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

Le profil d'expression aberrant des micro(mi)ARN est une caractéristique typique de nombreux cancers, dont le carcinome hépatocellulaire (CHC), une tumeur hépatique maligne primaire qui se classe seconde dans le monde en termes de mortalité par cancer. Notre équipe a récemment montré la baisse d’expression de miR-4510 dans des échantillons de patients atteints de CHC et son activité « suppresseur de tumeur ». L'analyse de données protéomiques recueillies à partir de cellules Huh7 transfectées par miR-4510 a révélé une diminution importante de plusieurs oncogènes, dont la sérine / thréonine protéine kinase RAF1. J’ai également découvert que le taux de protéine RAF1 était significativement surexprimé chez les patients atteints de CHC. Le rôle de RAF1 et de miR-4510 dans le CHC étant mal compris, j’ai étudié la fonction du couple RAF1/miR-4510 dans la tumorigenèse du foie. Mes analyses ont montré que miR-4510 régule négativement les taux de protéine RAF1 et d'ARNm. Une analyse par le système de double fluorescence-FunREG a révélé que miR-4510 interagit directement avec la région 3’ non-traduite de l’ARN de RAF1 via un site unique. La déplétion de RAF1 dans deux lignées tumorales de CHC par miR-4510 ou ARN interférant désactive leur caractère tumorigène in vitro et in vivo. Collectivement, mes données suggèrent que miR-4510 participe à la carcinogenèse du foie via son action directe sur RAF1 et la régulation de la voie MAPK/ERK. En conclusion, mon étude soutient l’hypothèse selon laquelle un traitement à base de miR-4510 pourrait être efficace pour traiter les patients atteints de CHC de type avancé ou réfractaire à la chimiothérapie. / Aberrant micro(mi)RNA expression signature is a hallmark of many cancers including hepatocellular carcinoma (HCC), a primary malignant liver disease which ranks second in cancer mortality worldwide. Our team previously reported the downregulation of miR-4510 in HCC samples and identified this miRNA as a strong tumor suppressor in liver. Proteomic data analysis collected from Huh7 cells transfected by miR-4510 showed a significant decrease of multiple oncogenes including RAF1 serine/threonine protein kinase. I also found that RAF1 protein level is significantly increased in HCC patients. The role of RAF1 and miR-4510 in HCC being poorly understood, I studied the function of RAF1/miR-4510 pair in tumorigenesis of the liver. My results showed that miR-4510 overexpression significantly decreases both RAF1 protein and mRNA levels and inhibits MAPK/ERK signaling. The dual fluorescence-FunREG assay revealed that miR-4510 directly interacts with RAF1 3’-untranslated region through a unique site. Silencing of RAF1 in two hepatic cell lines by miR-4510 or a specific small interfering RNA suppressed important tumorigenic features (proliferation, migration….) both in vitro and in vivo. Collectively, my data suggest that miR-4510 participates in liver carcinogenesis through RAF1 targeting and MAPK/ERK signaling inactivation. In addition, my study suggests that miR-4510-based therapy may represent a promising strategy to treat patients with advanced or refractory HCC.

There are multiple immune mechanisms identified for persistence of hepatitis B virus (HBV) infection. This thesis considers the vital role that inhibitory receptors play in contributing to impairment of the adaptive immune system in chronic hepatitis B (CHB), and the potential role they play in the innate immune system, focusing on the inhibitory receptor leucocyte-associated immunoglobulin-like receptor (LAIR)-1. The unique aspect of this work is that for the first time LAIR-1 expression has been investigated on natural killer (NK) cells in CHB. Our striking findings of increased LAIR-1 expression on peripheral NK cells in CHB and an inverse correlation between expression and effector function suggest this inhibitory receptor could have a potential role in exhaustion of NK cells in CHB. We therefore additionally explored the expression of LAIR-1 on circulating NK cells from patients with hepatocellular carcinoma (HCC) and non-alcoholic fatty liver disease (NAFLD). The particular relevance of LAIR-1 to liver disease is that one of its major ligands is collagen. We demonstrated a downregulation of LAIR-1 expression on intrahepatic NK cells, which we postulate might occur following repetitive engagement with abundant collagen within the liver. In line with this, intrahepatic NK cells with a liver-resident (CXCR6+) phenotype had even lower LAIR-1 expression than liver infiltrating (non-resident, CXCR6-) NK cells. Furthermore, preliminary experiments display attenuation of the cytotoxic degranulation capacity (CD107a) by circulating NK cells from CHB patients upon exposure to plate-bound collagen. We demonstrate differential expression of LAIR-1 on NK cells in viral hepatitis, HCC and NAFLD and between peripheral and intrahepatic NK cells. Preliminary experiments demonstrate a role in inhibiting NK cell function suggesting this as a novel therapeutic target to harness the capacity of NK cells to control chronic infection and cancer.

L’infection chronique par le virus de l’hépatite C (VHC) conduit au développement de la fibrose et de la cirrhose qui risque d’évoluer vers le carcinome hépatocellulaire (CHC). La protéine de capside du VHC interagit avec de nombreuses protéines de l’hôte et en particulier avec Smad3, protéine majeure de la voie de signalisation du transforming growth factor beta (TGF-Β). Mon travail de thèse consistait à étudier les conséquences biologiques de l’interaction entre la protéine de capside avec la voie de signalisation du TGF-Β. Le VHC présente une grande variabilité génétique et des travaux du laboratoire ont montré l’existence de séquences différentes de protéines de capside du virus entre les régions tumorales (cT) et cirrhotiques (cNT) d’un même sujet. Nous avons montré que ces différentes protéines de capside exprimées dans des hépatocytes orientent les réponses biologiques du TGF-Β vers la promotion tumorale en diminuant l’apoptose et en augmentant la transition épithelio-Mésenchymateuse (TEM) en particulier le variant cT. Cet effet est attribué à la capacité de la protéine de capside de diminuer l’activité transcriptionnelle de Smad3. De plus, les variants de la protéine de capside activent le TGF-Β latent via l’augmentation de l’expression de la trombospondine. L’un des marqueurs classiquement exprimé au cours d’une TEM est l’alpha-Actine musculaire lisse (αSMA). Nous avons montré qu’une autre isoforme, la γSMA, était polymérisée dans les cellules hépatiques développant une TEM. L’expression de γSMA a été retrouvée sur des coupes de CHC et a pu être significativement corrélée à la fois avec des marqueurs de la TEM, des marqueurs progéniteurs et avec l’agressivité de la tumeur.Ce travail apporte une meilleure compréhension du rôle de la protéine de capside dans la fibrose hépatique liée à l’infection virale. En effet, la protéine de capside du VHC agit à la fois de façon autocrine dans les hépatocytes en modulant les réponses du TGF-Β vers la promotion tumorale et de façon paracrine, en affectant l’activation des cellules étoilées en myofibroblastes par le TGF-Β activé. / Chronic HCV infection) may progress to liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV core binds several cellular proteins and in particular Smad3, a major protein of transforming growth factor beta (TGF-Β) signalling.. The aim of this study was to determine the implication of HCV core protein in TGF-Β responses. High genetic variability is a characteristic of HCV and it was previously shown that HCV core protein isolated from tumour (cT) or adjacent non-Tumour (cNT) livers displayed different sequences. Both were able to shift TGF-B responses from tumour suppressor to tumour promotor by decreasing hepatocyte apoptosis and increasing epithelial-Mesenchymal transition (EMT). Core cT was more potent than core cNT to promote this effect that was mainly attributed to the capacity of HCV core to alleviate Smad3 activity. Moreover, HCV core protein activated the latent form of TGF-Β through increased thrombospondin expression. It is commonly accepted that αSMA (alpha smooth muscle actin) is a hallmark of EMT. In the current study another SMA isoform, γSMA was found to be polymerized during hepatocyte EMT. γSMA was expressed in HCC tissues and correlated with EMT, stem cell and aggressiveness markers. In conclusion, this work contributed to a better understanding of the HCV core role in hepatitis fibrosis and HCC related to HCV. Indeed, HCV core might act both as an autocrine and paracrine way by modulating TGF-Β responses within hepatocytes and by activating hepatic stellate cells in stromal environment through its capacity to activate TGF-Β.

OBJETIVOS: Analisar a expressão de EGFR, proteínas da via de sinalização ou relacionadas aos seus efeitos em carcinoma hepatocelular (CHC) primários, metastáticos e em tecido hepático não tumoral em autópsias. Correlacionar os achados a dados clínico-patológicos e marcadores de classes moleculares. Avaliar a heterogeneidade de expressão em CHC metastáticos e fatores de disseminação extra-hepática. MÉTODOS: Oitenta autópsias de pacientes com CHC ao exame foram incluídas em estudo retrospectivo transversal. Foram analisados sexo, idade, raça, etilismo, infecção por vírus da hepatite B (VHB) e C (VHC), infecção pelo HIV, tratamento prévio, causas básica e imediata de óbito, peso do fígado, cirrose, número e tamanho dos nódulos, padrão macroscópico, grau histológico, variantes histológicas, padrão arquitetural, invasão de grandes veias e metástases extra-hepáticas. Imuno-histoquímica foi realizada em micromatrizes teciduais para pesquisa de EGFR, pEGFR(Tyr 1173), HER2, ERK1/2, MAPKAPK-2, pMAPK, Ag Ki67, caspase 3, citoqueratina 19 (CK19), mTOR, ciclina D1, Met, vimentina, p53 e beta-catenina. A expressão de EGFR foi avaliada em intensidade (0-3+) e distribuição (0-100%) em um sistema de escores de 0 a 300. Hiperexpressão foi definida para escores >= 200. Amostras de fígado normal foram incluídas como controles. Amostras de CHC primário foram pareadas às suas metástases e consideradas concordantes quando na mesma categoria de expressão. No tecido não tumoral foram semi-quantificadas a reação ductular expressando CK19 e a densidade da população de células estromais perissinusoidais pela vimentina. Estatística foi realizada através dos testes do qui-quadrado ou exato de Fisher ao nível de significância de 0,05. Para as correlações de escores e variáveis categóricas foi utilizado o coeficiente de Spearman. RESULTADOS: Foram incluídos 62 casos do sexo masculino e 18 do sexo feminino (58,1 ± 10,9 anos). Infecção pelo VHC foi a principal causa em 49% (39/80), seguida por etilismo em 30% (24/80) e infecção por VHB em 19% (15/80). Cirrose foi identificada em 90% (72/80) dos casos. Os tumores mostraram-se avançados em 95% (76/80). Invasão de grandes veias foi detectada em 19% (15/80) e metástases extra-hepáticas em 38% (30/80). MAPKAPK2, pEGFR (Tyr1173) e HER2 tiveram expressão fraca ou ausente. A expressão de EGFR foi mais frequente no fígado não neoplásico (26/26) (P < 0,05) - e nos controles normais (8/8) do que nas amostras tumorais primárias (60/75) e nas metástases (12/17). Nenhuma amostra dos controles apresentou hiperexpressão de EGFR, a qual foi mais frequente na cirrose (65% - 17/26) do que nos tumores avançados (36% - 26/72) (P < 0,05). EGFR hiperexpresso foi mais frequente nos tumores de grau 1/2 (P < 0,01) e nos casos com menos de quatro nódulos hepáticos (P = 0,014). A expressão de EGFR correlacionou-se à expressão de caspase 3 (P < 0,01). A expressão das quinases ERK1 e ERK2 foi correlacionada à proliferação celular pelo Ag Ki67 (P < 0,01), porém não ao escore de expressão de EGFR. CK19, p53 e beta-catenina nuclear foram correlacionaram-se às lesões de maior grau e a maiores taxas de proliferação celular (P<0,01). Met, EGFR e caspase 3 foram correlacionados a lesões mais diferenciadas. Vimentina teve forte correlação com CK19 (P < 0,01). A concordância de expressão entre tumores hepáticos e respectivas metástases variou de 50 a 85%. Para o EGFR foi de 61%. A expressão endotelial 2-3+ de pMAPK foi mais frequente nas metástases (P = 0,09). A disseminação extra-hepática foi mais frequente nos casos com baixa densidade de células perissinusoidais positivas para vimentina (P = 0,054) e nos casos sem reação ductular no tecido não neoplásico (P = 0,095). CONCLUSÕES: O EGFR tem papel relevante nas etapas iniciais e intermediárias do CHC, sendo sua expressão reduzida nas formas avançadas. Diferentes classes de CHC podem estar associadas a ativação da via do EGFR. A presente análise imuno-histoquímica ampla parece validar pelo menos dois grupos de CHC que nesta série de autópsias parecem ter sido separados pelo grau histológico. Confirma-se a hiperexpressão das quinases como evento importante na progressão tumoral, porém não necessariamente associada à hiperexpressão de EGFR. A heterogeneidade de expressão entre o CHC primário e suas metástases variou de 15 a 45% / OBJECTIVES: To analyze the expression of EGFR and proteins related to its signaling pathway or to its effects in hepatocellular carcinoma (HCC), metastases and surrounding liver tissue in a series of autopsies. To correlate expression patterns to clinicopathological data and other markers of molecular classification. To assess the heterogeneity of expression in metastatic HCC and factors related to extrahepatic spread. METHODS: Eighty autopsies of patients with HCC were included in a cross-sectional retrospective study. We analyzed gender, age, race, alcohol intake, infection with hepatitis B (HBV) and C virus (HCV), HIV infection, prior treatment, basic and immediate causes of death, the weight of the liver, cirrhosis, number and size of nodules, gross pattern, histological grade, histological variants, architectural pattern, invasion of large veins and extrahepatic metastases. Immunohistochemistry was performed on tissue microarrays to survey EGFR, pEGFR(Tyr 1173), HER2, ERK1/2, MAPKAPK-2, pMAPK, Ag Ki67, caspase 3, cytokeratin 19 (CK19), mTOR, cyclin D1, Met, vimentin, p53 and beta-catenin. EGFR expression was evaluated in intensity (0-3+) and distribution of membrane staining (0-100%) in a 0 - 300 score. Overexpression was defined for scores >= 200. Normal liver samples were included as controls. Intra-hepatic HCC samples were matched to their respective metastases and expression was considered concordant when they were assigned to the same category. Ductular reaction expressing CK19 and the density of perisinusoidal vimentin positive stromal cells were semi-quantified in non-tumor tissue. Statistics was performed using the chi- square or Fisher exact test at a significance level of 0.05. For the correlations of scores and categorical data we used the Spearman coefficient. RESULTS: Sixty-two males and eighteen females were included (age 58.1 ± 10.9). HCV was the major cause in 49% (39/80), followed by alcoholism in 30% (24/80) and HBV infection in 19% (15/80). Cirrhosis was identified in 90% (72/80) and advanced tumors in 95% (76/80). Large vein invasion was detected in 19% (15/80) and extra-hepatic metastases in 38% (30/80). MAPKAPK2, pEGFR (Tyr1173) and HER2 expression were weak or absent. The EGFR expression was more frequent in non-tumoral liver (26/26) (P <.05) and in normal controls (8/8) than in primary HCC tumor samples (60/75) and in metastatic HCC (12/17). No samples taken from the controls showed overexpression of EGFR, which was more common in cirrhotic tissue (65% - 17/26) than in advanced tumors (36% - 26/72) (P <0.05). EGFR overexpression was more frequent in grade 1/2 tumors (P <0.01) and in cases with less than four hepatic nodules (P = 0.014). EGFR expression was correlated to the expression of caspase 3 (P <0.01). The expression of the kinases ERK1 and ERK2 was correlated to Ag Ki67 cell proliferation index (P <0.01), but not to the EGFR expression score. CK19, p53 and nuclear beta- catenin were correlated to high grade lesions and to higher rates of cell proliferation (P <0.01). Met, EGFR and caspase 3 were correlated with more differentiated lesions. Vimentin was strongly correlated with CK19 (P <0.01). The concordance of expression between liver tumors and their metastases ranged from 50 to 85% (61% for EGFR). The 2-3+ expression of pMAPK in tumor endothelial cells was more common in metastases (P = 0.09). Extrahepatic dissemination was more frequent in cases with low density of vimentin positive perisinusoidal cells (P = 0.054) and in cases with no detectable ductular reaction in non-neoplastic tissue (P = 0.095). CONCLUSIONS: EGFR plays an important role in the early and intermediate stages of HCC progression, with lower expression in advanced tumors. Different classes of HCC may be associated with activation of EGFR. The present comprehensive immunohistochemical approach seems to validate at least two molecular classes of HCC, and histological grade seems to be able to discriminate these groups. We herein confirmed overexpression of kinases as a key event in tumor progression, but not necessarily associated with overexpression of EGFR. The heterogeneity of expression between primary HCC and its metastases ranged from 15 to 45%

Fu Zhenming. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 138-162). / Abstracts in English and Chinese. / List of abbreviations --- p.i / Abstract (in English) --- p.ii / 摘要（中文） --- p.iii / Acknowledgement --- p.iv / Chapter Chapter 1. --- Introduction and Objectives of Study --- p.1 / Chapter 1.1 --- Hepatocellular carcinoma in general --- p.2 / Chapter 1.1.1 --- "Epidemiology, risk factors" --- p.2 / Chapter 1.1.2 --- Pathology and staging --- p.4 / Chapter 1.1.3 --- Treatment --- p.6 / Chapter 1.1.4 --- Improvement of early detection and treatment of HCC --- p.7 / Chapter 1.2 --- General aspects of mitochondria and mitochondrial DNA (mtDNA) --- p.10 / Chapter 1.2.1 --- Structure and dynamics of mitochondria --- p.10 / Chapter 1.2.1.1 --- General introduction of mitochondria --- p.10 / Chapter 1.2.1.2 --- Respiration chain of mitochondria --- p.11 / Chapter 1.2.2 --- The mitochondrial genome --- p.14 / Chapter 1.2.2.1 --- Strucure --- p.14 / Chapter 1.2.2.2 --- Genes for structure proteins --- p.16 / Chapter 1.2.2.3 --- Genes for translation --- p.17 / Chapter 1.2.2.4 --- Imported proteins and RNAs --- p.17 / Chapter 1.2.3 --- Mitochondrial DNA maintenance --- p.19 / Chapter 1.2.4 --- Mitochondrial DNA replication --- p.25 / Chapter 1.2.5 --- Mitochondrial DNA transcription --- p.30 / Chapter 1.2.6 --- Mitochondrial DNA translation --- p.32 / Chapter 1.3 --- MtDNA diseases --- p.35 / Chapter 1.4 --- MtDNA mutation and HCC --- p.35 / Chapter 1.5 --- Aims of the study --- p.39 / Chapter Chapter 2. --- Materials and Methods --- p.41 / Chapter 2.1 --- Materials --- p.42 / Chapter 2.1.1 --- Chemicals --- p.42 / Chapter 2.1.2 --- Primers --- p.42 / Chapter 2.1.3 --- Enzymes --- p.45 / Chapter 2.1.4 --- Cell line --- p.45 / Chapter 2.1.5 --- Collection of specimens --- p.46 / Chapter 2.2 --- Methodology --- p.47 / Chapter 2.2.1 --- "DNA extraction from hcc tissues, cell line Hep3B and PBMCs" --- p.47 / Chapter 2.2.1.1 --- DNA extraction from HCC tissues --- p.47 / Chapter 2.2.1.2 --- DNA extraction from cell line Hep3B --- p.49 / Chapter 2.2.1.3 --- DNA extraction from and PBMCs --- p.50 / Chapter 2.2.1.3.1 --- Preparation of PBMCs --- p.50 / Chapter 2.2.1.3.2 --- DNA extraction from and PBMCs --- p.51 / Chapter 2.2.2 --- Detection of mt whole genome mutation by direct sequencing --- p.51 / Chapter 2.2.2.1 --- Design of mtDNA primers --- p.51 / Chapter 2.2.2.2 --- PCR amplification of the whole mt genome --- p.51 / Chapter 2.2.2.3 --- Direct sequencing of the whole mt genome --- p.52 / Chapter 2.2.2.3.1 --- Primer used in sequencing --- p.52 / Chapter 2.2.2.3.2 --- Purification of the PCR products of the whole mt genome --- p.53 / Chapter 2.2.2.3.3 --- Dye terminator cycle sequencing reaction --- p.53 / Chapter 2.2.2.3.4 --- Purification of extension products --- p.54 / Chapter 2.2.3 --- Detection of mtDNA control region mutation --- p.55 / Chapter 2.2.3.1 --- PCR amplification of D310 in the mtDNA control region --- p.55 / Chapter 2.2.3.2 --- Screening of D310 mutation by PFLDA --- p.55 / Chapter 2.2.3.2.1 --- Making 8% denatured gel mixture --- p.55 / Chapter 2.2.3.2.2 --- Setting up and Pouring the denatured gel --- p.56 / Chapter 2.2.3.2.4 --- Preparing and Loading the PCR products --- p.57 / Chapter 2.2.3.2.5 --- Electrophoresis --- p.57 / Chapter 2.2.3.2.6 --- "Gel fixing, silver staining and color development " --- p.58 / Chapter 2.2.3.3 --- Direct sequencing of D310 in the mtDNA control region --- p.59 / Chapter 2.2.4 --- Detection of mt DNA coding region mutation --- p.60 / Chapter 2.2.4.1 --- PCR amplification of the 5 respiratory chain subunit genes --- p.60 / Chapter 2.2.4.2 --- Restriction enzyme digestion of 5 genes in mtDNA coding region --- p.60 / Chapter 2.2.4.3 --- Screening of mtDNA coding region mutation by SSCP --- p.61 / Chapter 2.2.4.3.1 --- Making 6% 49:1 acrylamide/Bis SSCP gel mixture --- p.61 / Chapter 2.2.4.3.2 --- "Setting up the SSCP gel, loading sample, fixing, staining and developing of the gel " --- p.62 / Chapter 2.2.4.4 --- Sequencing conformation of the mtDNA coding region mutation --- p.62 / Chapter 2.2.5 --- Statistics --- p.63 / Chapter 2.2.5.1 --- The chi-square test --- p.63 / Chapter 2.2.5.2 --- The Friedman test --- p.63 / Chapter 2.2.5.3 --- Wilcoxon signed ranks test --- p.63 / Chapter Chapter 3. --- Results --- p.64 / Chapter 3.1 --- Detection mt DNA whole genome mutation --- p.65 / Chapter 3.1.1 --- Identification of mtDNA whole genome by direct sequencing --- p.65 / Chapter 3.2 --- Detection mt DNA D-loop mutation --- p.76 / Chapter 3.2.1 --- Screening of C-tract alteration in HCC tissus by PCR fragments length detection assay (PFLDA) --- p.76 / Chapter 3.2.2 --- Screening of coding region alteration in HCC tissues by SSCP --- p.77 / Chapter 3.2.2.1 --- Identification of C-tract alterations in HCC and non-tumorous tissues by direct sequencing --- p.77 / Chapter 3.2.3 --- Identification of C-tract alterations by direct sequencing --- p.82 / Chapter 3.2.3.1 --- Identification of C-tract alterations in HCC tissues by direct sequencing --- p.82 / Chapter 3.2.3.2 --- Identification of C-tract alteration in PBMC of normal subjects by direct sequencing --- p.82 / Chapter 3.2.3.3 --- Identification of C-tract alteration in PBMC of HCC patients by direct sequencing --- p.82 / Chapter 3.2.4 --- Statistics of the analysis of C-tract alterations --- p.82 / Chapter 3.3 --- Detection mt DNA mutation in the coding region --- p.87 / Chapter Chapter 4. --- Discussion --- p.98 / Chapter 4.1 --- Detection mtDNA whole genome mutation --- p.99 / Chapter 4.2 --- Detection mtDNA D-loop mutation --- p.107 / Chapter 4.3 --- Detection mtDNA mutation in the coding region --- p.119 / Chapter 4.4 --- Possible mechanisms of mtDNA mutation in HCC carcinogenesis --- p.125 / Chapter 4.5 --- Proposals for prospective studies --- p.126 / Chapter 4.5.1 --- Function of C7 in D310 --- p.128 / Chapter 4.5.2 --- Function changes of mtDNA coding region mutation --- p.130 / Chapter 4.5.3 --- Detection of D310 C-tract mutation in patients' plasma --- p.131 / Chapter 4.5.4 --- Relationship between nMSl and mtMSI --- p.132 / Chapter 4.6 --- Summary --- p.134 / References --- p.137

肝癌是造成癌症相關死亡的主要原因之一。而常規化療受耐藥性的發展和各種副作用的限制。由於無毒性和鲜明的生物药物能力，從蘑菇提取的代謝物在癌症治療中獲得更多的注意和关注。我們以前的研究已經證明來自平菇香菇多醣蛋白複合物的抗癌作用。本研究的目的是探討一種含有多醣蛋白複合物的秀珍菇（PP）熱水提取物在肝癌細胞中抗癌活性的分子機制。 / 我們的研究結果表明，用PP处理过的肝癌細胞，不僅顯著的显示出降低的體外腫瘤細胞的增殖和侵襲，也增強化療藥物順鉑的藥物敏感性。無論是口服和腹腔注射都顯著抑制移植免疫BALB / c裸小鼠的腫瘤生長。同时，PP也能在體外和體內实验顯著抑制PI3K/Akt信號通路在肝癌細胞。有趣的是，当过表达AKT时，Myr-AKT，PP的這種抑制癌细胞生长的效果有减弱的趋势，同时也反映在PP对癌细胞侵襲抑制的作用上。印跡和酶聯免疫吸附試驗結果表明，在PP处理过的肝癌細胞中，血管內皮生長因子（VEGF）的表達和分泌減少了。此外， rhVEGF的加入减弱了 PP对PI3K/Akt通路和肝癌细胞表型的抑製作用。 / 我們的研究結果表明，PP能在體外和體內试验中抑制肝癌細胞增殖，侵襲和耐藥性，通过抑制分泌血管內皮生長因子誘導PI3K/Akt的信號通路。這項研究表明了PP的潛在治療肝癌的治療意義。 / Liver cancer or hepatocellular carcinoma is one of the leading causes of cancer-related deaths. Conventional chemotherapies are limited by the development of drug resistance and various side effects. Because of its non-toxicity and potent biopharmacological activity, metabolites derived from mushrooms have received more attention in cancer therapy. Our previous studies have demonstrated the anti-cancer effects of polysaccharide-protein complexes derived from the Pleurotus mushrooms. The aim of this study was to investigate the underlying molecular mechanism of the anti-cancer activity of a hot water extract containing a polysaccharide-protein complex isolated from Pleurotus pulmonarius (PP) in liver cancer cells. / Our results indicated that exposure of liver cancer cells to PP not only significantly reduced the in vitro cancer cell proliferation and invasion but also enhanced the drug-sensitivity to the chemotherapeutic drug Cisplatin. Both oral administration and intraperitoneal injection of PP significantly inhibited the tumor growth in xenograft BALB/c nude mice. PP triggered a marked suppression of the PI3K/AKT signaling pathway in liver cancer cells in vitro and in vivo, and overexpression of the constitutively active form of AKT, Myr-AKT, abrogated this effect and the inhibited proliferation and invasion by PP. Both western blot and ELISA results showed that PP-treated liver cancer cells had reduced expression and secretion of vascular endothelial growth factor (VEGF). Addition of recombinant human VEGF attenuated the inhibitory effects of PP on PI3K/AKT pathway and the cancer phenotypes. / Our results demonstrated that PP suppressed the proliferation, invasion, and drug-resistance of liver cancer cells in vitro and in vivo, mediated by the inhibition of autocrine VEGF-induced PI3K/AKT signaling pathway. All these results suggest the potential therapeutic implication of PP in the treatment of human liver cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xu, Wenwen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 83-99). / Abstracts also in Chinese. / Thesis Committee --- p.i / English Abstract --- p.ii / Chinese Abstract --- p.iv / Acknowledgements --- p.v / List of Tables --- p.vi / List of Figures --- p.vii / Abbreviations --- p.x / Content page --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Mushroom as functional foods --- p.1 / Chapter 1.1.1 --- Introduction of functional food --- p.1 / Chapter 1.1.2 --- Functional food and cancer --- p.1 / Chapter 1.1.3 --- Edible Mushroom as functional food --- p.4 / Chapter 1.1.4 --- Pleurotus pulmonarius and its function --- p.7 / Chapter 1.2 --- Hepatocellular carcinoma --- p.9 / Chapter 1.2.1 --- Liver and hepatocellular carcinoma --- p.9 / Chapter 1.2.2 --- Carcinogenesis of liver cancer --- p.12 / Chapter 1.2.2.1 --- Hallmarks of cancer --- p.12 / Chapter 1.2.2.2 --- Cell cycle --- p.13 / Chapter 1.2.2.3 --- Apoptosis --- p.15 / Chapter 1.2.2.4 --- Angiogenesis --- p.17 / Chapter 1.2.2.5 --- Invasion and metastasis --- p.19 / Chapter 1.2.2.6 --- Drug resistance --- p.21 / Chapter 1.2.3 --- The role of PI3K/AKT pathway --- p.23 / Chapter 1.2.4 --- The role of growth factor Vascular endothelial growth factor (VEGF) in HCC --- p.25 / Chapter 1.3 --- Research objectives --- p.27 / Chapter 1.3.1 --- Hypothesis and objectives --- p.27 / Chapter 1.3.2 --- Experimental design --- p.28 / Chapter Chaper 2 --- Materials and Methods --- p.29 / Chapter 2.1 --- Materials --- p.29 / Chapter 2.1.1 --- Mushroom Pleurotus pulmonarius --- p.29 / Chapter 2.1.2 --- Drugs and cell lines --- p.29 / Chapter 2.1.3 --- Antibodies list --- p.30 / Chapter 2.1.4 --- Animal models --- p.32 / Chapter 2.2 --- Sample preparation and structure investigation --- p.32 / Chapter 2.2.1 --- Polysaccharide extraction from mushroom --- p.32 / Chapter 2.2.2 --- Endotoxin test --- p.32 / Chapter 2.2.3 --- Determination of monosaccharide profile by gas chromatography and mass spectrometry (GC/MS) --- p.33 / Chapter 2.2.3.1 --- Sample preparation for gas chromatography analysis --- p.33 / Chapter 2.2.3.1.1 --- Acid depolymerisation --- p.33 / Chapter 2.2.3.1.2 --- Neutral sugar derivatization --- p.33 / Chapter 2.2.3.1.3 --- External monosaccharide standard preparation --- p.34 / Chapter 2.2.3.2 --- Gas chromatography-mass spectrometry (GC/MS) --- p.34 / Chapter 2.2.4 --- Determination of total sugar by phenol-sulfuric acid method (Dubois, 1956) --- p.36 / Chapter 2.2.5 --- Determination of protein content by Lowry-Folin method (Lowry et al.,1951) --- p.37 / Chapter 2.3 --- Biological assays --- p.38 / Chapter 2.3.1 --- In vitro assays --- p.38 / Chapter 2.3.1.1 --- MTT assay --- p.38 / Chapter 2.3.1.2 --- Colony formation assay --- p.38 / Chapter 2.3.1.3 --- Plasmid transfection --- p.39 / Chapter 2.3.1.4 --- In vitro cell invasion assay --- p.39 / Chapter 2.3.1.5 --- Cell cycle analysis --- p.39 / Chapter 2.3.1.6 --- Western blot analysis --- p.40 / Chapter 2.3.1.7 --- VEGF ELISA Kit --- p.42 / Chapter 2.3.2 --- In vivo assays --- p.43 / Chapter 2.3.2.1 --- Tumor xenograft nude mouse model --- p.43 / Chapter 2.3.2.2 --- Immunohistochemistry --- p.45 / Chapter 2.3.2.3 --- H&Estaining --- p.45 / Chapter 2.3.3 --- Statistical analysis --- p.45 / Chapter Chaper 3 --- Results and discussion --- p.46 / Chapter 3.1 --- The yield and chemical characteristic of PP --- p.46 / Chapter 3.1.1 --- The yield of PP from mushroom Pleurotus pulmonarius --- p.46 / Chapter 3.1.2 --- Total carbohydrate and protein content --- p.47 / Chapter 3.1.3 --- Monosaccharide composition by GC-MS --- p.48 / Chapter 3.2 --- Toxicity of the PP water by Limulus amebocyte lysate (LAL) test --- p.48 / Chapter 3.2.1 --- Limulus amebocyte lysate (LAL) test --- p.48 / Chapter 3.3 --- Effects of PP on the proliferation of liver cancer cell lines --- p.50 / Chapter 3.3.1 --- MTT assay --- p.50 / Chapter 3.3.2 --- Colony-formation assay --- p.51 / Chapter 3.3.3 --- Cytotoxic effects of PP against normal liver cell --- p.52 / Chapter 3.3.4 --- The anti-proliferative effect of PP on other cancer types --- p.53 / Chapter 3.3.5 --- Cell cycle analysis by flow cytometry of PP treated liver cancer cells --- p.54 / Chapter 3.3.6 --- Protein expression by western blot analysis of P treated liver cancer cells --- p.56 / Chapter 3.4 --- Anti-cancer effect of PP on liver cancer cells through inactivation of PI3K/AKT signaling pathway --- p.57 / Chapter 3.4.1 --- Effect of PP on inactivation of PI3K/AKT pathway --- p.57 / Chapter 3.4.2 --- The abrogated inhibitory effect of PP on Huh7 with overexpression of AKT. --- p.59 / Chapter 3.4.3 --- The abrogated inhibitory effect of PP on PI3K/AKT signal pathway with overexpression of the constitutively active form of AKT, Myr-AKT --- p.60 / Chapter 3.5 --- Inhibition of VEGF expression and secretion by PP --- p.62 / Chapter 3.5.1 --- ELISA result of PP on VEGF secretion --- p.62 / Chapter 3.5.2 --- The attenuated inhibitory effect of PP on cell proliferation with addition of rhVEGF --- p.63 / Chapter 3.5.3 --- The attenuated inhibitory effect of PP on PI3K/AKT signal pathway with addition of rhVEGF --- p.64 / Chapter 3.6 --- Effect of PP on enhancing the chemosensitivity of liver cancer cells to Cisplatin --- p.66 / Chapter 3.6.1 --- Synergistic effect of PP with cisplatin (DDP) in liver cancer cells --- p.66 / Chapter 3.6.2 --- The abrogated drug-resistant effect by PP by overexpression of the constitutively active form of AKT, Myr-AKT --- p.67 / Chapter 3.6.3 --- The abrogated drug-resistant effect of PP with addition of rhVEGF --- p.68 / Chapter 3.7 --- The anti-invasive potential of PP on liver cancer cells. --- p.69 / Chapter 3.7.1 --- Boyden chamber assay --- p.69 / Chapter 3.7.2 --- The attenuated anti-invasive effect of PP on liver cancer cells with overexpression of constitutively activated AKT --- p.71 / Chapter 3.7.3 --- The attenuated anti-invasive effect of PP on liver cancer cells with addition of rhVEGF --- p.72 / Chapter 3.8 --- The anti-tumor effect of PP in vivo --- p.73 / Chapter 3.8.1 --- The anti-tumor effect of PP by using tumor xenograft model --- p.73 / Chapter 3.8.2 --- Body weight of nude mice treated with PP --- p.75 / Chapter 3.8.3 --- Harmful effect of PP on nude mice --- p.76 / Chapter 3.8.4 --- Immunohistochemist analysis of mice tumor xenograft treated with PP --- p.77 / Chapter 3.8.5 --- Western blot anaylysis using the tumor tissues harvested from mice xenograftes treated with PP --- p.78 / Chapter Chapter 4 --- Conclusion and future Plan --- p.81 / Reference --- p.83 / Related Publication List --- p.100

BTBD7 is a newly identified candidate gene for HCC using a high-throughput cDNA/EST microassay. This gene encodes for a protein of 410 amino acid residues. This protein was previously named as the function unknown protein 1 (FUP1) because the biological function of this protein was unknown at that time. Bioinformatics analysis revealed that this protein contains two bric-a-brac, tramtrack, broad-complex (BTB) domains located at amino acid positions 143 to 230 and 274 to 342. In order to reflect its structure and functions, and to be consistent with the GeneBank database (Accession No. NM_018167), we rename it as BTBD7 (BTB domain containing 7). / In conclusion, our study demonstrated that BTBD7 is a novel oncogene, which is associated with hepatocellular carcinoma and is essential for the inhibition of cell growth and tumorigenesis. To our knowledge, BTBD7 is the first identified regulator of p16INK4A through inhibiting the promoter activity of p16INK4A. BTBD7 may thus serve as a new tumor marker or as a potential target of treating hepatocellular carcinoma. / In previous studies, the expression of BTBD7 was shown to be tissue-specific as demonstrated by Northern blot. Furthermore, we collected 18-paired HCC samples to further reveal the correlation of BTBD7 gene expression profiles with tumorigenesis. Our data showed that BTBD7 was significantly elevated in 44.4% of the HCC samples. Compared with immortalized hepatocyte cell lines MIHA or LO2, both mRNA level and protein level of BTBD7 were also elevated in the hepatoma cell lines HepG2, BEL7404, Hep3B and Huh7. This gave a due that the expression of BTBD7 may be correlated with carcinogenesis of liver cells. / In the present study, the function of BTBD7 was investigated. We used RNAi approach to silence BTBD7. Compared with the control, siBTBD7 induced cell cycle arrest at G1 phase and later caused obvious cell death. The cell death was further demonstrated to be apoptosis through activation of caspase 3. Furthermore, we carried out candidate gene search using knockdown of BTBD7. The mRNA level of tumor suppresser p16INK4A was upregulated and hTERT was downregulated in BTBD7 knocked down cells. The other key genes involved in cell growth, cell cycle control, cell death and survival (c-myc, c-fos, c-jun, p21CIP1, p27KIP1, p53, Survivin, E2F, NF-kappaB, Bax, p14ARF, p16INK4A and hTERT) did not respond to the reduced BTBD7 levels. On the other hand, double knockdown of p16INK4A and BTBD7 markedly reduced the effects of cell cycle arrest and the death ratio caused by dysfunction of BTBD7 or overexpression of p16INK4A, suggesting that p16 INK4A is a downstream target of BTBD7. We further adopted a dominant negative approach to confirm these results. / Liu, Zheng. / Advisers: C. H. K. Cheng; Mingliang He. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3449. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 120-161). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

"Epigenetic" refers to a heritable change in the gene expression pattern that is not mediated by any alterations in the primary nucleotide sequence of a gene in the genome. This change involves methylation of DNA in the gene promoter regions, modification of histone residues and chromatin remodeling. Among them, methylation of DNA promoter region is an essential step in epigenetic gene silencing and is known to be closely related to carcinogenesis and cancer progression. / Our preliminary study on effect of treatments of some potential anti-cancer drug candidates, namely Pheophorbide A (Pa), Pa combining with photodynamic therapy, Polyphyllin D (designated as HK-18), and its derivative designated as HK-27 on human breast cancer cell lines MCF-7 and MDA-MB-231 showed that the promoter methylation of CDH1 was decreased in response to treatments of Pa, HK-18, and HK-27 in MDA-MB-231 cells. / The aim of this study was to explore whether any methylation of DNA promoters mechanism is involved in drug resistance of a doxorubicin-induced human multidrug resistant hepatocellular carcinoma sub-linage R-HepG2 which was established from the doxorubicin sensitive HepG2 cell line in our laboratory. In this project, it was observed that the DNA promoter methylations of ESR1, Rassf2A, CDH1 and MDR1 in R-HepG2 were higher than those in HepG2 cells respectively by methylation specific polymerase chain reaction method. Bisulfite sequencing showed that the total 32 CpGs of CDH1 promoter region in R-HepG2 cells were hypermethylated while they were hypomethylated in HepG2 cells. CDH1 is the encoding gene of E-cadherin. The promoter hypermethylation induced CDH1 silencing in R-HepG2 cells was confirmed by reverse transcription polymerase chain reaction and Western blotting that CDH1 transcription and E-cadherin expression were maintained in HepG2 cells but both were lost in R-HepG2 cells. RT-PCR of 10 multidrug resistant related genes revealed that transcription of MDR1 was obviously increased in R-HepG2 cells, transcription of MRP1 and MRP5 were slightly increased in R-HepG2 cells, transcription of MRP6 and BCRP were slightly decreased in R-HepG2 cells comparing to those in the parental HepG2 cells. This result suggests that up-regulation of P-glycoprotein expression which is the protein product of MDR1 may be one of the major causes of multidrug resistance in R-HepG2 cells. Transient transfection of CDH1 cDNA increased the CDH1 transcription and E-cadherin expression in R-HepG2 cells. I also found that the CDH1 transfected R-HepG2-CDH1 cells showed increased amount of doxorubicin uptake, increased apoptotic population of cells exposed to doxorubicin, suppressed cell migration, and decreased P-glycoprotein expression comparing to those in R-HepG2 cells. It was also found that the transcription levels of SNAI2, TWIST1, ASNA1 and FYN were obviously higher in R-HepG2 cells than those in HepG2 cells. The transcription of FYN and TWIST1 were obviously decreased in CDH1 cDNA transfected R-HepG2-CDH1 cells which displayed a negative correlation with the transcription level of CDH1 and these results imply a suppressive role of CDH1 in regulating these genes which were involved in cancer metastasis and multidrug resistance. / Jiang, Lei. / Adviser: Kwok-Pui, Fang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 144-171). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lam Mei Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 172-181). / Abstracts in English and Chinese. / Abstract --- p.i / 論文撮要 --- p.iv / Acknowledgement --- p.vii / Table of contents --- p.viii / List of tables --- p.xi / List of figures --- p.xii / Abbreviations --- p.xvii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- The facilitative glucose transporter family --- p.2 / Chapter 1.2 --- Overexpression of glucose transporters in tumor cells --- p.5 / Chapter 1.3 --- Antisense strategy --- p.8 / Chapter 1.3.1 --- Modifications of oligonucleotides --- p.9 / Chapter 1.3.2 --- Delivery system for oligonucleotides --- p.13 / Chapter 1.3.3 --- Factors influencing antisense activity --- p.16 / Chapter 1.3.4 --- Mechanism of action of antisense oligonucleotides --- p.17 / Chapter 1.3.5 --- Clinical trials of antisense treatment --- p.21 / Chapter 1.4 --- Objective of present study --- p.23 / Chapter Chapter 2: --- Materials and Methods --- p.24 / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Reagents and buffers --- p.25 / Chapter 2.1.2 --- Reagents for Western blot analysis --- p.26 / Chapter 2.1.3 --- Culture medium --- p.28 / Chapter 2.1.4 --- Chemicals --- p.29 / Chapter 2.1.5 --- Culture of cells --- p.31 / Chapter 2.1.5.1 --- Differentiated Human Hepatoblastoma cell line (HepG2) --- p.31 / Chapter 2.1.5.2 --- "Multi-drug resistant hepatoma cell line, R-HepG2 cells" --- p.32 / Chapter 2.1.6 --- Animal Studies --- p.33 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- In vitro studies --- p.34 / Chapter 2.2.1.1 --- Design of oligonucleotide sequence --- p.34 / Chapter 2.2.1.2 --- Transfection --- p.35 / Chapter 2.2.1.3 --- MTT assay --- p.36 / Chapter 2.2.1.4 --- Flow cytometry --- p.37 / Chapter 2.2.1.5 --- H-thymidine incorporation assay --- p.45 / Chapter 2.2.1.6 --- 2-Deoxy-D-[l-3H] glucose uptake assay --- p.46 / Chapter 2.2.1.7 --- Adenosine-5'-triphosphate (ATP) assay --- p.47 / Chapter 2.2.1.8 --- Western blot analysis --- p.50 / Chapter 2.2.2 --- In vivo studies --- p.55 / Chapter 2.2.2.1 --- Animal studies --- p.55 / Chapter (i) --- Lactate dehydrogenase (LDH) assay --- p.58 / Chapter (ii) --- Creatine kinase (CK) assay --- p.60 / Chapter (iii) --- Aspartate transaminase (AST) assay --- p.62 / Chapter (iv) --- Alanine transaminase (ALT) assay --- p.64 / Chapter Chapter 3: --- Results --- p.67 / Chapter 3.1 --- In vitro studies --- p.68 / Chapter 3.1.1 --- Characteristics of the multi-drug resistant cell line (R-HepG2) developed in our laboratory --- p.68 / Chapter 3.1.2 --- Effect of lipofectin on cell viability --- p.77 / Chapter 3.1.3 --- Cellular uptake of antisense oligonucleotide --- p.82 / Chapter 3.1.4 --- Effect of Glut 2 antisense oligonucleotides on human hepatoma HepG2 and its multidrug resistant (R-HepG2) cells by MTT assay --- p.87 / Chapter 3.1.5 --- Suppression of Glut 2 protein expression by antisense oligonucleotides as revealed by Western blot analysis --- p.96 / Chapter 3.1.6 --- Uptake of glucose in HepG2 and R-HepG2 after Glut 2 antisense treatment --- p.100 / Chapter 3.1.7 --- ATP content in HepG2 and R-HepG2 was lowered after treating the cells with antisense oligonucleotides --- p.108 / Chapter 3.1.8 --- Antisense oligonucleotides against Glut 2 exhibited antiproliferative effect on HepG2 and R-HepG2 cells --- p.117 / Chapter 3.1.9 --- Change in cell cycle pattern after antisense treatment --- p.125 / Chapter 3.1.10 --- Glut 2 antisense oligonucleotides did not induce apoptosis --- p.131 / Chapter 3.2 --- In vivo studies --- p.135 / Chapter 3.2.1 --- Effect of antisense oligonucleotides on the tumor weight in nude mice bearing HepG2 cells or R-HepG2 cells --- p.135 / Chapter 3.2.2 --- Assessment of any side effect of antisense drug done on normal tissues of nude mice --- p.139 / Chapter 3.2.2.1 --- Treatment on tumor bearing nude mice with Glut 2 antisense or sense oligonucleotides did not cause myocardial injury --- p.139 / Chapter 3.2.2.2 --- Liver injury was not detected in Glut 2 antisense or sense oligonucleotides treated tumor bearing nude mice --- p.147 / Chapter Chapter 4: --- Discussion --- p.151 / Chapter 4.1 --- In vitro study of the effect of antisense oligonucleotides against Glut 2 on HepG2 and its multi-drug resistant R-HepG2 cell lines --- p.152 / Chapter 4.1.1 --- Design of antisense oligonucleotides against Glut 2 --- p.154 / Chapter 4.1.2 --- Conditions for antisense inhibition by oligonucleotides --- p.155 / Chapter 4.1.3 --- Biological effects of antisense oligonucleotides --- p.158 / Chapter 4.2 --- In vivo study of the effect of antisense oligonucleotides against Glut 2 on HepG2 or R-HepG2 cells bearing nude mice --- p.166 / Chapter 4.2.1 --- Effect of Glut 2 antisense oligonucleotides on tumor weight --- p.167 / Chapter 4.2.2 --- In vivo side effects of oligonucleotides --- p.168 / Chapter 4.3 --- Conclusion --- p.169 / Bibliography --- p.172