Effects of polyphyllin D on the induction of apoptosis in human hepatocellular carcinoma HepG2 cells and its multidrug resistant derivative RHepG2 cells.

Ong Chik Ying Rose.Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.Includes bibliographical references (leaves 181-195).Abstracts in English and Chinese.Acknoledgements --- p.iList of Abbreviations --- p.iiAbstract --- p.iiiAbstract in Chinese --- p.vList of Publications and Abstracts --- p.viiList of Figures --- p.viiiList of Tables --- p.xiContents --- p.xiiChapter Chapter 1 --- Introduction --- p.1Chapter 1.1 --- Saponins --- p.3Chapter 1.1.1 --- Structure of saponins --- p.3Chapter 1.1.2 --- Occurrence of saponins --- p.7Chapter 1.1.3 --- Bioactivities of saponins --- p.8Chapter 1.2 --- Paris Polyphylla --- p.9Chapter 1.2.1 --- Chonglou --- p.11Chapter 1.3 --- Polyphyllin D --- p.12Chapter 1.4 --- Apoptosis --- p.15Chapter 1.4.1 --- Apoptosis and necrosis --- p.16Chapter 1.4.2 --- Initiation phase of apoptosis --- p.16Chapter 1.4.2.1 --- Extrinsic apoptotic pathway --- p.18Chapter 1.4.2.2 --- Intrinsic apoptotic pathway --- p.19Chapter 1.4.3 --- Execution phase of apoptosis --- p.19Chapter 1.4.4 --- Termination phase of apoptosis --- p.20Chapter 1.5 --- Multi-drug resistance (MDR) --- p.21Chapter 1.5.1 --- MDR mediated by decreased drug accumulation --- p.21Chapter 1.5.2 --- MDR mediated by enhanced anti-oxidant enzyme activities --- p.25Chapter 1.5.3 --- MDR mediated by enhanced detoxification of drugs --- p.25Chapter 1.5.4 --- MDR mediated by enhanced DNA repair system --- p.26Chapter 1.5.5 --- MDR mediated by altered apoptotic pathway --- p.26Chapter 1.5.6 --- Current strategies for overcoming multidrug resistance in cancer --- p.27Chapter 1.6 --- Hepatocellular carcinoma (HCC) --- p.30Chapter 1.7 --- Objectives of project --- p.32Chapter Chapter 2 --- Materials and Methods --- p.33Chapter 2.1 --- Materials --- p.34Chapter 2.1.1 --- Culture of Cells --- p.34Chapter 2.1.1.1 --- Cell lines --- p.34Chapter 2.1.1.2 --- Preservation of Cells --- p.35Chapter 2.1.2 --- Culture Media --- p.36Chapter 2.1.2.1 --- RPMI 1640 (Phenol Red Medium) --- p.36Chapter 2.1.2.2 --- RPMI 1640 (Phenol Red-free Medium) --- p.36Chapter 2.1.3 --- Buffers and Reagents --- p.37Chapter 2.1.3.1 --- Buffer for Common Use --- p.37Chapter 2.1.3.2 --- Reagents for Annexin-V-FITC/PI Assay --- p.37Chapter 2.1.3.3 --- Reagents for Western Blotting Analysis --- p.37Chapter 2.1.4 --- Chemicals --- p.40Chapter 2.1.4.1 --- Fluorescence Dyes --- p.40Chapter 2.1.4.2 --- Antibodies --- p.41Chapter 2.1.4.3 --- Other Chemicals --- p.42Chapter 2.1.5 --- Summary of chemicals used in this study --- p.43Chapter 2.2 --- Methods --- p.48Chapter 2.2.1 --- In vitro Cell Cytotoxicity Assay --- p.48Chapter 2.2.1.1 --- AlamarBlue Assay --- p.48Chapter 2.2.2 --- Flow Cytometry --- p.50Chapter 2.2.2.1 --- Analysis by Flow Cytometry --- p.50Chapter 2.2.2.2 --- Determination of Apoptotic and Late Apoptotic/Necrotic Cells with Annexin-V-FITC/PI Cytometric Analysis --- p.50Chapter 2.2.2.3 --- Determination of Mitochondrial Membrane Potential in cells --- p.51Chapter 2.2.2.4 --- Determination of Hydrogen Peroxide (H2O2) Release in cells --- p.52Chapter 2.2.2.5 --- Measurement of doxorubicin accumulation in cells --- p.53Chapter 2.2.2.6 --- Determination of P-glycoprotein expression level in cells --- p.54Chapter 2.2.2.7 --- Determination of mitochondrial depolarization and swellingin isolated mitochondria --- p.54Chapter 2.2.3 --- Methods involved in DNA sequencing of MDRl promoter --- p.56Chapter 2.2.3.1 --- DNA extraction --- p.56Chapter 2.2.3.2 --- DNA and Gel Band Purification --- p.56Chapter 2.2.3.3 --- Assessment of DNA amount --- p.57Chapter 2.2.3.4 --- Polymerase Chain Reaction --- p.57Chapter 2.2.3.5 --- Agarose Gel Electrophoresis --- p.59Chapter 2.2.3.6 --- Preparation for DNA sequencing --- p.59Chapter 2.2.4 --- Western Blotting Analysis --- p.61Chapter 2.2.4.1 --- Preparation of Proteins from Cells --- p.61Chapter 2.2.4.2 --- Preparation of proteins from isolated mitochondria --- p.63Chapter 2.2.4.3 --- Protein analysis with Western analysis --- p.63Chapter 2.2.5 --- Confocal laser scanning microscopy (Confocal microscopy) --- p.66Chapter 2.2.5.1 --- Analysis with confocal microscopy --- p.66Chapter 2.2.5.2 --- Determination of mitochondrial changes in cells by confocal microscopy --- p.66Chapter 2.2.5.3 --- Determination of lysosomal rupture in cells by confocal microscopy --- p.67Chapter 2.2.6 --- Mitochondrial isolation --- p.68Chapter Chapter 3 --- Results 一 Resistance Mechanisms in RHepG2 cells --- p.69Chapter 3.1 --- Resistance of RHepG2 cells towards various chemical agents --- p.70Chapter 3.1.1 --- RHepG2 cells are resistant to doxorubicin --- p.70Chapter 3.1.2 --- RHepG2 cells are resistant to taxol --- p.72Chapter 3.1.3 --- RHepG2 cells are resistant to valinomycin --- p.74Chapter 3.2 --- Resistance mechanism in RHepG2 cells --- p.76Chapter 3.2.1 --- Reduced doxorubicin accumulation is observed in RHepG2 cells --- p.76Chapter 3.2.2 --- More P-glycoproteins on the cell surface was observed in RHepG2 cells --- p.80Chapter 3.2.3 --- Inhibition of P-glycoprotein activity increased doxorubicin accumulation in RHepG2 cells --- p.82Chapter 3.2.4 --- HepG2 and RHepG2 cells contain the same P-glycoprotein promoter region --- p.86Chapter 3.2.5 --- RHepG2 over-expressed Bcl-2 --- p.91Chapter 3.2.6 --- HepG2 and RHepG2 cells had the same level of Bax protein --- p.93Chapter Chapter 4 --- Results - Effects of Polyphyllin D in HepG2 and RHepG2 cells --- p.95Chapter 4.1 --- Cytotoxicity of Polyphyllin D in HepG2 and RHepG2 cells --- p.96Chapter 4.1.1 --- Polyphyllin D exhibited cytotoxic effect in both HepG2 and RHepG2 cells --- p.96Chapter 4.2 --- Apoptotic mechanism caused by Polyphyllin D in HepG2 and RHepG2 cells --- p.93Chapter 4.2.1 --- Polyphyllin D caused apoptosis in HepG2 and RHepG2 cells --- p.98Chapter 4.2.2 --- Polyphyllin D did not activate caspase8 --- p.103Chapter 4.2.3 --- Polyphyllin D did not concentrate on the plasma membrane of cells --- p.106Chapter 4.2.4 --- Polyphyllin D did not change Bcl-2 level in HepG2 and RHepG2 cells --- p.109Chapter 4.2.5 --- Polyphyllin D treatment enhanced Bax protein expression in both HepG2 and RHepG2 cells --- p.111Chapter 4.2.6 --- Polyphyllin D caused cytochrome c and AIF release in HepG2 and RHepG2 cells --- p.113Chapter 4.2.7 --- Polyphyllin D induced mitochondrial membrane depolarization in HepG2 and RHepG2 cells --- p.118Chapter 4.2.8 --- Polyphyllin D caused mitochondrial swelling in HepG2 and clustering of mitochondriain RHepG2 cells --- p.122Chapter 4.2.9 --- "Polyphyllin D caused H202 release in HepG2 and RHepG2 cells, and the cytotoxic effects of Polyphyllin D could be reduced by NAC" --- p.127Chapter 4.2.10 --- Polyphyllin D caused permeabilization of lysosomes --- p.132Chapter 4.3 --- Site of action of Polyphyllin D in cells --- p.135Chapter 4.3.1 --- Purity of isolated mitochondria --- p.135Chapter 4.3.2 --- Polyphyllin D caused cytochrome c release from the HepG2 and RHepG2 isolated mitochondria --- p.137Chapter 4.3.3 --- Polyphyllin D induced mitochondrial depolarization in HepG2 and RHepG2 isolated mitochondria --- p.139Chapter 4.3.4 --- Polyphyllin D caused mitochondrial swelling in HepG2 and RHepG2 isolated mitochondria --- p.142Chapter 4.4 --- Resistance reversal effects of Polyphyllin D in RHepG2 cells --- p.144Chapter 4.4.1 --- Polyphyllin D increased doxorubicin accumulation in RHepG2 cells --- p.144Chapter 4.4.2 --- P-glycoprotein expression was not down-regulated after Polyphyllin D treatment --- p.146Chapter 4.4.3 --- Co-treatment of doxorubicin with Polyphyllin D had enhanced cytotoxic effect --- p.148Chapter Chapter 5 --- Discussion - Resistance mechanisms in RHepG2 cells --- p.150Chapter 5.1 --- Resistance of RHepG2 cells towards various chemical reagents --- p.151Chapter 5.2 --- Resistance mechanisms in RHepG2 cells --- p.154Chapter Chapter 6 --- Discussion - Effects of Polyphyllin D in HepG2and RHepG2 cells --- p.159Chapter 6.1 --- Cytotoxicity of Polyphyllin D in HepG2 and RHepG2 cells --- p.160Chapter 6.2 --- Apoptotic mechanisms caused by Polyphyllin D in HepG2 and RHepG2 cells --- p.162Chapter 6.3 --- Site of action of Polyphyllin D in HepG2 andRHepG2 cells --- p.172Chapter 6.4 --- Resistance reversal effects of Polyphyllin D in RHepG2 cells --- p.175Chapter Chapter 7 --- Future Perspectives --- p.177Chapter Chapter 8 --- Conclusion --- p.179Reference