Thesis (Ph.D. (Biochemistry)) -- University of Limpopo, 2021 / Cancer remains one of the leading causes of morbidity and mortality worldwide with an estimated 9.9 million deaths in 2020. Cancer treatment regimens such as chemotherapy and radiotherapy have over the years fallen short due to drug resistance, toxicity, damage to normal healthy cells and tissues surrounding the treatment area. Moreover, they have shown very limited survival benefits for most advanced staged cancers such as colorectal cancer, which in 2020 was responsible for 3 728 deaths with a 6.8% incidence rate. Despite the many efforts in developing alternative chemotherapeutic strategies, cancer of the colon and cancer, in general, remains a burden. For centuries, plants and plant derivatives have been exploited for their nutritional and medicinal properties and now serve as chemical scaffolds or templates for designing and synthesising products with pharmacological importance. Herbal medicines are claimed to enhance therapeutic effects and are often used in combination with chronic medication. However, the concurrent use of herbal medicines and synthetic drugs may affect the pharmacokinetic profile of therapeutic drugs or trigger unexpected and undesirable effects. This study aimed to characterise the leaf extracts (crude water and crude methanol) of Momordica balsamina and investigate their potential anticancer activity on HT-29 colon cancer cells. The study also aimed to asses the effect of the extracts on drug metabolising enzymes (CYP450), specifically those which metabolise 5-Fluorouracil (5-FU) prodrugs or are inhibited by 5-FU since it is one of the first-line treatments for colon cancer. Dried powdered leaves were extracted using water and absolute methanol to obtain crude water and crude methanol extracts, respectively. For characterisation, the extracts were spotted on thin-layer chromatography (TLC) plates and further screened using chemical tests. The ferric ion reducing power assay and Liquid chromatographymass spectrometry were used to determine the antioxidant activity of the extracts and to identify prominent or abundant compounds in each extract, respectively. To assess the cytotoxic effect of the extracts and 5-FU, HT-29 colon cancer cells and C2C12 muscle cells, which were used as a model for normal cells, were exposed to concentrations that ranged from 0 to 2000 µg/ml for the water (H2O) extract, 0 to 300 µg/ml for the methanol (MeOH) extract or 0 to 100 µg/ml of 5-FU for 24 and 72 hours, and subjected to the MTT assay. The effect of the extracts on the efficacy of 5-FU was xxi assessed using the MTT assay by combined treatments of the extract and 5-FU. Genotoxicity of the extracts was assessed on the C2C12 cells using the Muse™ MultiColour DNA Damage kit. The generation of intracellular reactive oxygen species (ROS) was assessed by flow cytometry using the DCFH-DA assay. The JC-1 and acridine orange (AO)/propidium iodide (PI) staining assays were used to assess the effect of the extracts on the mitochondrial potential as well as cell and nuclear morphology, respectively. Apoptosis was quantified by flow cytometry using annexin V/PI and caspase activation assessed using the Caspase-8 and Caspase-9 colourimetric assay kits. The pro-apoptotic mechanism(s) was determined by assessing the expression profiles of selected apoptosis regulatory proteins using the human apoptosis antibody array kit. Cell cycle analysis by flow cytometry was conducted to determine the effect of the extract on the cell division cycle. Moreover, to determine the potential of herb-drug interactions, the Vivid® CYP450 Screening kits and P-gp-GloTM Assay Systems with P-glycoprotein were used to assess the effect on the activity of drug metabolising enzymes and drug transportation, respectively. The results showed that the MeOH extract possessed fewer polar compounds, higher ferric iron-reducing power, and a relative abundance of flavonol glycosides, cucurbitane-type triterpenoid aglycones, and cucurbitane-type glycosides than the H2O extract. The MeOH extract was further selectively cytotoxic to the HT-29 colon cancer cells at 24 hours of treatment and selectively induced genotoxicity in HT-29 cells. The H2O extract, however, was not cytotoxic to the HT-29 cells at all the tested concentrations at 24 and 72 hours of treatment. Analysis of nuclear and cell morphology suggested that the decrease in the percentage viability of MeOH extracttreated cells was associated with apoptotic cell death. Apoptosis was further confirmed by the loss of mitochondrial potential, increase in ROS production, caspase-8 and -9 activities as well as Annexin-V/PI-stained cells. Cell cycle analysis revealed cell cycle arrest at the S phase in MeOH extract-treated cells. Analysis of protein expression profiles revealed that the extract modulated various proteins that play a role in the promotion or inhibition of apoptosis. Moreover, the MeOH extract was shown to inhibit the activity of CYPs 1A2, 2A6, 2C8, and 2C9, while the H2O extract showed no significant inhibitory effects on the activity of all tested CYPs and 5-FU only significantly inhibited the activity of CYP2C9. However, combinatory treatments with 5-FU and the MeOH extract were shown to have no additive or diminishing effects on the efficacy of 5-FU on the activity of all the tested CYP enzymes. Treatment with 5FU (0.008 – 32 μg/ml) and the H2O extract (0.02 – 200 μg/ml) was shown to stimulate the ATPase activity of P-gp, while the MeOH extract significantly inhibited its activity with concentrations of 0.2, 2, and 20 μg/ml. In conclusion, the MeOH extract selectively induced cancer cell toxicity, genotoxicity as well as S phase cell cycle arrest and apoptosis via the intrinsic and extrinsic pathways. The anticancer activity of the MeOH leaf extract of M. balsamina as well as its antioxidant potential may be attributed to the presence and relative abundance of flavonol glycosides, cucurbitane-type triterpenoid aglycones, and cucurbitane-type glycosides. Although the MeOH extract may potentially reverse the effects of P-gp multidrug resistance by decreasing its activity, its inhibition of the activity of CYPs 1A2, 2A6, 2C8 and, 2C9, which are involved in the metabolism of more than 80% of the drugs in clinical use may suggest that co-administration of the MeOH extract may still result in increased plasma levels of drugs, thereby resulting in toxicity. The H2O extract, although not pro-apoptotic as the MeOH extract may still have the potential to be developed as a nutraceutical as it was shown to exhibit no adverse drug interactions and because this species is known to possess a wide variety of nutritional and medicinal values. / South African Medical Research Council (SAMRC) Research Capacity Development Initiative.

Indiana University-Purdue University Indianapolis (IUPUI) / Although cancer and treatment-associated cognitive dysfunction has been well-documented in the literature, much work remains to elucidate the biological mechanisms driving this effect, hampering current therapeutic efforts. To address this gap, we first reviewed studies utilizing neuroimaging to characterize cognitive dysfunction in cancer, as studies of neurodegenerative diseases point to neuroimaging as a sensitive measure of cognitive dysfunction. This review highlighted the need for longitudinal imaging studies of cancer and treatment-related changes in cerebral structure and function. Subsequently, we utilized multimodal neuroimaging techniques in a female breast cancer cohort to investigate the longitudinal impact of cancer and chemotherapy treatment on cerebral perfusion and gray matter. Our findings indicate that chemotherapy is associated with elevated perfusion, primarily in posterior brain regions, as well as depressed frontal perfusion associated with decreased frontal gray matter density. This pattern of results suggests the involvement of multiple mechanisms of chemotherapy-induced cognitive dysfunction. We also investigated the relationship of cognitive dysfunction and chemotherapy-induced peripheral neuropathy (CIPN), another type of chemotherapy-related nervous system sequelae, again utilizing multimodal, longitudinal neuroimaging, and found that peripheral neuropathy symptoms following chemotherapy were associated with changes in cerebral perfusion and gray matter density. Together, these findings support the hypothesis that multiple biological mechanisms drive cancer and treatment-related cognitive dysfunction. Interestingly, although cancer is associated with cognitive dysfunction, epidemiological studies have shown that cancer and Alzheimer's disease (AD) are inversely correlated. To extend our imaging analysis beyond breast cancer, we leveraged the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort to investigate the inverse relationship of cancer and AD and investigate the impact of both of these diseases on gray matter density. We found that though the inverse relationship of these diseases was replicated in the ADNI cohort, cancer history was associated with lower gray matter density, similar to findings from breast cancer studies, independent of AD diagnostic group. Finally, we reviewed microRNA studies, as microRNAs are important regulators of many cell signaling pathways and have been actively investigated in relation to both diseases. This review suggests several pathways that may be driving the inverse association and may contribute to cognitive dysfunction.

Indiana University-Purdue University Indianapolis (IUPUI) / Targeting uncontrolled cell proliferation and resistance to DNA damaging chemotherapeutics using small molecule inhibitors of proteins involved in these pathways has significant potential in cancer treatment. Several proteins involved in genomic maintenance and stability have been implicated both in the development of cancer and the response to chemotherapeutic treatment. Replication Protein A, RPA, the eukaryotic single-strand DNA binding protein, is essential for genomic maintenance and stability via roles in both DNA replication and repair. Xeroderma Pigmentosum Group A, XPA, is required for nucleotide excision repair, the main pathway cells employ to repair bulky DNA adducts. Both of these proteins have been implicated in tumor progression and chemotherapeutic response. We have identified a novel small molecule that inhibits the in vitro and cellular ssDNA binding activity of RPA, prevents cell cycle progression, induces cytotoxicity and increases the efficacy of chemotherapeutic DNA damaging agents. These results provide new insight into the mechanism of RPA-ssDNA interactions in chromosome maintenance and stability. We have also identified small molecules that prevent the XPA-DNA interaction, which are being investigated for cellular and tumor activity. These results demonstrate the first molecularly targeted eukaryotic DNA binding inhibitors and reveal the utility of targeting a protein-DNA interaction as a therapeutic strategy for cancer treatment.

No / Objectives Previous studies have demonstrated that intravesical administration of apaziquone (EOquin) has ablative activity against superficial bladder cancer marker lesions with 8 out of 12 complete responses recorded. We present a comparison between the rates of tumor recurrence before and after treatment with apaziquone. Methods The rate of tumor recurrence after treatment with apaziquone was compared with each patient's historical record of recurrences obtained from a retrospective analysis of the patients' case notes. The time to each recurrence event before apaziquone treatment and the time to the first recurrence after apaziquone treatment were recorded, and the data were analyzed using a population-averaged linear regression model using Stata Release, version 9.2, software. Results Of the eight complete responses obtained in the Phase I study, tumor recurrence occurred in 4 patients and the remaining 4 patients remained disease free after a median follow-up of 31 months. The time to the first recurrence after apaziquone treatment was significantly longer (P <0.001) compared with the historical pattern and recurrence interval before apaziquone. Before apaziquone instillation, the mean ± SE recurrence rate and tumor rate per year was 1.5 ± 0.2 and 4.8 ± 1.2, respectively, and these decreased to 0.6 ± 0.25 and 1.5 ± 0.8, respectively, after apaziquone treatment (P <0.05). Conclusions The results of this study indicate that early recurrences after treatment with apaziquone are infrequent and the interval to recurrence is significantly greater compared with the historical recurrence times for these patients. Larger prospective randomised trials are warranted to confirm these results. Aapaziquone (EOquin, USAN, E09, 3-hydroxy-5-aziridinyl-1-methyl-2[indole-4,7-dione]¿prop-¿-en-¿-ol) belongs to a class of anticancer agents known as bioreductive drugs that require metabolism by cellular reductases to generate a cytotoxic species.1 Although it is chemically related to mitomycin C, apaziquone has a distinctly different mechanism of action and preclinical activity profile.1 and 2 The initial optimism generated by its preclinical activity profile rapidly evaporated after the demonstration that intravenously administered apaziquone was clinically inactive against a range of solid tumors in Phase II clinical trials.3 and 4 Several possible explanations were considered for its lack of efficacy, but poor drug delivery to the tumor because of the rapid pharmacokinetic elimination of apaziquone in conjunction with relatively poor penetration through avascular tissue was considered to be the principal reason.5 On the basis of the rationale that intravesical administration would circumvent the problem of drug delivery and any apaziquone absorbed into the blood stream would be rapidly cleared,6 a Phase I-II clinical pilot study of intravesical administration of apaziquone to superficial bladder tumors was established.7 The results of that trial demonstrated that intravesically administered apaziquone has ablative activity against superficial bladder transitional cell carcinoma (TCC) marker lesions.7 These results were confirmed and extended in a Phase II clinical trial of 47 patients with superficial bladder TCC, in which complete responses were obtained in 67% of patients.8 Because all the enrolled patients in the original trial7 had had multiple recurrences after previous intravesical chemotherapy and/or immunotherapy, the purpose of the present study was, first, to report the recurrences that occurred after apaziquone treatment and, second, to study the effect of apaziquone instillation on the recurrence rate by statistically comparing these results with the historical pattern of recurrences for each patient before treatment with apaziquone.

Ng, Po Yan. / Thesis submitted in: November 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 109-120). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 摘要 --- p.iii / Table of Contents --- p.iv / List of Figures --- p.viii / List of Tables --- p.xi / List of Abbreviation --- p.xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- A General Introduction to the Development and Clinical Activities of Platinum Drugs --- p.1 / Chapter 1.1.1 --- Platinum Drugs used in a Clinical Setting --- p.4 / Chapter 1.1.2 --- Platinum Drugs under Clinical Trials --- p.5 / Chapter 1.1.3 --- Platinum Compounds with Dual Mechanisms --- p.7 / Chapter 1.2 --- Platinum Drug Antitumor Mechanism --- p.9 / Chapter 1.3 --- Limitations of Platinum Drugs --- p.12 / Chapter 1.3.1 --- Toxicity --- p.12 / Chapter 1.3.2 --- Drug Resistance or Cross Resistance --- p.15 / Chapter 1.3.2.1 --- Reduced Drug Accumulation or Increased Drug Efflux --- p.16 / Chapter 1.3.2.2 --- Drug Inactivation --- p.18 / Chapter 1.3.2.3 --- Enhanced DNA Repair --- p.19 / Chapter 1.4 --- Why Combinational Therapy? --- p.21 / Chapter 1.4.1 --- Antimetabolites --- p.20 / Chapter 1.4.2 --- Topoisomerase Inhibitors --- p.22 / Chapter 1.4.3 --- Tubulin-Active Antimitotic Agents --- p.24 / Chapter 1.4.4 --- Demethylcantharidin as a potential candidate for drug combination --- p.28 / Chapter 1.5 --- Study Objectives --- p.31 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Cell Lines --- p.33 / Chapter 2.2 --- Cancer Cell Preparation / Chapter 2.2.1 --- Chemicals and Reagents --- p.33 / Chapter 2.2.2 --- Cell Culture Practice --- p.34 / Chapter 2.2.2.1 --- Subcultures --- p.35 / Chapter 2.2.2.2 --- Cryopreservation --- p.37 / Chapter 2.2.2.3 --- Thawing Cryopreservated Cells --- p.38 / Chapter 2.2.3 --- Development of Drug-Resistant Cell Lines --- p.39 / Chapter 2.3 --- Growth Inhibition Assay / Chapter 2.3.1 --- Evaluation of Cytotoxicity in vitro --- p.40 / Chapter 2.3.2 --- Drug Pretreatment --- p.43 / Chapter 2.3.3 --- Drug Pre-sensitization with Concurrent Treatment --- p.44 / Chapter 2.4 --- Calculations for Drug Combinations --- p.46 / Chapter 2.5 --- Statistical Analysis --- p.49 / Chapter Chapter 3 --- Results and Discussions / Chapter 3.1 --- In vitro Cytotoxicity and Evaluation of Drug Resistance --- p.50 / Chapter 3.2 --- Role of Leaving Ligand in a Platinum Complex --- p.58 / Chapter 3.3 --- Priority in Selecting the Most Effective Drug Combination --- p.66 / Chapter 3.4 --- Drug Combination Studies / Chapter 3.4.1 --- Drug Combination Prescreening --- p.68 / Chapter 3.4.1.1 --- Comparison of the effectiveness of the three Drug Combinations --- p.72 / Chapter 3.4.1.2 --- Rationale for Drug Combination Studies presented in Section 3.4.2 & 3.4.3 --- p.73 / Chapter 3.4.2 --- Drug Pre-sensitization Studies in Colorectal Cancer Cell Lines --- p.74 / Chapter 3.4.2.1 --- Comparison of Drug Pre-sensitization Treatment in Sensitive Colorectal Cancer Cell Lines --- p.84 / Chapter 3.4.2.2 --- Comparison of Drug Pre-sensitization Treatment in Sensitive and Oxaliplatin Resistant HCT116 Colorectal Cancer Cell Lines --- p.87 / Chapter 3.4.3 --- Drug Pre-sensitization Studies in Liver Cancer Cell Lines --- p.89 / Chapter 3.4.3.1 --- Comparison of Drug Pre-sensitization Treatment in Sensitive Liver Cancer Cell Lines --- p.99 / Chapter 3.4.3.2 --- Comparison of Drug Pre-sensitization Treatment in Sensitive and Cisplatin Resistant SK-Hepl Liver Cancer Cell Line --- p.101 / Chapter 3.5 --- Possible Explanation to the Observed Drug Combination Effect --- p.103 / Chapter 3.6 --- General Protocols for Drug Combinations --- p.105 / Chapter Chapter 4 --- Conclusions / Reference --- p.109 / Appendices --- p.121 / Chapter I a. --- "Raw Data of Pre-screening for HCT116 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.122 / Chapter I b. --- "Raw Data of Pre-screening for HCT116 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.123 / Chapter II a. --- "Raw Data of Pre-screening for SK-Hepl (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.124 / Chapter II b. --- "Raw Data of Pre-screening for SK-Hepl ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.125 / Chapter III a. i) --- "Isobolograms for HCT116 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.126 / Chapter III a. ii) --- "Raw Data for HCT116 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.127 / Chapter III b. i) --- "Isobolograms for HCT116 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.128 / Chapter III b. ii) --- "Raw Data for HCT116 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.129 / Chapter IV a. i) --- "Isobolograms for HCT1160xaR (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.130 / Chapter IV a. ii) --- "Raw Data for HCT1160xaR (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.131 / Chapter IV b. i) --- "Isobolograms for HCT1160xaR ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.132 / Chapter IV b. ii) --- "Raw Data for HCT1160xaR ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.133 / Chapter V a. i) --- "Isobolograms for HT29 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.134 / Chapter V a. ii) --- "Raw Data for HT29 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.135 / Chapter V b. i) --- "Isobolograms for HT29 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.136 / Chapter V b. ii) --- "Raw Data for HT29 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.137 / Chapter VI a. i) --- Isobolograms for Hep G2 (Cisplatin and [Pt(DMC)(NH3)2]) --- p.138 / Chapter VI a. ii) --- Raw Data for Hep G2 (Cisplatin and [Pt(DMC)(NH3)2]) --- p.139 / Chapter VI b. i) --- "Isobolograms for Hep G2 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.140 / Chapter VI b. ii) --- "Raw Data for Hep G2 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.141 / Chapter VII a. i) --- "isobolograms for SK Hep 1 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.142 / Chapter VII a. ii) --- "Raw Data for SK Hep 1 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.143 / Chapter VII b.i) --- "Isobolograms for SK Hep 1 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.144 / Chapter VII b. ii) --- "Raw Data for SK Hep 1 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.145 / Chapter VIII a. i) --- "Isobolograms for SK Hep ICisR (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.146 / Chapter VIII a. ii) --- "Raw Data for SK Hep ICisR (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.147 / Chapter VIII b. i) --- "Isobolograms for SK Hep ICisR ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.148 / Chapter VIII b. ii) --- "Raw Data for SK Hep ICisR ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.149

Tang Angie. / Thesis submitted in: November 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 151-172). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / Abstract in Chinese --- p.vi / Table of Contents --- p.vii / List of Publications --- p.xi / Awards --- p.xii / List of Abbreviations --- p.xiii / List of Tables --- p.xv / List of Figures --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Gastric cancer-overview --- p.3 / Chapter 2.1.1 --- Epidemology --- p.3 / Chapter 2.1.2 --- Pathology --- p.3 / Chapter 2.1.3 --- Etiologies and Risk Factors --- p.4 / Chapter I. --- Environmental factors --- p.4 / Chapter a. --- Helicobacter pylori infections --- p.4 / Chapter b. --- Epstein-Barr virus (EBV) --- p.6 / Chapter c. --- Dietary factors --- p.6 / Chapter d. --- Smoking --- p.6 / Chapter II. --- Genetic Factors --- p.7 / Chapter a. --- Hereditary Gastric Cancer --- p.7 / Chapter b. --- Genetic polymorphism --- p.8 / Chapter III. --- Cyclooxygenases (COX) enzymes --- p.10 / Chapter IV. --- Molecular carcinogenesis --- p.11 / Chapter a. --- Activation of proto-oncogenes --- p.11 / Chapter b. --- Candidate tumor suppressor genes --- p.12 / Chapter 1. --- Gene mutation and deletion --- p.12 / Chapter 2. --- Epigenetic Silencing --- p.13 / Chapter 2.2 --- Epigenetics --- p.14 / Chapter 2.2.1 --- DNA methylation --- p.15 / Chapter 2.2.2 --- Histone modification --- p.28 / Chapter I. --- Histone acetylation and deacetylation --- p.32 / Chapter II. --- Histone methylation --- p.32 / Chapter III. --- Histone phosphorylation --- p.34 / Chapter IV. --- Histone ubiquitylation --- p.34 / Chapter 2.3 --- "HAT, HDAC and HDAC inhibitors" --- p.36 / Chapter 2.3.1 --- HAT --- p.38 / Chapter 2.3.2 --- HDAC --- p.39 / Chapter (a) --- Class I --- p.40 / Chapter (b) --- Class II --- p.41 / Chapter (c) --- Class III --- p.42 / Chapter (d) --- Mammalian HDAC and their mechanism of deacetylation --- p.44 / Chapter 2.3.3 --- HDAC inhibitors --- p.45 / Chapter I. --- Class I/II natural inhibitors --- p.47 / Chapter II. --- Class I/II synthetic inhibitors --- p.48 / Chapter III. --- Sirtuins inhibitors --- p.49 / Chapter IV. --- Activity of HDAC inhibitors in vitro --- p.50 / Chapter a. --- Effect in the gene expression --- p.50 / Chapter b. --- Non-transcriptional effects --- p.55 / Chapter c. --- Activity of HDAC inhibitors with other agents --- p.57 / Chapter d. --- Effects in xenograft tumor models --- p.57 / Chapter V. --- Clinical trials of HDAC inhibitors --- p.59 / Chapter Chapter 3 --- Aims of the study --- p.63 / Chapter Chapter 4 --- Materials and Methods --- p.64 / Chapter 4.1 --- Cell culture --- p.64 / Chapter 4.2 --- Drug treatment --- p.64 / Chapter 4.2.1 --- Suberoylanilide Hydroxamic Acid treatment --- p.64 / Chapter 4.2.2 --- Trichostatin A treatment --- p.65 / Chapter 4.3 --- Cell proliferation assay --- p.66 / Chapter 4.4 --- Apoptotic assay --- p.67 / Chapter 4.5 --- Flow cytometry --- p.67 / Chapter 4.5.1 --- Cell preparation --- p.67 / Chapter 4.5.2 --- Propidium Iodide staining --- p.68 / Chapter 4.5.3 --- Annexin V-FITC staining --- p.68 / Chapter 4.5.4 --- Flow cytometer analysis --- p.69 / Chapter 4.6 --- Total RNA extraction --- p.70 / Chapter 4.7 --- DNA extraction --- p.71 / Chapter 4.8 --- Protein extraction --- p.72 / Chapter 4.9 --- Western blottng --- p.72 / Chapter 4.10 --- Microarray analysis --- p.74 / Chapter 4.10.1 --- Sample preparation for microarray --- p.74 / Chapter 4.10.2 --- Hybridization --- p.75 / Chapter 4.10.3 --- Scanning and data processing --- p.75 / Chapter 4.10.4 --- Data analysis --- p.76 / Chapter 4.11 --- Primer design --- p.77 / Chapter 4.12 --- RT-PCR --- p.77 / Chapter 4.12.1 --- Reverse transcription --- p.77 / Chapter 4.12.2 --- Quantitative RT-PCR --- p.78 / Chapter 4.13 --- Methlyation study --- p.79 / Chapter 4.13.1 --- Demethylation by 5-aza-2'deoxycytidine --- p.79 / Chapter 4.13.2 --- Bisulfite modification --- p.79 / Chapter 4.13.3 --- Methylation-specific PCR (MSP) --- p.79 / Chapter Chapter 5 --- Results --- p.81 / Chapter 5.1 --- Morphological changes in AGS cells --- p.81 / Chapter 5.2 --- Anti-cancer effects of HDAC inhibitors --- p.81 / Chapter 5.2.1 --- Effect of HDAC inhibitors on cell growth --- p.81 / Chapter a. --- SAHA inhibits cell proliferation --- p.82 / Chapter b. --- TSA inhibits cell proliferation --- p.82 / Chapter 5.2.2 --- Cell cycle analysis --- p.87 / Chapter a. --- Effect of SAHA on cell cycle --- p.87 / Chapter b. --- Effect of TSA on cell cycle --- p.88 / Chapter 5.2.3 --- Induction of apoptosis on AGS cells --- p.92 / Chapter a. --- SAHA induces apoptotic cell death --- p.92 / Chapter b. --- TSA induces apoptotic cell death --- p.94 / Chapter 5.3 --- Induction of histone expression on AGS cells --- p.102 / Chapter 5.3.1 --- HDAC inhibitors induced acetylation of histone H3 --- p.102 / Chapter 5.3.2 --- HDAC inhibitors induced acetylation of histone H4 --- p.103 / Chapter 5.4 --- SAHA- and TSA-induced gene expression profiles --- p.106 / Chapter 5.5 --- Verification of gene expression by quantitative RT-PCR --- p.108 / Chapter 5.6 --- Methylation study --- p.113 / Chapter Chapter 6 --- Discussion --- p.116 / Chapter 6.1 --- Improved treatment strategy is needed for gastric cancer. --- p.116 / Chapter 6.2 --- HDAC inhibitors as potential anti-cancer agents --- p.117 / Chapter 6.3 --- Potential anti-cancer effect of TSA and SAHA on AGS cells --- p.120 / Chapter I. --- Morphological changes of AGS gastric cancer cells --- p.120 / Chapter II. --- Inhibition of cell proliferation --- p.120 / Chapter III. --- Induction of cell cycle arrest --- p.121 / Chapter IV. --- Induction of apoptosis --- p.122 / Chapter 6.4 --- Expression of acetylated histones upon treatment with TSA and SAHA --- p.124 / Chapter 6.5 --- Identify potential target genes upon treatment with TSA and SAHA --- p.125 / Chapter 6.5.1 --- Candidate genes involved in cell cycle --- p.126 / Chapter a. --- P21WAF1 --- p.126 / Chapter b. --- p27kip1. --- p.128 / Chapter c. --- Cyclin E & Cyclin A --- p.128 / Chapter d. --- Signal-induced proliferation-associated gene 1 (SIPA1) .… --- p.129 / Chapter 6.5.2 --- Candidate genes involved in apoptosis and anti-proliferation --- p.130 / Chapter a. --- BCL2-interacting killer (apoptosis-inducing) (BIK) (Pro-apoptotic gene) --- p.131 / Chapter b. --- Thioredoxin interacting protein (TXNIP) (Proapoptotic gene) / Chapter c. --- Cell death-inducing DFFA-like effector b (CIDEB) (apoptosis induction) --- p.132 / Chapter d. --- B-cell translocation gene 1 (BTG1) - (anti-proliferation) --- p.133 / Chapter e. --- Quiescin 6 (QSCN6) (anti-proliferation) --- p.133 / Chapter f. --- "Cysteine-rich, angiogenic inducer, 61 (CYR61) (anti-proliferative)" --- p.134 / Chapter g. --- Metallothionein 2A (MT2A) (apoptosis induction and anti-proliferative) --- p.134 / Chapter 6.5.3 --- Other genes reported to be up-regulated with HDAC inhibitors treatment --- p.135 / Chapter a. --- Glia maturation factor-gamma (GMFG) --- p.135 / Chapter b. --- v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) / Chapter c. --- Interleukin 8 (IL-8) --- p.136 / Chapter d. --- Insulin-like growth factor binding protein- 2 (IGFBP2) --- p.137 / Chapter e. --- Integrin alpha chain 7 (ITGA7) --- p.138 / Chapter 6.5.4 --- Selected highly up-regulated genes with HDAC inhibitors treatment --- p.139 / Chapter a. --- Aldo-keto reductase family 1，member C3 (AKR1C3) --- p.139 / Chapter b. --- GPI-anchored metastasis-associated protein homolog (C4.4A) --- p.139 / Chapter c. --- "Serine (or cysteine) proteinase inhibitor，clade I (neuroserpin), member 1 (SERPINI1)" --- p.140 / Chapter d. --- "Serine (or cysteine) proteinase inhibitor，clade E (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1)" --- p.140 / Chapter e. --- Adrenomedullin (ADM) --- p.141 / Chapter f. --- Dehydrogenase/reductase (SDR family) member 2 (HEP27) --- p.142 / Chapter g. --- Cholecystokinin (CCK) --- p.142 / Chapter h. --- Silver homolog (mouse) (SILV) --- p.143 / Chapter 6.6 --- Genes regulated by gene promoter hypermethylation in AGS cells --- p.143 / Chapter Chapter 7 --- Conclusion --- p.147 / Chapter Chapter 8 --- Further Studies --- p.150 / References --- p.151 / Appendix I --- p.151 / Appendix II --- p.III / Appendix III --- p.IV / Appendix IV --- p.VI

慢性乙型肝炎病毒（HBV）感染是肝癌發生的重要因素，其中乙肝病毒X蛋白（HBx）在這一過程起著關鍵作用。研究發現，一些HBV變體和HBx突變具有更高致癌風險，而且這些變體和突變存在地區差異。香港是HBV感染高發地帶，因此本研究目的是從這一地區120個肝癌組織標本中篩查出HBx突變位點。我們用巢式PCR從84.16% (101/120)的標本中提取和擴增了HBx，並進行基因測序。三種HBx突變被檢測出，包括點突變，遠端羧基端截斷和缺失突變。其中點突變位點有39個，特別的是在50%的標本中檢測出A1630G/G1721A 和 A1762T/G1764A雙突變。在31.68% (32/101)的標本中發現遠端羧基端截斷，以及在2.97% (3/101)的標本中檢測出缺失突變。總之，大多數突變集中在HBx轉錄啟動域，表明這些突變在肝癌發生中可能起著重要作用。 / 缺氧誘導因數-1α（HIF-1α）在肝癌的發生和發展中也起著重要作用。研究發現，野生型HBx可以啟動HIF-1α，但是變異型HBx和HIF-1α的關係還沒有研究清楚。我們研究表明HBx轉錄啟動域是必需而且足夠啟動HIF-1α的。在這個區域的突變中，雙突變K130M/V131Z增強HBx對HIF-1α的活性，但遠端羧基端截斷和缺失突變削弱其功能。進一步研究發現，羧基端特別是119-140氨基酸對HBx的穩定和功能非常重要。肝癌標本中，我們也發現HBx和HIF-1α的表達呈正相關。因此，雖然不同的突變對於HBx的功能有不同的影響，但總的來說這些突變可以促進HIF-1α的表達和啟動，進而導致肝癌患者的預後不良。 / 靶向治療在肝癌綜合治療中扮演重要角色。索拉菲尼（Sorafenib）是一種多激酶抑制劑，臨床實驗發現它對晚期肝癌治療有效，但其抑制腫瘤血管生成機制還不完全清楚。我們研究發現Sorafenib明顯而且劑量依賴性地降低HIF-1α的表達和活化，進而抑制血管內皮生長因數（VEGF）的表達。Sorafenib抑制mTOR, ERK, p70S6K, RP-S6, eIF4E和4E-BP1等翻譯起始因數的磷酸化，從而抑制HIF-1α的合成而不影響其降解。體外實驗進一步發現Sorafenib降低HIF-1α和VEGF的表達，從而抑制腫瘤的血管形成和生長。總之，我們的研究表明sorafenib可能通過阻斷mTOR/p70S6K/4E-BP1 和 ERK 信號通路來抑制HIF-1α的合成，從而發揮其抗腫瘤血管生成作用。 / Chronic HBV infection is the leading cause of hepatocellular carcinoma (HCC) and HBx plays a crucial role in the molecular pathogenesis of HBV-related HCC. Previous investigations have indicated that some variations of HBV or mutations of HBx are associated with higher risk of HCC development, whereas the mutations profiles may be disparate in different regions. In the present studies, we thus aim to screen and identify the HBx mutation hotspots in 120 HCC tissues from Hong Kong, a region with HBV hyper-endemic. HBV DNAs were successfully isolated and amplified in 84.16% (101/120) HCC specimens via nest-PCR, and then subjected to gene sequencing. Three types of HBx mutations, including point mutations, distal carboxyl-terminal truncations and deletion mutations, were discovered. Among the point mutations, 39 mutation hotspots were indentified, with two double mutations (A1630G/G1721A and A1762T/G1764A) occurring in approximate 50% of 101 HCC cases. Distal C-terminal truncated mutations were discovered in 31.68% (32/101) of HCC cases, whereas deletion mutations were detected in 2.97% (3/101) of them. Overall, majority of identified mutations were located at the transactivation domain of HBx, suggesting the crucial roles of these mutations in HCC development. / Hypoxia-inducible factor-1α (HIF-1α) also closely involves in the development and progression of HCC. Wild-type HBx has been shown to activate HIF-1α. But the relationship between HBx mutants and activation of HIF-1α has not been fully elucidated. We here revealed that the transactivaiton domain of HBx was necessary and sufficient to activate HIF-1α. Double mutations K130M/V131Z in this domain enhanced the functionality of HBx in upregulating the expression and the activation of HIF-1α, whereas C-terminal truncations and deletion mutations weakened this prosperity of HBx. We further uncovered that the C-terminus, especially the region of amino acids 119-140, was essential for the stability and transactivation of HBx. The positive association between the HBx mutants and HIF-1α was found in the HCC tissue samples. Therefore, although mutations exerted different effects on the functionality of HBx, the overall activity of HBx mutants was suggested to upregulate HIF-1α, whose level is related to poor prognosis of HCC patients. / The therapy targeting a critical molecule in the development of HCC such as HIF-1α may be a potential and effective treatment regimen for HCC patients. Sorafenib, a multikinase inhibitor, has demonstrated promising results for the treatment of advanced HCC in clinical trials, but the mechanism that accounts for the anti-angiogenic efficiency of this agent has not been fully elucidated. We here revealed that sorafenib remarkably and dose-dependently decreased the expression and the transcriptional activity of HIF-1α, and its target gene, vascular endothelial grow factor (VEGF). Further analysis revealed that this reduction of HIF-1α by sorafenib was caused by the inhibition of HIF-1α protein synthesis rather than by the promotion of HIF-1α protein degradation. Moreover, the phosphorylated levels of mTOR, ERK, p70S6K, RP-S6, eIF4E and 4E-BP1 were significantly suppressed by sorafenib. In vivo studies further confirmed the inhibitory effect of sorafenib on the expression of HIF-1α and VEGF proteins, leading to a decrease of tumor vascularisation and growth. Collectively, our data suggest that sorafenib may exhibit anti-angiogenic activity by inhibiting HIF-1α synthesis, which is likely to be achieved through suppressing the phosphorylation of mTOR/p70S6K/4E-BP1 and ERK. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Liping. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 133-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.I / 摘要 --- p.IV / Publications --- p.VI / Acknowledgements --- p.VII / Abbreviations --- p.IX / List of Figures --- p.XI / List of Tables --- p.XIII / Table of Contents --- p.XIV / Chapter Chapter I --- General Introduction --- p.1 / Chapter 1.1 --- Overview of Hepatocellular Carcinoma --- p.1 / Chapter 1.2 --- HBV Infection and HCC Development --- p.6 / Chapter 1.3 --- Overview on Hepatitis B virus X Protein --- p.10 / Chapter 1.4 --- Roles of Hypoxia-inducible Factors in HCC --- p.17 / Chapter 1.5 --- Targeted Therapies and Sorafenib --- p.27 / Chapter Chapter II --- Identification of HBx Mutation Hotspots in HCC Tissues --- p.31 / Chapter 2.1 --- Abstract --- p.31 / Chapter 2.2 --- Introduction --- p.32 / Chapter 2.3 --- Materials and Methods --- p.35 / Chapter 2.4 --- Results --- p.40 / Chapter 2.5 --- Discussion --- p.53 / Chapter Chapter III --- The Relationship between HBx Mutants and HIF-1α --- p.59 / Chapter 3.1 --- Abstract --- p.59 / Chapter 3.2 --- Introduction --- p.60 / Chapter 3.3 --- Materials and Methods --- p.63 / Chapter 3.4 --- Results --- p.70 / Chapter 3.5 --- Discussion --- p.91 / Chapter Chapter IV --- The Effects of Sorafenib on Hypoxia-inducible Factor-1α --- p.96 / Chapter 4.1 --- Abstract --- p.96 / Chapter 4.2 --- Introduction --- p.98 / Chapter 4.3 --- Materials and Methods --- p.101 / Chapter 4.4 --- Results --- p.108 / Chapter 4.5 --- Discussion --- p.124 / Chapter Chapter V --- Conclusion and Future Plans --- p.129 / Chapter 5.1 --- Conclusion --- p.129 / Chapter 5.2 --- Future Plans --- p.131 / References --- p.133

Aim: Cisplatin is the first platinum drug that shows promising anti-tumor effect clinically. Oxaliplatin, a third-generation platinum drug that incorporates a diaminocyclohexane (DACH) structural entity, can overcome cisplatin resistance. R,R-5, a novel platinum compound that integrates the DACH entity with a demethylcantharidin (DMC) component that is derived from a traditional Chinese medicine (TCM) , can also overcome cisplatin resistance. The principal objectives of this study was to investigate in detail, the effect of these compounds at the antephase and G2 checkpoints of the cell cycle, and to establish the relationship (if any) between different structural entities with checkpoint activation. The ultimate aim of the study was to ascertain the potential for the development of novel checkpoint abrogators as anti-tumor agents. / Background: A common procedure in current cancer chemotherapy is to induce genomic stress in cancer cells, leading to irreparable DNA damage and eventually cell death. However, there are several DNA repair mechanisms in cancer cells to maintain genomic stability, which require cell cycle checkpoints to stop cell proliferation for DNA damage repair, thereby avoiding errors in cellular events like DNA replication, transcription and mitosis. Among these cell cycle checkpoints, antephase and G2 checkpoints are two gate checkpoints for mitosis. Abrogation of G2 checkpoint has been reported to give rise to synergistic cytotoxic effect with DNA damaging agents, representing a means of circumventing drug resistance in chemotherapy. / Conclusions: Acute stress to cisplatin can activate the MMR/c-Abl/MEKK1/p38MAPK pathway, leading to the activation of antephase checkpoint, and stop cells from entering mitosis immediately. DACH-containing platinum compound oxaliplatin fails to activate this antephase checkpoint. However, both cisplatin and oxaliplatin can activate the G2 checkpoint, which can be abrogated by DMC. In contrast, RR-5 can bypass both the antephase and G2 checkpoints. In summary, novel TCM-platinum compound R,R-5 can bypass mitotic DNA damage checkpoints in cancer cells and thus has the potential for further development as an anti-cancer drug. / Methods: Microarray analysis was used to detect gene transcription profiles after drug treatments. The activation of mitotic checkpoints was inspected by counting mitotic cells and utilizing flow cytometry. Using Western blotting, the activation of certain key players in the antephase and G2 checkpoint was revealed. MTT assays were performed to show the outcome of checkpoint activation. / Results: In HCT116 cells, 35 genes that facilitate G2/M transition were found to be up-regulated after R,R-5 treatment compared with oxaliplatin in the microarray analysis, implying the bypass of mitotic checkpoints by R,R-5 rather than oxaliplatin. Acute stress (2 hour) of cisplatin activated the antephase checkpoint, resulting in a rapid decrease in mitotic index and phosphorylation of histone H1, which avoided mitotic catastrophe and promoted cell survival in HeLa cells. Further experiments demonstrated that this antephase checkpoint could be abrogated by c-Abl and p38MAPK inhibitors, or siRNAs against c-Abl or MEKK1, suggesting that this checkpoint may be controlled by an MMR/c-Abl/MEKK1/p38MAPK pathway. In contrast, oxaliplatin and R,R-5 did not activate this antephase checkpoint. Moreover, after 24 hour oxaliplatin treatment in HeLa cells, the mitotic index and CDK1 activity were decreased, which could be restored by concomitant treatment with ATM/ATR inhibitor and DMC. This indicated the activation of G2 checkpoint by oxaliplatin and implied that DMC can abrogate oxaliplatin-activated G2 checkpoint by restoring CDK1 activity. Cisplatin could also activate G2 checkpoint, whereas R,R-5 apparently bypassed this G2 checkpoint. / Guan, Huaji. / Adviser: Vincent Hon Leung Lee. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 212-249). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Pang Siu Kwong. / "August 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 181-191) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

背景和目的：胰腺導管腺癌（簡稱胰腺癌）是世界範圍內惡性程度最高的癌癥之一，目前它的5 年生存率不到5%。大部分的病人在診斷初期就已經發展到了局部浸潤或遠處轉移的階段，因此失去了根治性手術切除的机会。輔助性化療對於胰腺癌病人來說是一個首選的治療方案，但是目前只有一小部分病人對化療藥物有良好的反應，而臨床化療失敗常與腫瘤細胞對化療藥物產生耐藥有關。吉西他濱是目前臨床上常用的一線抗癌藥物，但是它的耐藥現象在胰腺癌病人中廣泛存在，也是阻礙其臨床應用的主要原因之一。盡管已經有很多研究致力於揭示吉西他濱在胰腺癌細胞中的耐藥機理，目前臨床上仍然沒有有效的方法應對吉西他濱耐藥。我們的研究主要是為了探討一些以前沒有报道過的參與吉西他濱耐藥機理的基因，借此揭示胰腺癌細胞的吉西他濱耐藥的深層機制，為臨床上的治療提供理論依據。 / 實驗方法：我們實驗室之前在胰腺癌細胞株Capan2 中用全基因組RNAi篩選的方法確定LLGL1 作為抑癌基因能增強吉西他濱在胰腺癌細胞中的細胞毒性。我們隨後用體外細胞毒性分析實驗和皮下腫瘤動物模型來驗證LLGL1 是否能增強吉西他濱的細胞毒性，用蘇木素-伊紅染色和原味末端轉移酶標記技術分析抑制LLGL1 的表達是否會影響吉西他濱誘導的細胞雕亡反應。我們還應用微陣列分析技術進一步探尋LLGL1 的下遊靶蛋白，用實時定量PCR(qRT-PCR) 、蛋白印跡法（western blotting）、熒光素酶檢測等技術來進一步證實LLGL1 與下遊靶蛋白的關系，用免疫組織化學方法探究LLGL1 下遊靶蛋白在胰腺癌組織中的表達情況，以及該蛋白與LLGL1 的表達相關性，還應用染色體免疫共沈澱的方法探討轉錄因子Sp1(pThr453) 和RNA 聚合酶 II 在LLGL1 下遊靶蛋白的啟動子上的富集情況。 / 實驗結果：LLGL1 能增強吉西他濱在胰腺癌中的細胞毒性，抑制該基因的表達能誘導胰腺癌細胞對吉西他濱的耐藥，而上調該基因的表達則會增強胰腺癌細胞對吉西他濱的細胞毒性反應。OSMR 是LLGL1 的下遊靶蛋白, 其在胰腺癌組織中的表達與LLGL1 呈負性相關，抑制OSMR 的表達可以逆轉由LLGL1表達下調引起的吉西他濱耐藥現象。OSMR 表達上調可以增強腫瘤幹細胞標記物CD44 和CD24 的表達。另外，在胰腺癌細胞中，抑制LLGL1 的表達能激活ERK2/Sp1 信號通路，導致磷酸化Sp1(pThr453)的表達升高。OSMR 啟動子既沒有TATA 元件也沒有INR 元件，但是有Sp1 结合元件可供Sp1 結合。磷酸化Sp1(pThr453)可以結合到OSMR 啟動子的Sp1 结合元件上，從而促使RNA 轉錄酶II 結合到該啟動子上，啟動OSMR 基因的轉錄。 / 結論：我們的研究發現：1，LLGL1 能增強吉西他濱在胰腺癌中的細胞毒性，抑制該基因在胰腺癌細胞中的表達能上調OSMR 的表達，並誘導吉西他濱耐藥；2，OSMR 的表達在胰腺癌組織中與LLGL1 呈負性相關；3，下調LLGL1的表達能激活ERK2/Sp1 信號通路，進一步導致磷酸化Sp1(pThr453)和RNA 轉錄酶II 在OSMR 啟動子上的聚集，最終促使OSMR 的高表達，而下調LLGL1的表達能抑制該調節通路，從而抑制OSMR 的轉錄。 / Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant cancers worldwide. Its 5-year survival rate is less than 5%, because most patients have already developed to the advanced stage of local invasion or distant metastasis once diagnosed, and missed the chances of curable surgical resection. Adjuvant chemotherapy is an alternative therapeutic strategy against PDAC. Yet, only very small proportion of patients could benefit from chemotherapy due to the innate and easily-acquired chemo-resistance in PDAC cells, especially to the first-line chemotherapeutic drug, gemcitabine. Many studies have been conducted to exploring the mechanisms underlying gemcitabine resistance in PDAC cells, but gemcitabine resistance is still the major obstacle impeding PDAC patients benefits from chemotherapy. Our studies aimed to investigate novel genes involved in gemcitabine response and to explore the undefined mechanisms generating gemcitabine resistance in PDAC cells. / Methods: Our colleagues previously performed genome-wide RNAi screening in gemcitabine-sensitive Capan2 cells. Lethal giant larvae homolog 1 (LLGL1) was identified as a potential gemcitabine-sensitizing gene which was then validated by our subsequent in-vitro drug cytotoxicity assay in LLGL1-inhibited Capan2 and SW1990 cells and in vivo subcutaneous xenograft mouse model. Hematoxylin & Eosin staining and terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling were applied for the assessment of apoptotic effects induced by gemcitabine in subcutaneous xenografts. We did gene expression microarray analysis to explore the potential downstream targets of LLGL1. Western blotting, qRT-PCR, and luciferase assay were applied to validate the downstream target of LLGL1 that were figured out by microarray analysis. We also did immunohistochemical staining to investigate the expression levels and correlationship of LLGL1 and its downstream target in PDAC specimens. Chromatin immunoprecipitation was performed to explore the enrichment of the transcriptional factor Sp1(pThr453) and RNA polymerase II (Pol II) at the promoter of the downstream targets of LLGL1. / Results: LLGL1 was identified as a gemcitabine-sensitizing gene, whose inhibition remarkably reduced gemcitabine response in gemcitabine-sensitive Capan2 and SW1990 cells, and ectopic expression induced gemcitabine response in gemcitabine-resistant PANC1 cells. Oncostatin M receptor (OSMR) was identified as a downstream target of LLGL1, whose expression was negatively correlated with LLGL1, and knockdown of OSMR significantly reversed gemcitabine resistance induced by LLGL1 inhibition in Capan2 and SW1990 cells. Additionally, activation of OSMR signaling was associated with the elevated expression of cancer stem cell markers, CD44 and CD24, both of which had already been identified to contribute to gemcitabine resistance in PDAC cells. Moreover, OSMR up-regulation induced by LLGL1 inhibition in SW1990 cells depended on the activation of ERK2/Sp1 signaling and subsequent accumulation of Sp1(pThr453) and Pol II at the TATA-less, INR-less but Sp1-binding-site-rich promoter of OSMR, while ectopic expression of LLGL1 in PANC1 cells inactivated ERK2/Sp1 signaling and subsequently reduced the enrichment of Sp1(pThr453) and Pol II at OSMR promoter. / CONCLUSIONS: Our studies revealed the novel tumor suppressive role of LLGL1 as a gemcitabine-sensitizing gene in PDAC cells. Loss of LLGL1 resulted in the activation of ERK2/Sp1 signaling and up-regulation of OSMR expression, and ultimately desensitized gemcitabine response in PDAC cells. More importantly, ectopic expression of LLGL1 disrupted such regulatory axis and improved gemcitabine response. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhu, Yinxin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 154-183). / Abstracts also in Chinese.