by Wai-Tong Leung. / Thesis (M.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 282-315). / Title Page --- p.i / Dedication --- p.ii / Table of Contents --- p.iii / Precis to the thesis --- p.7 / Glossary of abbreviation used in the thesis --- p.20 / List of Figures and Pictures --- p.22 / List of Tables --- p.26 / Acknowledgments --- p.30 / Statement of Originality --- p.32 / Chapter PART ONE --- Introduction / Chapter Chapter 1 --- Background for Hepatocellular Carcinoma / Chapter 1. --- History --- p.33 / Chapter 2. --- "Epidemiology - World wide, China,Hong Kong" --- p.36 / Chapter 3. --- Pathology --- p.55 / Chapter 4. --- Aetiology --- p.62 / Chapter 5. --- Clinical Manifestation and Diagnosis --- p.68 / Chapter 6. --- Natural History --- p.74 / Chapter 7. --- Conventional Treatment --- p.76 / Chapter 8. --- Prognosis --- p.85 / Chapter Chapter 2 --- Hepatocellular Carcinoma in Hong Kong / Chapter 1. --- Clinical Study of 1119 cases of Hepatocellular Carcinoma --- p.88 / Chapter PART TWO --- Conventional Treatment for Hepatocellular Carcinoma / Chapter Chapter 3 --- Chemotherapy for Hepatocellular Carcinoma / Chapter 1. --- Review of the Literature in Systemic Chemotherapy --- p.108 / Chapter 2. --- Phase II Trials of Systemic Chemotherapy --- p.114 / Chapter a. --- VP16-213 --- p.115 / Chapter b. --- Low Dose 4'-Epidoxorubicin --- p.116 / Chapter c. --- High Dose 4'-Epidoxorubicin --- p.117 / Chapter d. --- 5_FU and High Dose Folinic Acid --- p.119 / Chapter e. --- High Dose Ifosfamide --- p.122 / Chapter 3. --- Review of the Literature in Intra-Arterial Chemotherapy --- p.125 / Chapter 4. --- Intra-Arterial Chemotherapy Trial Performed in PWH --- p.131 / Chapter a. --- An Early Phase II Trial on Treatment of Inoperable Hepatocellular Carcinoma by Intrahepatic Arterial Chemotherapy with Lipiodol and 4'-Epidoxorubicin --- p.131 / Chapter b. --- "Phase II Trial of Treatment of Inoperable Hepatocellular Carcinoma by Intra-Arterial Lipiodol and High Dose 4'-Epidoxorubicin, a Comparison with Intravenous 4'-Epidoxorubicin" --- p.137 / Chapter PART THREE --- Novel Treatment for Hepatocellular Carcinoma / Chapter Chapter 4 --- Selective Internal Radiation Treatment for Hepatocellular Carcinoma / Chapter 1. --- Review of the Literature in Radiotherapy for Liver Tumours --- p.145 / Chapter 2. --- Selective Internal Radiation with 131Iodine-Lipiodol in HCC / Chapter a. --- Background and Review of the Literature --- p.151 / Chapter b. --- Physical Aspects of 131Iodine-Lipiodol --- p.154 / Chapter c. --- Bio-distribution of 131I-L in one HCC Patient --- p.156 / Chapter d. --- Clinical Study on the Use of131I-Lipiodol in the Treatment of Inoperable HCC --- p.168 / Chapter 3. --- Selective Internal Radiation with 90Yttrium-Microspheres in HCC / Chapter a. --- Background and Review of the Literature --- p.182 / Chapter b. --- Physical Aspects of 90Yttrium-Microspheres --- p.187 / Chapter c. --- Treatment of Inoperable Hepatocellular Carcinoma with Intrahepatic-Arterial 90Yttrium Microspheres - An Early Phase II Study --- p.195 / Chapter d. --- Treatment of Inoperable Hepatocellular Carcinoma with Intrahepatic-arterial 90Yttrium Microspheres through Hepatic Angiography --- p.214 / Chapter 4. --- Selection of Patients for 90Yttrium -Microspheres Treatment --- p.230 / Chapter a. --- Diagnostic Scintigraphy with Hepatic Intra-Arterial Technetium-99m Macroaggregated Albumin in the Determination of Tumour to Non-Tumour Uptake Ratio in Hepatocellular Carcinoma --- p.230 / Chapter b. --- Measuring Lung Shunting in Hepatocellular Carcinoma with Intrahepatic-Arterial Technetium-99m Macroaggregated Albumin Scan --- p.245 / Chapter c. --- Correlation of Tumour Vascularity Grading by Selective Hepatic Angiography with T/N Ratio from Tc-MAA Scanin Hepatocellular Carcinoma --- p.260 / Chapter d. --- Pulmonary Complications from 90Yttrium-Microspheres Treatment --- p.266 / Conclusion of the Thesis --- p.280 / References to Chapter1 --- p.282 / References to Chapter2 --- p.296 / References to Chapter3 --- p.297 / References to Chapter4 --- p.308 / Appendix: / Chapter 1. --- Recommendations for Grading of Acute and Subacute Toxicityin Cancer Treatment --- p.316 / Chapter 2. --- Recommendations for Grading of Response Criteria --- p.318 / Selected Publications by the Author Relevant to the Thesis --- p.319

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer-related deaths. Systemic therapies are the main treatment options for HCC patients with advanced disease (∼ 80% of all cases). However, only very moderate clinical responses are achieved with most of the conventional therapies. Thus, more effective therapeutic strategies are much needed. The PI3K/Akt/mTOR signaling pathway, which plays a critical role in controlling cell proliferation and survival, is aberrantly activated in ∼ 45% HCC, suggesting it to be a potential target for HCC treatment. Moreover, emerging evidences indicate that activation of the PI3K/Akt/mTOR pathway may be associated with resistance to many cytotoxic chemotherapies, including microtubule targeting agents. In this study, by gene expression profiling and gene ontology analysis, "microtubule-related cellular assembly" was identified to be the major biological/functional process involved in HCC development, suggesting that microtubule is also an important therapeutic target for HCC. With these understandings, it is hypothesize in this thesis that combined targeting of a key component ofthe PI3K/Akt/mTOR pathway, namely the mammalian target of rapamycin (mTOR) and the microtubule would be an effective therapeutic strategy for HCC. The objectives of the thesis are to examine the therapeutic potential of microtubule targeting, mTOR targeting, and combined targeting of the microtubule and mTOR in both in vitro and in vivo models of HCC. / In summary, the PI3K/Akt/mTOR pathway and the microtubule represent promising therapeutic targets for HCC treatment. The findings from this thesis offer a rationale for combining mTOR inhibitors with microtubule targeting agents for effective HCC treatment. / In the second part, the effect of mTOR inhibition, either alone or in combination with an additional microtubule targeting agent (vinblastine) was investigated in HCC. Temsirolimus, an mTOR inhibitor, suppressed HCC cell proliferation in as early as 24 hrs with an IC50 of 1.27+/-0.06muM (Huh7), 8.77+/-0.76muM (HepG2), and 52.95+/-17.14muM (Hep3B). Vinblastine (1nM) alone caused 30--50% growth inhibition in 3 HCC cell lines. In these HCC cell lines, it was found that temsirolimus/vinblastine combination resulted in an additive to synergistic effect (when compared to single agents alone) with maximum growth inhibition of 80--90% as early as 24 hrs upon treatment. This marked growth inhibition was accompanied with cell cycle arrest at both G1 and G2/M phases, and PARP cleavage (a hallmark for apoptosis). Moreover, the combination specifically caused concerted down-regulation of several important anti-apoptotic and survival proteins (survivin, Bcl-2 and Mcl-1), which was not observed in single agent treatments. It was hypothesized that inhibition of these key anti-apoptotic/survival proteins may represent a novel mechanistic action of this highly effective combination approach of dual targeting of mTOR and microtubule by temsirolimus/vinblastine in HCC cells. Indeed, transient over-expression of each of these genes (survivin, Bcl-2 or Mcl-1) in HCC cells did partially rescue the growth inhibitory effect of the temsirolimus/vinblastine combination. More importantly, this novel combination significantly suppressed the growth of HCC xenografts in nude mice (when compared with single agents alone). / In the third part, the anti-tumor effect of another mTOR inhibitor everolimus in combination with microtubule targeting agents, vinblastine and patupilone (a microtubule-stabilizing agent), was investigated in HCC cells. Everolimus/vinblastine combination resulted in an additive to synergistic effect accompanied with cell cycle arrest at both G1 and G2/M phases, and PARP cleavage. The combination also caused concerted down-regulation of anti-apoptotic and survival proteins (survivin, Bel-2 and Mel-1) as observed with the temsirolimus/vinblastine combination. However, everolimus only moderately enhanced the sensitivity of patupilone for reasons unknown. / Taxanes are the major chemotherapeutic agents that target the microtubule. In the first part of the thesis, the anti-tumor activity of two taxanes, paclitaxel and docetaxel (which are known to stabilize microtubules) was examined and compared with doxorubicin (a DNA intercalating agent). Across all three HCC cell lines tested, it was found that the microtubule targeting agents, taxanes, were more efficacious than doxorubicin. This supports the initial finding that microtubule assembly process is functionally important in HCC. Recent studies demonstrated that using nanoparticles for drug delivery can greatly enhance therapeutic efficacy and reduce side-effects. Therefore, the nanoparticle albumin-bound (nab)-paclitaxel was employed to further evaluate the therapeutic efficacy of such a delivery strategy in HCC models. In all three HCC cell lines tested, nab-paclitaxel was found to be the most effective agent, with an average IC50 value of 0.16--10.42nM, when compared to non-conjugated taxanes (paclitaxel, docetaxel) and doxorubicin. In vitro analysis showed that nab-paclitaxel was able to induce cell cycle arrest at G2/M phase and apoptosis in HCC cells. In vivo study demonstrated that nab-paclitaxel readily inhibited the growth of HCC xenografts with lower toxicity when compared to paclitaxel, docetaxel and doxorubicin. Moreover, specific silencing of a key regulatory protein for microtubule dynamics, Stathmin 1, by siRNA significantly enhanced the effect of nab-paclitaxel in HCC cells, resulting in synergistic growth inhibition in vitro. / Zhou, Qian. / Advisers: Winnie Yeo; Vivian Lui; Nathalie Wong. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 148-164). / 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.

研究背景： / 肝癌是全球常見的惡性腫瘤之一，世界上每年大約有50萬死亡病例，並且呈逐年上升之勢， 是全球第3位的腫瘤死亡原因。慢性乙型和丙型肝炎病毒感染是肝癌的主要成因。肝癌惡性程度高、預後差，並且目前的治療手段非常有限，術後易復發和轉移，迄今尚無正式獲准有效治療藥物。現階段，治療肝癌的主要方法是手術切除，但是隨之引起的併發症以及較高的復發機率嚴重影響了治療的療效，大大降低肝癌病人的存活期。 / 研究目的： / 分析TDP對肝癌細胞和肝腫瘤旁細胞生長的影響；分析TDP抑癌的分子靶標蛋白及其分子機理；驗證TDP對肝癌動物模型的抑制效果。開發一種新型有效的肝癌治療藥物。 / 研究方法： / 首先用MTT法從102種來源於嶺南山竹子的純複合物中分離出了TDP,它是一種甾醇類化合物。採用MTT法檢測TDP對腫瘤細胞生長的影響；流式細胞實驗驗證TDP能否引起腫瘤細胞的凋亡；採用蛋白組學和質譜分析找出TDP抑癌的分子靶標；進一步的蛋白功能增加和缺失實驗證明Hsp27的功能和作用；生物資訊學驗證HSP27和TDP的作用結果；最後利用動物模型驗證TDP對肝腫瘤的治療效果。 / 結果： / TDP不但能效率極高的抑制肝癌細胞的生長而且可以大量誘發肝癌細胞的凋亡，而對正常的肝癌旁細胞沒有影響。二維電泳以及質譜分析TDP處理的肝癌細胞對比DMSO處理的肝癌細胞發現了具有不同表達水準的18種蛋白，Hsp27是其中一個在TDP誘導下調變化倍數較大並且與細胞凋亡有密切關係的蛋白，Hsp27的過表達以及Knock-down都充分驗證了TDP通過調節Hsp27的表達參與了依賴於caspase的線粒體凋亡途徑，在Western Blotting以及RT-PCR中得到了充分的驗證。生物資訊學預測TDP可以與Hsp27結合，實驗結果表明TDP可以誘導Hsp27的聚集並導致功能喪失。動物實驗腫瘤生長結果以及免疫組化結果證明，TDP可以在很大程度上對肝癌有抑製作用。 / 結論： / 本研究首次表明，TDP如果不是完全的,最起碼也是部分通過誘導依賴於caspase的線粒體凋亡的途徑來抑制肝癌細胞的增值和分化, 具有明顯的抗腫瘤的功效，特別是對Hsp27高表達的腫瘤細胞有比較明顯的作用，是一種值得繼續深入研究的有較高潛在價值的藥物。 / Background： / Hepatocellular carcinoma (HCC), the most common primary hepatic malignancy, is a global public health problem that accounts for approximately 500,000 deaths annually. Chronic hepatitis B and hepatitis C infections are the major risk factors for the development of HCC. Due to the high rate of these infections, the incidence of HCC remains alarmingly high globally. Although great advances have been made in HCC treatment, poor prognosis and high risk of recurrence have been major challenges to patients. Currently, surgical resection is the main treatment option for HCC patients; however, complications arising from surgery can threaten its therapeutic effect and patients’ survival. / Objectives： / To characterize the functions of 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b]Xanthone (TDP) in cell proliferation of HepG2 cells; to discover the molecular target genes and elucidate the underlying molecular mechanism of TDP; to examine the in vivo function of TDP in a nude mouse tumor model of HCC. Finally, to investigate TDP’s potential as an anti-HCC drug candidate. / Methods and Results： / In this study, we discovered that TDP, isolated from the Chinese medicinal herb, Garcinia oblongifolia, strongly inhibited cell growth and induced caspase-dependent mitochondrial apoptosis in HCC, as evidenced from MTT assay and flow cytometry analysis. Two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to find the molecular targets of TDP in HCC cells, and eighteen proteins were identified with altered expression, with Hsp27 protein being one of the proteins most significantly down-regulated by TDP. Further Hsp27-siRNA knockdown and Lenti-Hsp27 overexpression studies found that Hsp27 was involved in TDP induced mitochondrial apoptosis, with bioinformatics predictions and biological results revealing that TDP might cause Hsp27 protein form dimer and consequent degradation via the ubiquitin-proteasome system. Finally, subcutaneously injecting cancer cells with Hsp27 expression vector into the dorsal flank of nude mice tumor model also demonstrated the suppressive effect of TDP on HCC. / Conclusions： / In summary, our study discovered that TDP, a natural xanthone, was a potent inhibitor of Hsp27 in HCC. TDP inhibited cell growth and induced apoptosis by inducing Hsp27 degradation, which stimulated mitochondrial cytochrome C release which resultantly activated caspase-3 and caspase-9. These data combined with the results of the animal model strongly supported TDP’s potential as a novel anti-cancer drug candidate, especially for cancers with an abnormally high expression of Hsp27. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Fu, Weiming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 111-151). / Abstract also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iiv / Acknowledgment --- p.vi / Publications --- p.viii / List of Contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.1.1 --- Overview of HCC --- p.1 / Chapter 1.1.2 --- Epidemiology of HCC in China and Hong Kong --- p.3 / Chapter 1.2 --- Etiology of HCC --- p.7 / Chapter 1.2.1 --- Cirrhosis --- p.8 / Chapter 1.2.2 --- HBV infection --- p.9 / Chapter 1.2.3 --- HCV infection --- p.10 / Chapter 1.2.4 --- Viral Co-Infection --- p.11 / Chapter 1.2.5 --- Fatty Liver Disease and Cryptogenic Cirrhosis --- p.12 / Chapter 1.2.6 --- Alcohol --- p.13 / Chapter 1.2.7 --- Iron --- p.13 / Chapter 1.2.8 --- Aflatoxin --- p.14 / Chapter 1.2.9 --- Others --- p.14 / Chapter 1.3 --- Diagnosis of HCC --- p.14 / Chapter 1.4 --- Prognosis of HCC --- p.17 / Chapter 1.5 --- Treatment of HCC --- p.19 / Chapter 1.5.1. --- Early stage --- p.19 / Chapter 1.5.2. --- Intermediate and advanced stage --- p.24 / Chapter 1.5.3. --- Terminal stage --- p.28 / Chapter 1.6 --- Signaling pathways in HCC --- p.28 / Chapter 1.6.1 --- Proliferation signaling pathways --- p.29 / Chapter 1.6.2 --- Signaling pathways frequently dysregulated in HCC --- p.30 / Chapter 1.6.3 --- Pathways involved in liver development and cell differentiation --- p.34 / Chapter 1.6.4 --- Inflammation pathways involved in hepatocarcinogenesis --- p.35 / Chapter 1.6.5 --- Pathways involved in neoangiogenesis --- p.37 / Chapter 1.6.6 --- The P53 tumor suppressor --- p.38 / Chapter 1.6.7 --- Heat shock proteins in HCC --- p.39 / Chapter 1.7 --- The roles of microRNAs in liver cancer progression --- p.42 / Chapter 1.8 --- TCM in the treatment of HCC --- p.45 / Chapter 1.8.1 --- Introduction --- p.45 / Chapter 1.8.2 --- Garcinia --- p.49 / Chapter 1.9 --- Objectives of the study --- p.51 / Chapter Chapter 2 --- Materials and Methods --- p.52 / Chapter 2.1 --- Preparation of the pure compounds --- p.52 / Chapter 2.2 --- Liver cell lines and tissue culture --- p.52 / Chapter 2.3 --- Human tissue samples --- p.52 / Chapter 2.4 --- Cell viability assessment with MTT assay --- p.53 / Chapter 2.5 --- Apoptosis analysis --- p.53 / Chapter 2.6 --- Two-dimensional electrophoresis (2-DE), protein visualization and image analysis --- p.54 / Chapter 2.6.1 --- Materials --- p.54 / Chapter 2.6.2 --- Protein extraction --- p.54 / Chapter 2.6.3. --- 2-DE protein profiling --- p.55 / Chapter 2.6.4. --- Gel staining and image analysis --- p.55 / Chapter 2.6.5. --- In-gel protein digestion with trypsin --- p.56 / Chapter 2.6.6. --- MALDI-TOF mass spectrometric analysis --- p.56 / Chapter 2.6.7. --- Database search --- p.57 / Chapter 2.7.1 --- Sample preparation --- p.58 / Chapter 2.7.2 --- SDS-PAGE --- p.58 / Chapter 2.7.3 --- Protein transfer --- p.58 / Chapter 2.7.4 --- Blocking --- p.59 / Chapter 2.7.5 --- Incubation with primary and secondary antibodies --- p.59 / Chapter 2.7.6 --- Proteins Visualization --- p.59 / Chapter 2.8 --- Real-time PCR --- p.60 / Chapter 2.9 --- Vector construction and lentivirus production --- p.61 / Chapter 2.9.1 --- Lenti-vector construction for Hsp27 expression --- p.61 / Chapter 2.9.2 --- Lentivirus production --- p.62 / Chapter 2.9.3 --- Lentivirus infection --- p.63 / Chapter 2.10 --- SiRNAs transfection. --- p.63 / Chapter 2.11 --- Identification of potential protein targets for TDP --- p.64 / Chapter 2.12 --- In Vivo Tumorigenesis --- p.64 / Chapter 2.13 --- Assay of chaperone activity of Hsp27 using lysozyme as substrate --- p.65 / Chapter 2.14 --- Mitochondria and cytosolic proteins preparation --- p.66 / Chapter 2.15 --- Immunohistochemistry (IHC) --- p.67 / Chapter 2.15.1 --- Preparation of paraffin tissue sections --- p.67 / Chapter 2.15.2 --- Immunostaining --- p.67 / Chapter 2.16 --- Methodology of this study --- p.68 / Chapter 2.17 --- Statistical analysis --- p.68 / Chapter CHAPTER 3 --- Results --- p.69 / Chapter 3.1 --- Introduction --- p.69 / Chapter 3.2 --- TDP significantly suppressed cell growth and induced apoptosis in HCC cells. --- p.69 / Chapter 3.2.1 --- TDP was identified from 102 pure compounds by using MTT assay --- p.69 / Chapter 3.2.2 --- TDP significantly suppressed HCC cell growth --- p.73 / Chapter 3.2.3 --- TDP induced the apoptosis of HCC cells --- p.74 / Chapter 3.3 --- Study of the molecular mechanism of TDP on HCC --- p.76 / Chapter 3.3.1 --- The comparative proteomic profiling --- p.76 / Chapter 3.3.2 --- Hsp27 was one of the molecular targets of TDP in HepG2 cells. --- p.80 / Chapter 3.3.3 --- TDP induced apoptosis through the caspase-dependent mitochondrial pathway. --- p.82 / Chapter 3.3.4 --- Hsp27 involved in the mitochondrial apoptosis induced by TDP --- p.84 / Chapter 3.3.5 --- Enforced Hsp27 overexpression rescued the mitochondrial apoptosis induced by TDP in HepG2 cells --- p.87 / Chapter 3.3.6 --- The possible regulatory signaling by TDP --- p.91 / Chapter 3.4 --- TDP directly targeted Hsp27 and destroyed its chaperone action --- p.92 / Chapter 3.5 --- Degradation of Hsp27 aggregation stimulated by TDP was mediated by ubiquitin-proteasome system (UPS) pathway --- p.96 / Chapter 3.6 --- Nude mice model demonstrated the suppressive effect of TDP on HCC --- p.97 / Chapter Chapter 4 --- Discussion and Conclusions --- p.100 / Chapter 4.1 --- Discussion --- p.100 / Chapter 4.2 --- Conclusion --- p.110 / Reference --- p.111