by Cheung Yuen-Nei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 194-208). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Contents --- p.vi / List of Figures --- p.xii / List of Tables --- p.xv / Abbreviations --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1 --- Introduction --- p.2 / Chapter 1.1 --- Characteristic of Saponins --- p.3 / Chapter 1.1.1 --- Occurrence of Saponins --- p.3 / Chapter 1.1.2 --- General Properties of Saponins --- p.3 / Chapter 1.1.2.1 --- Emulsifying Agents --- p.3 / Chapter 1.2.2.2 --- Forming Complex with Cholesterol --- p.4 / Chapter 1.1.2.3 --- Hemolytic Property --- p.4 / Chapter 1.1.3 --- Structure of Saponins --- p.5 / Chapter 1.1.3.1 --- Categories of Saponins --- p.5 / Chapter 1.1.3.1.1 --- Triterpene Saponins --- p.5 / Chapter 1.1.3.1.2 --- Steroid Saponins --- p.5 / Chapter 1.1.3.2 --- Monodesmosidic and Bidesmosidic Saponins --- p.7 / Chapter 1.1.4 --- Biological and Pharmacological Properties of Saponins --- p.9 / Chapter 1.1.4.1 --- Anti-microbial Activity --- p.9 / Chapter 1.1.4.1.1 --- Anti-fungal Activities --- p.9 / Chapter 1.1.4.1.2 --- Anti-bacterial Activities --- p.10 / Chapter 1.1.4.1.3 --- Anti-viral Activities --- p.10 / Chapter 1.1.4.2 --- Insecticidal Activity --- p.10 / Chapter 1.1.4.3 --- Molluscicidal Activity --- p.10 / Chapter 1.1.4.4 --- Hypocholesterolemic Activity --- p.11 / Chapter 1.1.4.5 --- Anti-ulcer Activity --- p.11 / Chapter 1.1.4.6 --- Contraceptive Activity --- p.12 / Chapter 1.1.4.7 --- Immunomodulatory Activities --- p.12 / Chapter 1.1.4.7.1 --- Direct Immunostimulation --- p.12 / Chapter 1.1.4.7.2 --- Acting as Immuno-adjuvants --- p.13 / Chapter 1.1.4.8 --- Anti-tumor Activity --- p.14 / Chapter 1.1.4.8.1 --- Anti-carcinogenesis --- p.15 / Chapter 1.1.4.8.2 --- Suppression of Tumor Growth --- p.16 / Chapter 1.1.5 --- Anti-tumor Activity of Steroid Saponins --- p.18 / Chapter 1.1.5.1 --- Diosgenin Steroid Saponin --- p.18 / Chapter 1.1.5.2 --- Hong Kong Compounds --- p.18 / Chapter 1.1.5.3 --- Hong Kong18 --- p.21 / Chapter 1.2 --- Human Hepatocellular Carcinoma (HCC) --- p.24 / Chapter 1.2.1 --- The Incidence of Liver Cancer --- p.24 / Chapter 1.2.2 --- Classification of Liver Cancer --- p.24 / Chapter 1.2.3 --- Human Hepatocellular Carcinoma Cell Lines --- p.25 / Chapter 1.2.3.1 --- Human Hepatocellular Carcinoma Cell Line HepG2 --- p.25 / Chapter 1.2.3.2 --- Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 --- p.27 / Chapter 1.2.3.2.1 --- Mechanisms of Multidrug Resistance --- p.28 / Chapter 1.2.3.2.2 --- Structure and Characteristics of P-glycoprotein --- p.29 / Chapter 1.2.3.2.3 --- Methods in Dealing with P-glycoprotein Over-expressed MDR Cells --- p.31 / Chapter 1.3 --- Objectives of the Project --- p.32 / Chapter 1.3.1 --- Study of the Anti-tumor Activities of Hong Kong 18 on Human Hepatocellular Carcinoma Cell Line HepG2 and Unravel the Underlying Mechanisms --- p.32 / Chapter 1.3.2 --- Study of the Anti-tumor Activities of Hong Kong 18on Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 and Unravel the Underlying Mechanisms --- p.32 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.34 / Chapter 2.1.1 --- Cell Culture --- p.34 / Chapter 2.1.1.1 --- Cell Lines --- p.34 / Chapter 2.1.1.2 --- Culture Media --- p.35 / Chapter 2.1.2 --- Reagents and Buffers --- p.36 / Chapter 2.1.2.1 --- Phosphate Buffered Saline (PBS) --- p.36 / Chapter 2.1.2.2 --- Reagents and Buffers for DNA Fragmentation --- p.36 / Chapter 2.1.2.3 --- Reagents and Buffers for Western Analysis --- p.37 / Chapter 2.1.2.4 --- Reagents and Buffer for Caspases Activities --- p.39 / Chapter 2.1.2.5 --- Fluorescent Dyes used for Flow Cytometry --- p.39 / Chapter 2.1.3 --- Chemicals --- p.39 / Chapter 2.2 --- Methods --- p.46 / Chapter 2.2.1 --- MTT Assay --- p.46 / Chapter 2.2.2 --- Determination of Cell Viability --- p.46 / Chapter 2.2.3 --- Purification of Macrophages from balb/c Mice --- p.47 / Chapter 2.2.4 --- Hemolysis Assay --- p.47 / Chapter 2.2.5 --- In vivo Studies of the Toxicity of HK18 --- p.48 / Chapter 2.2.6 --- DNA Fragmentation Assay --- p.50 / Chapter 2.2.7 --- Detection of Apoptotic and Necrotic / Late Apoptotic Cells Death by Flow Cytometry with Annexin V-FITC / PI --- p.51 / Chapter 2.2.8 --- Detection of Mitochondrial Membrane Potential by JC-1 Fluorescent Dye --- p.52 / Chapter 2.2.9 --- Detection of Intracellular Ca Level by Flow Cytometry with Fluo-3 Fluorescent Dye --- p.52 / Chapter 2.2.10 --- Detection of Intracellular Hydrogen Peroxide Level by Flow Cytometry with DCF Fluorescence Dye --- p.53 / Chapter 2.2.11 --- Simultaneous Detection of Mitochondrial Membrane Potential and Intracellular Ca2+ or Mitochondrial Membrane Potential and Intracellular Hydrogen Peroxide --- p.54 / Chapter 2.2.12 --- Western Analysis --- p.55 / Chapter 2.2.12.1 --- Total Protein Extraction --- p.55 / Chapter 2.2.12.2 --- Extraction of Cytosolic Proteins --- p.59 / Chapter 2.2.13 --- Determination of Caspases Enzymatic Activity --- p.63 / Chapter 2.2.14 --- Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) --- p.67 / Chapter 2.2.14.1 --- RNA Extraction by TRIzol Reagent --- p.67 / Chapter 2.2.14.2 --- Reverse Transcription --- p.68 / Chapter 2.2.14.3 --- Polymerase Chain Reaction --- p.68 / Chapter 2.3 --- Statistic Analysis --- p.71 / Chapter Chapter 3 --- Cytotoxicity of HK18 --- p.72 / Chapter 3.1 --- Cytotoxicity of HK18 on HepG2 Cells --- p.73 / Chapter 3.1.1 --- Study of the Cytotoxic Activity of HK18 on HepG2 Cells by MTT Assay --- p.73 / Chapter 3.1.2 --- Study of the Cytotoxic Activity of HK18 on HepG2 Cells by Tryphan Blue Exclusion Assay --- p.76 / Chapter 3.2 --- Cytotoxicity of HK18 on R-HepG2 Cells --- p.78 / Chapter 3.2.1 --- Study of the Cytotoxic Activity of HK18 on R-HepG2 Cells by MTT Assay --- p.78 / Chapter 3.2.2 --- Study of the Cytotoxic Activity of HK18 on R-HepG2 Cells by Tryphan Blue Exclusion Assay --- p.81 / Chapter 3.3 --- Cytotoxicity of HK18 on Macrophages --- p.83 / Chapter 3.4 --- Hemolytic Activity of HK18 --- p.85 / Chapter 3.5 --- In vivo Study of the Toxicity of HK18 --- p.87 / Chapter Chapter 4 --- Mechanistic Study of HK18 on HepG2 Cells --- p.90 / Chapter 4.1 --- Hallmarks of Apoptosis Induced by HK18 on HepG2 Cells --- p.91 / Chapter 4.1.1 --- Induction of Phosphatidylserine Externalization by HK18 on HepG2 Cells --- p.91 / Chapter 4.1.2 --- Induction of DNA Fragmentation by HK18 of HepG2 Cells --- p.97 / Chapter 4.2 --- Study of the Underlying Mechanisms of HK18 Induced Apoptosis in HepG2 Cells --- p.99 / Chapter 4.2.1 --- The Role of Mitochondria in HK18 Induced Apoptosis of HepG2 Cells --- p.99 / Chapter 4.2.1.1 --- HK18 Induced Mitochondrial Membrane Depolarization in HepG2 Cells --- p.101 / Chapter 4.2.1.2 --- Addition of Bongkrekic Acid Reduced HK18 Cytotoxicity on HepG2 Cells --- p.105 / Chapter 4.2.1.3 --- Elevation of Intracellular Hydrogen Peroxide Level in HK18 Treated HepG2 Cells --- p.108 / Chapter 4.2.1.4 --- Elevation of Intracellular Ca2+ Level in HK18 Treated HepG2 Cells --- p.114 / Chapter 4.2.1.5 --- HK18 Induced Cytochrome c and AIF Released from Mitochondria of HepG2 Cells --- p.120 / Chapter 4.3 --- Downstream Biochemical Changes Induced by HK18 on HepG2 Cells --- p.123 / Chapter 4.3.1 --- Activation of Caspase 3 of HepG2 Cells by HK18 as Demonstrated by Western Blot --- p.123 / Chapter 4.3.2 --- Induction of Caspases Activities of HepG2 Cells by HK18 as Demonstrated by Enzymatic Activity Assays --- p.125 / Chapter 4.4 --- Down-regulation of Anti-apoptotic Bcl-2 Family Members of HepG2 Cells by HK18 --- p.129 / Chapter Chapter 5 --- Mechanistic Study of HK18 on R-HepG2 Cells --- p.133 / Chapter 5.1 --- Hallmarks of Apoptosis Induced by HK18 on R-HepG2 Cells --- p.134 / Chapter 5.1.1 --- Induction of Phosphatidylserine Externalization by HK18 on R-HepG2 Cells --- p.134 / Chapter 5.1.2 --- Induction of DNA Fragmentation by HK18 of R-HepG2 Cells --- p.137 / Chapter 5.2 --- Study of the Underlying Mechanisms of HK18 Induced Apoptosis in R-HepG2 Cells --- p.139 / Chapter 5.2.1 --- The Role of Mitochondria in HK18 Induced Apoptosis of R-HepG2 Cells --- p.139 / Chapter 5.2.1.1 --- HK18 Induced Mitochondrial Membrane Depolarization in R-HepG2 Cells --- p.139 / Chapter 5.2.1.2 --- Addition of Bongkrekic Acid Reduced HK18 Cytotoxicity on R-HepG2 Cells --- p.142 / Chapter 5.2.1.3 --- Elevation of Intracellular Hydrogen Peroxide Level in HK18 Treated R-HepG2 Cells --- p.144 / Chapter 5.2.1.4 --- Elevation of Intracellular Ca2+ Level in HK18 Treated R-HepG2 Cells --- p.146 / Chapter 5.3 --- Downstream Biochemical Changes Induced by HK18 on R-HepG2 Cells --- p.148 / Chapter 5.3.1 --- Activation of Caspase 3 of R-HepG2 Cells by HK18 as Demonstrated by Western Blot --- p.148 / Chapter 5.3.2 --- Induction of Caspases Activation of R-HepG2 Cells by HK18 as Demonstrated by Enzymatic Activity Assays --- p.150 / Chapter 5.4 --- Down-regulation of the Anti-apoptotic Bcl-2 Protein of R-HepG2 Cells by HK18 --- p.154 / Chapter 5.5 --- HK18 was Not a Substrate of P-glycoprotein Contents --- p.156 / Chapter Chapter 6 --- Discussion --- p.158 / Chapter 6.1 --- Cytotoxicity of HK18 --- p.159 / Chapter 6.1.1 --- HK18 was Cytotoxic to the Human Hepatocellular Carcinoma Cell Line HepG2 and Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 --- p.159 / Chapter 6.1.2 --- Study of the Toxicity of HK18 --- p.160 / Chapter 6.2 --- Mechanistic Studies of the Cytotoxic Effects of HK18 on HepG2 Cells --- p.161 / Chapter 6.2.1 --- Apoptotic Cell Death Induction of HK18 on HepG2 Cells --- p.161 / Chapter 6.2.2 --- Studies of the Underlying Mechanisms of HK18 Induced Apoptosis of HepG2 Cells --- p.162 / Chapter 6.3 --- Mechanistic Studies of the Cytotoxic Effects of HK18 on R-HepG2 Cells --- p.181 / Chapter 6.3.1 --- Apoptotic Cell Death Induction of HK18 on R-HepG2 Cells --- p.181 / Chapter 6.3.2 --- Studies of the Underlying Mechanisms of HK18 Induced Apoptosis of HepG2 Cells --- p.181 / Chapter Chapter 7 --- Future Perspectives --- p.190 / Chapter Chapter 8 --- References --- p.193