Cholesterol is an essential component of the cell, but excessive blood levels are a major risk factor for the development of atherosclerotic plaques that can lead to heart disease and stroke, the foremost cause of premature death in Western societies. Policosanol, a mixture of very long chain alcohols derived from sugarcane, has gained considerable attention among the public as safe and effective means to reduce blood cholesterol levels, a belief based on some early clinical studies. My research investigates one possible mechanism by which policosanol might decrease blood cholesterol levels: the inhibition of cholesterol synthesis in the liver. Previous studies with cultured hepatoma cells have indicated that policosanol suppresses HMG-CoA reductase activity, the regulatory step in cholesterol synthesis, by activation of AMP-kinase, which then inactivates HMG-CoA reductase by phosphorylation. My studies have confirmed this activation of AMP-kinase both in hepatoma cells and in whole animals after intragastric administration of policosanol. The present studies were also undertaken to identify the upstream signaling mechanism by which policosanol activates AMP-kinase. Treatment of rat hepatoma cells with policosanol increased the amount of phosphorylated CaMKK, which can directly activate AMP-kinase, but had only a small effect on LKB1, the principal activator of AMP-kinase. Intragastric administration to mice similarly activated CaMKK, but not LKB1, in the liver. To determine if metabolism of policosanol was necessary for activation of AMP-kinase, siRNA-mediated suppression of fatty aldehyde dehydrogenase, fatty acyl CoA synthase-4, or β-ketothiolase in hepatoma cells prevented the phosphorylation of AMP-kinase and HMG-CoA reductase by policosanol, indicating that metabolism of these very long chain alcohols to fatty acids and subsequent peroxisomal β-oxidation is necessary for the suppression of cholesterol synthesis. As the principal product of fatty acid -oxidation is acetyl-CoA, further studies demonstrated that addition of acetate to cells similarly activated AMP-kinase and inactivated HMG-CoA reductase. This finding argues that the activation of AMP-kinase by policosanol results from the generation of excess acetyl-CoA via peroxisomal metabolism. Finally, although the intestine is a significant source of circulating cholesterol, policosanol was unable to activate AMP-kinase in the small intestine. These findings open new perspectives for the control of cholesterol synthesis by activators of AMP-kinase.

Eleven known compounds were separated from Oldenlandia diffusa using the bioassay-guided methods. Among which, heptatriacontane and stearic acid (SA) were isolated from this herb for the first time. The anti-proliferative activities of ursolic acid (UA) and SA, as well as the anti-proliferative and immunomodulatory activities of quercetin, kaempferol, quercetin-3-O-D-glucoside, kaempferol-3-O-D-glucoside and kaempferol-3-O-D-galactoside, are responsible for the anti-hepatomatic effect of OD, to which UA might be the major contributor due to relatively high content in OD and potent cytotoxicity. / In conclusion, our findings provided a better elucidation on phytochemical basis responsible for the anti-cancer activities of OD and AU, and also suggested the potential of UA, SB and SD as new chemotherapeutic agents for the treatment of liver cancer in further studies. / Mechanistic study indicated that anti-proliferative effects of SB and SD due to induction of apoptosis on both HepG2 and R-HepG2 cells were established by sub-G1 accumulation in cell cycle profile and cell population with PS externalization, which were confirmed by activation of apoptosis mediators PARP and caspase-3. The induction of apoptosis was suggested to be mediated by both extrinsic and intrinsic pathways, as evidenced by activation of caspase-8 and -9, up-regulation of Bcl-XS, dysfunction of mitochondria and release of cytochrome c during SB and SD treatment. Besides, Bcl-2 and Bax expression levels were notably different on SB/SD-treated HepG2 and R-HepG2 cells, which implied that Bcl-2 and Bax might play a role in SB and SD modulation of drug resistance on R-HepG2 cells. / Motivated by the serious health hazard worldwide caused by hepatoma and side effects of chemotherapeutic agents in clinical treatment, we have initiated a research project to isolate and characterize bioactive compounds from Oldenlandia diffusa (OD) and Androsace umbellata (AU) as well as to study the molecular mechanisms of their anti-proliferative effects on human hepatoma cells. / On the other hand, phytochemical study of Androsace umbellata led to isolation of two novel triterpenoid sapogenins and five known compounds (3-O-D-glucosyl-(1→2)-L-arabinosyl cyclamiretin A, primulanin, saxifragifolin B, saxifragifolin C and saxifragifolin D). Their anti-tumor effects were firstly reported here, where saxifragifolin B (SB) and saxifragifolin (SD) showed the most potent cytotoxicities on human hepatoma cells. Structure-activity relationship study revealed that introduction of glucosyl moiety might be useful for the enhancement of cytotoxicity of this chemotype. / The action mechanism of UA has been intensively investigated. Our results showed that UA was not a substrate of p-glycoprotein, and it could bypass multidrug resistance of R-HepG2 cells. Furthermore, UA treatment also resulted in apoptotic cell death which was indicated by cell morphology observation, cell cycle analysis, DNA fragmentation and Annexin V-FITC/PI double staining assay. UA-induced apoptosis was associated with the extrinsic (death receptor-mediated) pathway, which was suggested by increase of FasL expression, activation of caspase-8 and caspase-3 as well as cleavage of PARP. Besides, changes implying the intrinsic (mitochondria-mediated) apoptotic pathway, including up-regulation of p53 and Bax, down-regulation of Bcl-2, cleavage of Bid, collapse of Deltapsi m, leakage of cytochrome c and AIF as well as activation of caspase-9, were also observed on R-HepG2 cells after UA treatment. Moreover, elevation of cytosolic calcium concentration, generation of reactive oxygen species and activation of MAPKs pathway were involved in UA-induced apoptosis. Proteomic analysis exhibited significant changes in the expression level of twelve proteins which were involved in tumor cell proliferation, invasion and apoptosis. / Zhang, Dongmei. / "September 2007." / Adviser: Kwok-Pui Fung. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4744. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 239-263). / 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.

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

Das Ziel dieser Arbeit war die Darstellung der Einflüsse verschiedener Interleukine auf die HDGF-Expression in verschiedenen Kolonzelllinien. HDGF stellt einen Wachstumsfaktor dar, der nicht nur physiologisch bei der Entwicklung einiger Gewebe wie der Niere, der Leber und des Darms von Be-deutung ist, sondern auch eine wichtige Rolle in der Karzinogenese verschie-dener Tumoren spielt. Hierzu zählen unter anderem das hepatozelluläre Karzi-nom, das NSCLC und das Melanom. Von besonderer Relevanz ist seine Rolle in der Pathogenese des kolorektalen Karzinoms. Die verwendeten Interleukine (1beta, 4, 5, 8 und 13) zeigen sowohl inhibierende als auch fördernde Eigenschaften in Bezug auf die Karzinogenese von kolorektalen Tumoren. Dies steht im Einklang mit früheren Resultaten der Literatur. Die vier verschiedenen Zelllinien, eine Adenomzelllinie, zwei Adenokarzinomzelllinien sowie eine Zelllinie aus Lymphknotenmetastasenzellen wurden mit den verschiedenen Interleukinen inkubiert und mittels REAL TIME-RT-PCR analysiert. Die Ergebnisdarstellung in Blockdiagrammen zeigt semiquantitativ die relative HDGF-Expression. So lassen sich Aussagen über Anstieg oder Abfall der Expression durch den Einfluss der verschiedenen Interleukine machen. Die hier gezeigten Ergebnisse lassen, wie auch schon teilweise in der Literatur beschrieben, für alle Interleukine außer für IL 1beta, sowohl hemmende als auch tumorunterstützende Effekte beobachten. Interleukin 1beta zeigt in Kongruenz der vorbeschriebenen Studien, im Gegensatz zu den anderen Zytokinen, in allen Zelllinien tumorfördernde Eigenschaften. Für IL 4 ist zunächst in den Adenomzellen ein antitumoröser Effekt zu erkennen, dieser kehrt sich in der Metastasenzelllinie in eine förderndene Wirkung um. In den Adenokarzinomzelllinien sind weder eindeutige suppressive noch unterstützende Wirkungen zu verzeichnen. Über einen Zusammenhang zwischen dem Grad der malignen Transformation und unterschiedlichem Ansprechen auf IL 4 lässt sich jedoch bisher nur spekulieren. Für IL 5 ist ein ähnliches Verhalten zu beobachten. Eine anfängliche inhibitorische Wirkung auf die HDGF-Expression in den Adenomzellen sowie Adenokarzinomzellen kehrt sich in der Metastasenzelllinie in den gegenteiligen Effekt um. Auch hier lässt sich eine Umkehr der ausgelösten Effekte mit fortschreitender maligner Transformation vermuten. IL 8 zeigt kongruente Effekte zu IL 4 und IL 5, jedoch lassen sich für IL 8 in der Literatur bisher nur tumorunterstützende Wirkungen finden. Hier lässt sich in den Adenomzellen eine suppressive Wirkung verzeichnen, wohingegen in den beiden Adenokarzinomzelllinien fördernde Effekte beobachtet werden. In der Metastasenzelllinie lassen sich jedoch weder positive noch negative Auswirkungen feststellen. Des Weiteren spiegeln auch die Ergebnisse des Einflusses von IL 13 die Vielgestaltigkeit der Wirkweisen dieses Interleukins dar, mit tumorhemmenden Effekten in den Adenom- sowie Metastasenzellen und fördernder Wirkung in den HT29-Zellen. Über die genauen Mechanismen, inwiefern ein Interleukin die Expression von HDGF hochreguliert oder supprimiert, kann zum momentanen Zeitpunkt nur spekuliert werden. Es kann jedoch vermutet werden, dass ein gewisser Zu-sammenhang zwischen dem Grad der malignen Transformation und der Wirk-weise der Interleukine existiert. Entscheidend sind hier sicherlich klonal erwor-bene Alterationen einzelner Signalkaskaden. Festzuhalten ist zum einen, dass bis auf IL 1beta für alle Zytokine der Einfluss auf HDGF vom jeweiligen Zellsystem abhängt. Diese Ergebnisse machen eine Schlüsselrolle von HDGF eher unwahrscheinlich, vielmehr scheint seine Regulation hier in teilweise komplexe Regulationsmechanismen mit eingebunden zu sein. Dass diese Alterationen möglicherweise auch im Rahmen der Karzinogenese bzw. der Akquise der Metastasierungsfähigkeit entstehen könnten, zeigen die teilweise bestehenden Unterschiede zwischen der verwendeten Adenomzelllinie und den Karzinomzelllinien respektive zwischen Karzinom- und Metastasenzelllinie. Die beschriebenen Ergebnisse geben einen Anhaltspunkt, in welche Richtung die einzelnen Interleukine wirken, zumindest in wie weit hier ein Einfluß auf die Transkription von HDGF als Surrogatmarker der Mitogenese erfolgt. Um die Komplexität und Vielfalt der Effekte von Interleukinen in Bezug zu Tumorstadium, Invasivität sowie Metastasierungsfähigkeit in Einklang zu bringen, bedarf es jedoch weiterführender Studien. Es lies sich zeigen, dass die angewendeten Interleukine generell Einfluss auf die Expressionshöhe von HDGF in verschiedenen Kolonzelllinien haben und als exogene Faktoren in die Regulation eingreifen können. Dies könnte ein weiterer Ansatz zur Etablierung immunmodulatorischer Therapieoptionen in soliden Neoplasien in der Zukunft sein. / Hepatoma-derived growth factor (HDGF) is a growth factor which plays a role in physiological development of some organ tissues and in the carcinogensis of a few tumors like colorectal cancer, hepatocellular cancer, NSCLC. For this study especially the role of HDGF with regard to colorectal cancer is important. The main focus is set on the influences that different interleukins have on the expression of HDGF in different gut-tissues and colon cancer-tissues. To this end, five interleukins (1beta, 4, 5, 8 and 13) with different effects on the carcinogenesis of colorectal cancer (inhibition/promotion) were investigated. It is known from the literature that all five interleukins show different behavior. Four cell lines–one adenoma cell line, two different cell lines of adenoma carcinoma of intestine, one cell line of lymph node metastase of adenoma carcinoma of intestine–were incubated with the five interleukins and analyzed with Real Time-RT-PCR. This method allows for an observation of changes of the relative HDGF-expression. The results show that all interleukins have an influence on the HDGF-expression. The most pronounced effects are observed in dependency of the concentration of the interleukins under investigation. Interleukin 1beta exhibits throughout a tumor supporting behavior. In contrast to this, all other interleukins showed that their influence depends on the probed cell line. This suggests that there is a connection between the effect of the interleukin and the degree of malign differentiation. This complex interplay manifests itself, for instance, in a totally inversion of the effects, with depression of the HDGF-expression in the cell line of adenoma and a promotion in the cell line of metastasis in some experimental runs. These findings are partly concurrent with known properties described in the literature related to colorectal cancer. ln summary the results show that interleukins as exogenous factor can influence the HDGF-expression. However, the data do not allow to derive final statements on the mechanism of regulation. It is imaginable that an alteration of the signal pathways, presumably acquired clonal, determine whether an interleukin shows effects of inhibition or promotion on the cell lines. Therefore, further studies are required to clarify in how far interleukins influence the HDGF-expression.

Metabolic alterations in hepatocellular carcinoma (HCC) are fundamental for the development of diagnostic screening and therapeutic intervention since energy metabolism plays a central role in differentiated hepatocytes. In HCC research, hepatoma cell lines (HCLs) like HepG2 and Huh7 cells are still the gold standard. In this study, we characterized the metabolic profiles of primary human hepatoma cells (PHCs), HCLs and primary human hepatocytes (PHHs) to determine their differentiation states. PHCs and PHHs (HCC-PHHs) were isolated from surgical specimens of HCC patients and their energy metabolism was compared to PHHs from non-HCC patients and the HepG2 and Huh7 cells at different levels (transcript, protein, function). Our analyses showed successful isolation of PHCs with a purity of 50–73% (CK18+). The transcript data revealed that changes in mRNA expression levels had already occurred in HCC-PHHs. While many genes were overexpressed in PHCs and HCC-PHHs, the changes were mostly not translated to the protein level. Downregulated metabolic key players of PHCs revealed a correlation with malign transformation and were predominantly pronounced in multilocular HCC. Therefore, HCLs failed to reflect these expression patterns of PHCs at the transcript and protein levels. The metabolic characteristics of PHCs are closer to those of HCC-PHHs than to HCLs. This should be taken into account for future optimized tumor metabolism research.

Although human liver tumor cells have reduced metabolic functions as compared to primary human hepatocytes (PHH) they are widely used for pre-screening tests of drug metabolism and toxicity. The aim of the present study was to modify liver cancer cell lines in order to improve their drug-metabolizing activities towards PHH. It is well-known that epigenetics is strongly modified in tumor cells and that epigenetic regulators influence the expression and function of Cytochrome P450 (CYP) enzymes through altering crucial transcription factors responsible for drug-metabolizing enzymes. Therefore, we screened the epigenetic status of four different liver cancer cell lines (Huh7, HLE, HepG2 and AKN-1) which were reported to have metabolizing drug activities. Our results showed that HepG2 cells demonstrated the highest similarity compared to PHH. Thus, we modified the epigenetic status of HepG2 cells towards ‘normal’ liver cells by 5-Azacytidine (5-AZA) and Vitamin C exposure. Then, mRNA expression of Epithelial-mesenchymal transition (EMT) marker SNAIL and CYP enzymes were measured by PCR and determinate specific drug metabolites, associated with CYP enzymes by LC/MS. Our results demonstrated an epigenetic shift in HepG2 cells towards PHH after exposure to 5-AZA and Vitamin C which resulted in a higher expression and activity of specific drug metabolizing CYP enzymes. Finally, we observed that 5-AZA and Vitamin C led to an increased expression of Hepatocyte nuclear factor 4α (HNF4α) and E-Cadherin and a significant down regulation of Snail1 (SNAIL), the key transcriptional repressor of E-Cadherin. Our study shows, that certain phase I genes and their enzyme activities are increased by epigenetic modification in HepG2 cells with a concomitant reduction of EMT marker gene SNAIL. The enhancing of liver specific functions in hepatoma cells using epigenetic modifiers opens new opportunities for the usage of cell lines as a potential liver in vitro model for drug testing and development.

Grace S.N. Lau. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 335-362). / List of Abbreviations --- p.i / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction and Study Objectives / Chapter 1.1 --- Metabolic activation - role in drug toxicity and carcinogenesis --- p.5 / Chapter 1.2 --- Hepatocellular carcinoma --- p.12 / Chapter 1.2.1 --- Epidemiology --- p.12 / Chapter 1.2.2 --- Aetiological factors --- p.17 / Chapter 1.2.2.1 --- Hepatitis B virus infection --- p.17 / Chapter 1.2.2.2 --- Cirrhosis --- p.24 / Chapter 1.2.2.3 --- Aflatoxins --- p.25 / Chapter 1.2.2.4 --- Other factors --- p.26 / Chapter 1.2.2.5 --- Summary --- p.29 / Chapter 1.3 --- Study objectives --- p.30 / Chapter Chapter 2 --- The Metabolism of Paracetamol in Healthy Subjects andin Patients with Liver Disease and Hepatocellular Carcinoma / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.1.1. --- History of paracetamol --- p.34 / Chapter 2.1.2 --- Pharmacology of paracetamol --- p.37 / Chapter 2.1.3 --- "Absorption, Distribution, Metabolism and Excretion" --- p.38 / Chapter 2.1.3.1 --- Absorption --- p.38 / Chapter 2.1.3.2 --- Distribution --- p.41 / Chapter 2.1.3.3 --- Metabolism --- p.42 / Chapter 2.1.3.4 --- Excretion --- p.57 / Chapter 2.1.4 --- Toxicity and Overdosage --- p.59 / Chapter 2.2 --- Estimation of paracetamol and its metabolites in plasma and urine by high performance liquid chromatography --- p.72 / Chapter 2.2.1 --- Introduction --- p.72 / Chapter 2.2.2 --- Analytical method --- p.76 / Chapter 2.2.2.1 --- Materials --- p.76 / Chapter 2.2.2.2 --- Instrumentation --- p.77 / Chapter 2.2.2.3 --- Collection and storage of samples --- p.79 / Chapter 2.2.2.4 --- Chromatographic conditions --- p.79 / Chapter 2.2.3 --- Urine assay --- p.79 / Chapter 2.2.3.1 --- Preparation of standards and test samples for urine assay --- p.79 / Chapter 2.2.3.2 --- Calculation of results for urine assay --- p.80 / Chapter 2.2.3.3 --- Results of urine assay --- p.81 / Chapter 2.2.3.4 --- Validation of urine assay --- p.81 / Chapter 2.2.4 --- Plasma assay --- p.83 / Chapter 2.2.4.1 --- Preparation of standards and test samples for plasma assay --- p.83 / Chapter 2.2.4.2 --- Calculation of results for plasma assay --- p.91 / Chapter 2.2.4.3 --- Results of plasma assay --- p.91 / Chapter 2.2.4.4 --- Validation of plasma assay --- p.93 / Chapter 2.2.5 --- Summary --- p.99 / Chapter 2.3 --- The pharmacokinetics of paracetamol in healthy subjects --- p.103 / Chapter 2.3.1 --- Introduction --- p.103 / Chapter 2.3.2 --- Study protocol --- p.103 / Chapter 2.3.3 --- Methods --- p.103 / Chapter 2.3.3.1 --- Subjects --- p.103 / Chapter 2.3.3.2 --- Drug administration and sampling --- p.104 / Chapter 2.3.3.3 --- Drug analysis --- p.108 / Chapter 2.3.3.4 --- Calculations --- p.108 / Chapter 2.3.4 --- Pharmacokinetic analysis --- p.109 / Chapter 2.3.5 --- Statistical analysis --- p.113 / Chapter 2.3.6 --- Results --- p.114 / Chapter 2.3.6.1 --- Plasma Results --- p.114 / Chapter 2.3.6.2 --- Urine Results --- p.118 / Chapter 2.3.6.3 --- Pharmacokinetic Results --- p.125 / Chapter 2.3.6.4 --- Statistical Results --- p.134 / Chapter 2.3.7 --- Discussion --- p.145 / Chapter 2.4 --- "The pharmacokinetics of paracetamol in healthy subjects, patients with liver disease and hepatocellular carcinoma" --- p.155 / Chapter 2.4.1 --- Introduction --- p.155 / Chapter 2.4.2 --- Study protocol --- p.156 / Chapter 2.4.3 --- Methods --- p.156 / Chapter 2.4.3.1 --- Subjects --- p.156 / Chapter 2.4.3.2 --- Drug administration and sampling --- p.157 / Chapter 2.4.3.3 --- Drug analysis --- p.160 / Chapter 2.4.3.4 --- Calculations --- p.160 / Chapter 2.4.4 --- Pharmacokinetic analysis --- p.161 / Chapter 2.4.6 --- Results --- p.162 / Chapter 2.4.6.1 --- Plasma Results --- p.162 / Chapter 2.4.6.2 --- Urine Results --- p.162 / Chapter 2.4.6.3 --- Pharmacokinetic Results --- p.179 / Chapter 2.4.7 --- Discussion --- p.194 / Chapter 2.4.8 --- Summary --- p.203 / Chapter Chapter 3 --- Metabolic Activation of Aflatoxin B1 in Healthy Subjects and in Patients with Liver Disease and Hepatocellular Carcinoma / Chapter 3.1 --- General introduction --- p.206 / Chapter 3.1.1 --- Chemical structures and properties --- p.207 / Chapter 3.1.2 --- Contamination of food by aflatoxins --- p.209 / Chapter 3.1.3 --- Metabolism of aflatoxins --- p.210 / Chapter 3.1.4 --- Human diseases possibly related to exposure to aflatoxins --- p.226 / Chapter 3.1.4.1 --- Acute aflatoxicosis --- p.226 / Chapter 3.1.4.2 --- Reye's syndrome --- p.227 / Chapter 3.1.4.3 --- Kwashiorkor --- p.228 / Chapter 3.1.4.4 --- Impaired immune function --- p.229 / Chapter 3.1.4.5 --- Hepatocellular carcinoma --- p.230 / Chapter 3.1.5 --- Biochemical and molecular epidemiology of aflatoxins --- p.232 / Chapter 3.2 --- Development of an ELISA method to monitor AFB1 exposure in human serum --- p.237 / Chapter 3.2.1 --- Introduction --- p.237 / Chapter 3.2.2 --- Preparation of all the components necessary for analysing AFB1-albumin adducts by ELISA --- p.243 / Chapter 3.2.2.1 --- Materials --- p.243 / Chapter 3.2.2.2 --- Preparation of rabbit AFB1 antiserum --- p.244 / Chapter 3.2.2.3 --- Preparation of the rat monoclonal antibody --- p.244 / Chapter 3.2.2.4 --- Concentration of cell culture supernatant by ammonium sulphate precipitation --- p.246 / Chapter 3.2.2.5 --- Preparation of the BSA-AFB1 conjugate --- p.248 / Chapter 3.2.2.6 --- Preparation of the immunoaffinity gel --- p.250 / Chapter 3.2.2.7 --- Preparation of the ELISA plates --- p.251 / Chapter 3.2.3 --- General procedures used in the analysis of AFB1- albumin adducts --- p.252 / Chapter 3.2.3.1 --- Competitive ELISA binding assay --- p.253 / Chapter 3.2.3.2 --- Sep-pak C18 cartridge --- p.254 / Chapter 3.2.3.3 --- Immunoaffinity column --- p.255 / Chapter 3.2.3.4 --- Evaporation process --- p.255 / Chapter 3.2.3.5 --- HPLC --- p.256 / Chapter 3.2.3.6 --- Radioactive counting --- p.256 / Chapter 3.2.3.7 --- Albumin isolation --- p.257 / Chapter 3.2.3.8 --- Digestion of albumin --- p.257 / Chapter 3.2.3.9 --- Animal procedures --- p.258 / Chapter 3.2.4 --- Validations --- p.259 / Chapter 3.2.4.1 --- Analysis of standard AFB1 and AFB1- lysine in ELISA --- p.259 / Chapter 3 2.4.2 --- Optimisation of antiserum dilution and concentration of coating antigenin ELISA --- p.259 / Chapter 3 2.4.3 --- Elution characteristics and capacity of the immunoaffinity column --- p.261 / Chapter 3.2.4.4 --- Comparison of immunoaffinity gels prepared with different affinity gels --- p.261 / Chapter 3.2.4.5 --- Immunoaffinity column experiment of AFB1-lysine --- p.263 / Chapter 3.2.4.6 --- HPLC Analysis of fractions from immunoaffinity column --- p.263 / Chapter 3.2.4.8 --- HPLC analysis of fractions from Sep- Pak C18 cartridge --- p.264 / Chapter 3.2.4.9 --- Digestion of serum albumin by proteinase K --- p.264 / Chapter 3.2.4.10 --- Effect of ethanol in samples to be loaded onto Sep-Pak C18 cartridge --- p.265 / Chapter 3.2.4.11 --- Effect of drying in a vacuum concentrator on recovery of radioactivity of 3H-AFB1 --- p.266 / Chapter 3.2.4.12 --- Evaluation of the overall procedure for the analysis of serum albumin adducts of AFB1 --- p.267 / Chapter 3.2.4.13 --- HPLC analysis of samples obtained after digestion and all clean-up procedures --- p.268 / Chapter 3.2.5 --- Results and discussion --- p.268 / Chapter 3.2.5.1 --- BSA-AFB1 conjugate --- p.268 / Chapter 3.2.5.2 --- Treatment of experimental animals with 3H-AFB1 --- p.270 / Chapter 3.2.5.3 --- Optimisation of antiserum dilution and concentration of coating antigenin ELISA --- p.272 / Chapter 3.2.5.4 --- Analysis of standard AFB1 and AFB1- lysine in ELISA --- p.275 / Chapter 3.2.5.5 --- Sep-Pak C18 cartridge - elution characteristics and capacity --- p.279 / Chapter 3.2.5.6 --- Elution characteristics of immunoaffinity columns --- p.282 / Chapter 3.2.5.7 --- Immunoaffinity column experiment of AFB1-lysine --- p.290 / Chapter 3.2.5.8 --- Digestion of serum albumin by proteinase K --- p.295 / Chapter 3.2.5.9 --- Effect of ethanol in samples to be applied onto Sep-Pak C18 cartridges --- p.297 / Chapter 3.2.5.10 --- Recovery of radioactivity after dryingin a vacuum concentrator --- p.300 / Chapter 3.2.5.11 --- Recovery of the overall clean-up procedure for the analysis of serum albumin adducts of AFB1 --- p.300 / Chapter 3.2.5.12 --- HPLC analysis of samples obtained after all clean-up procedures --- p.305 / Chapter 3.2.5.13 --- The use of rabbit anti-AFB1 anti-serum and rat anti-AFB1 monoclonal antibody --- p.308 / Chapter 3.2.6 --- Summary --- p.309 / Chapter 3.3 --- Monitoring of AFBralbumin adducts in plasma of patients with liver disease and hepatocellular carcinoma --- p.311 / Chapter 3.3.1 --- Introduction --- p.311 / Chapter 3.3.2 --- Material and methods --- p.314 / Chapter 3.3.2.1 --- Subject --- p.314 / Chapter 3.3.2.2 --- Sample collections --- p.315 / Chapter 3.3.2.4 --- Assay for AFB1-albumin adducts --- p.315 / Chapter 3.3.2.5 --- Statistical analysis --- p.318 / Chapter 3.3.3 --- Results and discussion --- p.318 / Chapter Chapter 4 --- Summary and Ideas for Further Studies --- p.330 / Acknowledgements --- p.333 / References --- p.335 / Appendices --- p.364

Both mRNA and protein expression levels of P-glycoprotein, one of the major factors involved in drug resistance, was decreased in Pa-treated R-HepG2 cells. The chemo-sensitivity of these MDR cells towards doxorubicin would be enhanced by pretreatment of Pa. / In the study, 35 TCMs with historical background in treating liver diseases were screened. S. barbata was chosen for intensive studies based on its significant anti-hepatoma activity. Using bioassay-guided purification approach, an active component, pheophorbide a (Pa) - a chlorophyll derivative, was isolated from Scutellaria barbata. / Motivated by the severe health hazards worldwide caused by liver cancer, and the pronounced side effects of some recent anti-hepatoma agents in clinical treatment, we have initiated a research project in screening safe and effective agents from Traditional Chinese Medicine (TCM) for the treatment of hepatoma. The main objective of this research is to define the in vitro and in vivo anti-proliferative activities and to identify the action mechanisms of a TCM, the aerial part of Scutellaria barbata , in human hepatoma cells (HepG2 and Hep3B cells). / Pa exhibited anti-proliferative effects on HepG2 and Hep3B cells, through cell-cycle arrest and apoptosis, with IC50 values being 12.5 and 25.7 muM respectively. However, Pa produced insignificant cytotoxic effect on WRL-68 cells, a normal hepatic cell line. Pa also caused cell death in R-HepG2 cells, a multi-drug resistant (MDR) cell line developed from HepG2 cells. Microarray analysis indicated that a hypothetical protein FLJ10803 was found to be down-regulated upon the treatment of Pa on HepG2 cells. The sub-cellular localization of FLJ10803 was demonstrated by over-expression of the GFP fusion protein in HepG2 cells. / The anti-tumor effects of Pa could be enhanced by photodynamic therapy (PDT) approach, presumably due to the rapid generation of reactive oxygen species in the drug-binding site. Pa-PDT showed potent cytotoxicity on hepatoma cell lines, HepG2 and Hep3B, with IC50 values being 0.4 and 1.5 muM, respectively. The antitumor effects were confirmed by studies using animal model, where Pa treatment (300mug/kg/day, s.c.) could significantly inhibit the growth of Hep3B cells in nude mice after PDT treatment in vivo. Fluorescent imaging showed that Pa was located at the mitochondria, and the induction of cell death was found to be initiated by the mitochondrial dependent apoptotic pathway. Results of 2D-gel analysis suggested that Pa-PDT activated an immune-marker expression pathway that results in an over expression of HLA class I proteinsin Pa-PDT treated HepG2 cells. / To conclude, Pa may be a candidate for further development into an anti-hepatomic agent for clinical application. / Tang, Ming Kuen. / "September 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4742. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 227-243). / 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.

Interferons (IFNs) perform a wide range of biological activities: anti-microbial, anti-proliferative, immunomodulatory etc. The IFN family includes three main classes: Type I, Type II and the recently identified Type III. The two main members of Type I class are IFNα and IFNβ, which are well known for their anti-viral roles. IFNλ, a member of the Type III class of IFNs, also exhibits antiviral activity. IFNγ, also known as immune IFN, is a Type II IFN which is secreted, primarily, by activated T cells, NK cells and macrophages. IFNγ is a potent immunomodulator which plays important roles in host defense. The diverse functions of this cytokine are demonstrated in Ifnγ-/- mice which display increased sensitivity to several pathogens, high incidences of tumors, reduced inflammatory response etc. IFNγ binds to its cognate receptors, which consist of two subunits, IFNγ receptor (IFNGR) 1 and IFNGR2. IFNγ mediates its multifarious biological actions by activating the Janus activated kinase (Jak)-Signal transducer and activator of transcription (Stat) 1 signaling pathway. Jaks belong to a family of non-receptor protein tyrosine kinases and phosphorylate the IFNγ receptor and the transcriptional co-activator, Stat. IFNGR1, the larger subunit, is required for ligand binding and its carboxyl terminus is involved in binding to Jak1, which in turn phosphorylates Stat1. The smaller subunit, IFNGR2, is required for signaling and contains the Jak2 binding site. After binding of IFNγ to its receptor, a series of phosphorylation events occur, resulting in Stat1 phosphorylation and homodimerization of Stat1 to form the gamma activating factor (GAF). These activated molecules translocate to the nucleus and bind to gamma activating sequence (GAS) present in the promoters of several IFNγ-modulated genes. Thus, the cellular responses mediated by IFNγ are, primarily, due to modulation of gene expression. Therefore, the identification and study of IFNγ stimulated genes, signaling mediators and their cross talk with other cellular pathways is an active area of research. The system of our study was a hepatoma cell line, H6, which is derived from a spontaneous tumor from B10.A mice and selected for in vitro cell culture. It is an IFNγ inducible system and has been used to study IFNγ-induced gene expression and functional responses. Treatment of H6 cells with IFNγ greatly enhanced MHC class I levels but also reduced cell growth. High amounts of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) play crucial roles in the growth suppressive effect of IFNγ. To better understand the signaling pathways involved in the generation of ROS and RNI, the involvement of Ras was investigated. Ras-GTP levels were determined by pull down assays using GST-Raf1-Ras binding domain fusion protein bound to glutathione agarose. Ras activation (conversion of Ras-GDP to Ras-GTP) was observed in H6 cells upon IFNγ treatment by ~12 hr. To assess the functional role of Ras activation, studies with Manumycin A, a farnesyl transferase inhibitor (FTI), were performed. The formation of functional Ras requires farnesylation, a post-translational modification, which is inhibited by FTIs. Treatment with Manumycin A blocked Ras activation but did not significantly modulate the IFNγ-induced MHC class I. However, the inhibitor reduced ROS amounts leading to increased cell growth in the presence of IFNγ. Together, these results delineated the role of Ras and ROS in modulating some functions of IFNγ. To further understand the mechanisms by which Ras mediates its functions during IFNγ mediated growth suppression, the activation and function of Ras effectors was evaluated. In particular, the role of Ras-like (Ral) guanyl nucleotide-binding proteins, RalA and RalB, was investigated. IFNγ induced transcripts of RalA but not RalB. Also, the induction of RalA and IFNγ induced growth suppression were Stat1-dependent. Studies involving chemical inhibitors and genetic studies revealed that Ras played a role in the induction of RalA during IFNγ treatment. The role of c-Jun N-terminal kinase (JNK), a stress induced kinase, was also elucidated in this system. Together, IFNγ induced activation of Ras and its effectors RalA and JNK, leading to high amounts of ROS that suppressed cell growth. To evaluate the physiological significance of Ras activation during inflammatory responses, the mouse model of acetaminophen (APAP) induced liver injury was established. Hepatotoxicity due to overdose of the analgesic and antipyretic, APAP, is a major cause of liver failure in adults. APAP is metabolized into a reactive metabolite which binds to glutathione. Consequently, the depletion of intracellular glutathione stores leads to oxidative stress and liver injury. Notably, Ifnγ-/- mice are resistant to APAP-induced liver damage demonstrating a crucial role for this cytokine. The role of Ras activation was evaluated after oral dosing of BALB/c mice with APAP. Ras-GTP was induced early and decreased amounts were observed upon treatment with L-methionine, which replenished glutathione amounts. Injection with L-methionine or Manumycin A rescued liver injury as assessed by lowered serum alanine aminotransferase amounts and histological analysis. Kinetic studies were also performed, under different treatment conditions, to estimate different biochemical parameters: glutathione amounts, JNK activation, protein carbonylation, ROS amounts, serum amounts of cytokines, TNFα and IFNγ etc. This study reveals a role of Ras activation in stimulating proinflammatory responses and demonstrates the therapeutic efficacy of FTIs during APAP-induced liver injury. In addition the role of RalA during APAP-induced liver injury was also studied. In summary, this study, involving in vitro cell culture and in vivo liver injury model systems, sheds light on the significant contributions of Ras and its effector, RalA, during IFNγ mediated growth suppression and APAP-induced liver injury.

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