE: --- BIOASSAY-GUIDED ISOLATION AND CHARACTERISATION OF BIOACTIVE COMPOUNDS FROM SOPHORA FLAVESCENS --- p.54Chapter 3.1 --- Bioassay-guided Isolation of Bioactive Compounds from Sophora flavescens --- p.55Chapter 3.2 --- Structure Identification of the Bioactive Compounds Isolated from Sophora flavescens --- p.64Chapter 3.3 --- In Vitro Anti-tumor Effect of the Bioactive Compounds Isolated from Sophora flavescens --- p.71Chapter CHAPTER FOUR: --- MECHANISTIC STUDY OF SOPHORAFLAVANONE G IN THE INDUCTION OF APOPTOSIS IN HEPATOCELLULAR CARCINOMA CELLS --- p.76Chapter 4.1 --- In Vitro Anti-tumor Effect of Sophoraflavanone G --- p.77Chapter 4.2 --- Cell Cycle Analysis of Human Hepatocellular Carcinoma Cells and Multidrug Human Hepatocellular Carcinoma Cells --- p.81Chapter 4.3 --- Induction of Apoptosis in Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.88Chapter 4.3.1 --- Induction of Phosphatidylserine Externalization in Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.89Chapter 4.3.2 --- Induction of DNA Fragmentation in Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.94Chapter 4.3.3 --- Induction of Caspase-3 activation in Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.97Chapter 4.4 --- Underlying Mechanisms of Sophoraflavanone G-induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.102Chapter 4.4.1 --- Involvement of Death Receptor Pathway in Sophoraflavanone G- induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.103Chapter 4.4.2 --- Involvement of Bid protein in Sophoraflavanone G-induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.105Chapter 4.4.3 --- Involvement of Mitochondrial Pathway in Sophoraflavanone G- induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.108Chapter 4.4.4 --- Induction of Mitochondrial Membrane Depolarization in Human Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.112Chapter 4.4.5 --- Involvement of Caspase-independent Pathway in Sophoraflavanone G-induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.116Chapter CHAPTER FIVE: --- MECHANISTIC STUDY OF SOPHORAFLAVANONE G ON HUMAN HEPATOCELLULAR CARCINOMA CELLS BY USING cDNA MICROARRAY ANALYSIS --- p.119Chapter 5.1 --- Identification of Differentially Expressed Genes in Sophoraflavanone G- treated Human Hepatocellular Carcinoma Cells by cDNA Microarray Analyasis --- p.120Chapter CHAPTER SIX: --- MECHANISTIC STUDY OF SOPHORAFLAVANONE G ON HEPATOCELLULAR CARCINOMA CELLS BY USING TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS --- p.136Chapter 6.1 --- Identification of Differentially Expressed Proteins in Sophoraflavanone G- treated Human Hepatocellular Carcinoma Cells by Two-Dimensional Polyacrylamide Gel Electrophoresis --- p.137Chapter CHAPTER SEVEN: --- DISCUSSION --- p.150Chapter 7.1 --- Bioassay-guided Isolation of Bioactive Compounds from Sophora flavescens --- p.151Chapter 7.2 --- Induction of Apoptosis in Human Hepatocellular Carcinoma cells and Multidrug Human Hepatocellular Carcinoma Cells --- p.154Chapter 7.3 --- Differential Gene Expression Induced by Sophoraflavanone G in Human Hepatocellular Carcinoma Cells --- p.161Chapter 7.4 --- Differential Protein Expression Induced by Sophoraflavanone G in Human Hepatocellular Carcinoma Cells and Multidrug Human Hepatocellular Carcinoma Cells --- p.164Chapter 7.5 --- Toxicity of Sophoraflavanone G against Normal Liver Cells --- p.170Chapter CHAPTER EIGHT: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.173Chapter 8.1 --- Conclusion --- p.174Chapter 8.2 --- Future Prospects --- p.176REFERENCES --- p.17