published_or_final_version / Anatomy / Master / Master of Philosophy

 Hepatocellular carcinoma (HCC) is the primary cancer of liver. It is the fifth common malignant tumor in men while seventh common in women. Aetiology of HCC is complex; however, it is now believed that sustained chronic liver injury and fibrosis are critically involved in the development of HCC. Prevention and treatment of HCC is far from desirable and prognosis remains poor. Coptis is a Chinese herbal Medicine which has been used for more than thousands years for clearing heats, dampness and toxics. Recently, studies from our group reported the hepatoprotective effect of Coptis and its major active component, berberine, on acute liver injury and berberine was extensively studied for their anti-tumor effect. However, there’s no comprehensive investigation focusing on the preventive and curative potential of berberine on HCC. Hence, here we hypothesized Coptis and berberine exhibits both preventive and curative effects on HCC. The prevention of HCC by berberine and Coptis may rely on their effects on chronic liver damage and fibrosis, and the curative action may depend on their actions on the angiogenesis, tumor growth and invasion of HCC. Both in vitro cell models and in vivo animal system were used in our study and some molecular events were investigated. We found that berberine and Coptis could significantly attenuate the chronic liver injury and fibrosis by restoring the anti-oxidative enzyme SOD activity in CCl4-, bile duct ligation- and alcohol-induced liver injury and fibrosis model. Recovery of SOD activity prevents the hepatocytes from apoptosis by inhibiting the oxidative stress-induced Erk1/2 signaling activation. The prevention of berberine and Coptis on chronic liver injury and fibrosis may contribute to its preventive effect against HCC. Then we found that berberine (as representative to Coptis) could suppress the angiogenesis of HCC, in which berberine does not directly act on the blood vessel formation, but suppress the expression and secretion of pro-angiogenic factors VEGF in HCC cells, and Id-1 inhibition by berberine plays a central role in the suppression of HIF-1α/VEGF and NF-κB pathways. We also found that berberine could induce both apoptotic and autophagic cell death in HCC, and the mitochondria related-caspases activation confers the apoptosis while mTOR inhibition initiates autophagy in berberine treated- cells. We found that berberine could suppress the migration and invasion of HCC cells as well, and Rho-GTPases/ROCK signaling is the particular target in berberine’s anti-invasive action. Finally, to dig out some molecular events involved in berberine’s action on HCC, we studied critically the mechanism underlying berberine’s inhibition on Cyclin D1 in HCC. We found berberine may promote the IKKα-induced Cyclin D1 phosphorylation at T286, and this may initiate the ubiquitination-dependent proteasomal degradation of Cyclin D1 in berberine-treated HCC cells and contribute to berberine’s anti-HCC action. Critical clinical trials and OMICS techniques were planned to further our comprehensive study on Coptis and berberine’s effects on HCC. In all, we found that berberine targets on different stages and molecules and exerts preventive and curative potential against HCC. Our study sheds light on the clinical application of berberine in HCC treatment. / published_or_final_version / Chinese Medicine / Doctoral / Doctor of Philosophy

abstract / Pathology / Master / Master of Philosophy

published_or_final_version / abstract / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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.

丹參為雙子葉植物唇形科鼠尾草族植物的乾燥根及根莖。在中國，丹參為廣泛用於治療心血管疾病的藥用植物，而在西方，丹參也常作為一種輔助性藥物。《中國藥典2010版》收錄了35個以上含有丹參的複方或者方劑。在這些複方中，採用了富含丹酚酸和丹參酮的丹參水提物、乙醇提取物或兩者的混合物。丹參提取物具有較強的抗氧化作用，被認為在化學預防和化療的輔助治療中有一定用途。作為主要的丹參水溶性成分，熱敏感的丹酚酸在提取與加熱過程中可能會降解為其他丹酚酸。丹參水提取物的化學組成可能會在不同熱水提取溫度下有所不同，進而影響其藥理活性。在本研究中，通過加熱回流提取和在不同溫度下的微波提取（MAE-W）獲得了6種丹參水提取物，並對這些提取物進行化學成分和藥理分析，考察它們的抗氧化、抗凋亡和血管舒張作用。在這些提取物中，第三輪的微波提取物（100 oC）含有最多的丹酚酸和丹參酮，在1，1-二苯基-2-三硝基苯肼（DPPH）法和鐵還原/抗氧化能力（FRAP）法中具有最強的抗氧化活性，在2,2'-偶氮二(2-脒基丙烷)二鹽酸鹽（AAPH）誘導人血紅細胞的溶血實驗和過氧化氫誘導大鼠心肌細胞H9c2凋亡實驗中還顯示了最強的抑制作用，對大鼠腦基底動脈有最強的鬆弛效應。這些丹參水提取物的抗氧化作用與它們的血管舒張效應呈一定的線性關係（回歸係數r = 0.895 - 0.977）。通過多元線性回歸分析發現，丹參素可以作為丹參水提物的抗氧化和血管舒張功能的顯著性標記物，而丹參酮IIA則是抑制過氧化氫誘導大鼠心肌H9c2細胞凋亡的標記物。 / 作為丹參中主要的脂溶性成分，丹參酮在不同的腫瘤細胞系和荷瘤小鼠模型中展示了抗癌潛力。這些丹參酮的抗癌機制包括細胞週期阻滯，觸發半胱天冬酶（Caspase）依賴的內源性和外源性的凋亡途徑和絲裂原激活的蛋白激酶（MAPK）信號通路等。丹參新酮（miltirone）是從丹參中分離得到的松香烷型二萜醌類化合物，具有多種的藥理活性，如抗氧化，抗焦慮和抗腫瘤等。本研究評估了丹參新酮在人肝癌HepG2細胞系和P-糖蛋白（P-gp）過表達的阿霉素耐藥HepG2細胞系（R-HepG2）中的凋亡作用及其機制。丹參新酮在HepG2細胞中顯示了細胞毒性（EC₅₀值為7.06 微摩），而丹參新酮在抑制HepG2和R-HepG2細胞增殖中的濃度依賴性沒有顯著性差異。丹參新酮（1.56 - 6.25微摩）與阿霉素（DOX）對R-HepG2細胞的增殖具有協同效應，在達到50的生長抑制時，它們的聯合用藥指數為0.3至0.5。流式細胞術分析表明，丹參新酮降低了R-HepG2細胞中P-gp介導的阿霉素外排，分子對接研究表明該效果是通過抑制P-gp的藥物結合位點。在非壞死濃度（25微摩或以下），丹參新酮在HepG2和R-HepG2細胞中活化了Caspase依賴的凋亡途徑，誘導產生活性氧（ROS）和氧化應激，且觸發ROS介導的包括p38 MAPK，應激活化蛋白激酶/c-Jun氨基末端激酶（SAPK / JNK）以及細胞外調節激酶1和2在內的MAPK信號通路。綜上所述，在R-HepG2中丹參新酮是P-gp和細胞增殖的雙重抑制劑，顯示了其在治療肝癌（HCC）的潛力。 / 為了增加藥物開發的成功率，在藥物發現的早期階段應考察新化學實體（NCEs）的蛋白結合率，清除率，藥動學參數，以及藥物代謝相互作用等體內代謝參數。以往的研究已經顯示了從丹參中分離得到的四種主要丹參酮對人和大鼠的細胞色素P450酶介導的探針底物的代謝具有不同程度的抑制作用，需要注意丹參和其他藥物間的相互作用。本研究的另一目的是在人類肝微粒體中探討丹參新酮與探針底物間的細胞色素P450酶介導的代謝相關的相互作用。人肝微粒體孵育實驗結果表明丹參新酮對CYP1A2（IC₅₀值為 1.73微摩）和CYP2C9（IC₅₀值為8.61微摩）有中等強度的抑制，對CYP2D6（IC₅₀值為30.20微摩）和CYP3A4（IC₅₀值為33.88微摩）有弱的抑制。酶動力學和分子對接研究的結果進一步表明，丹參新酮為CYP1A2（Ki值為3.17微摩）的中等強度混合型抑制劑，是CYP2C9（Ki值為1.48微摩）的中等強度競爭型抑制劑，也是CYP2D6（Ki值為24.25微摩）和CYP3A4（Ki值為35.09微摩）的弱的混合型抑制劑。這些結果表明，應考慮丹參新酮與CYP1A2和CYP2C9代謝的藥物間的相互作用，但是可認為其與CYP2D6及CYP3A4代謝的藥物間幾乎不存在相互作用。 / 總之，本研究考察了不同提取方法對丹參提取物成份及其藥效的影響，確定了不同用途的丹參提取物的質控標記物。本研究還考察了丹參新酮體外抗肝癌的能力及其藥物代謝相互作用為基礎的類藥性,為其進一步的體內試驗提供了依據。 / Danshen, the dried root and rhizome of Salvia miltiorrhiza Bg. (Fam. Labiatae), is a widely used medicinal plant for the treatment of cardiovascular diseases in China and also a complementary medicine in the West. Danshen is indexed in the Pharmacopoeia of People’s Republic of China (2010 Edition), with more than 35 formulations and concoctions containing Danshen water-extracts, ethanolic extracts or their combination which are rich in phenolic acids and tanshinones with various contents. Danshen extracts have been considered for the use as an adjunct in chemoprevention and chemotherapy due to their strong antioxidant effects. Phenolic acids, the major water-soluble components in Danshen, are thermosensitive and may degrade to other phenolic acids during extractions upon heating. The chemical profiles of Danshen water-extracts may vary with different heat water extraction at different temperatures, affecting the composition and bioactivity of the extracts obtained. In this study, six water-extracts of Danshen obtained from heat reflux water extraction and microwave-assisted extraction with water (MAE-W) at different temperatures were prepared for evaluation of their composition and pharmacological effects such as antioxidant, anti-apoptosis and vascular relaxation. Among these extracts obtained, the third-round MAE-W (100 °C) product, which was the last round product obtained by extracting the same crude material three times, had the highest contents of phenolic acids and tanshinones, with the strongest antioxidant activity estimated by 2, 2-diphenyl-1-(2, 4, 6-trinitrophenyl) hydrazyl (DPPH) assay and ferric reducing / antioxidant potential (FRAP) assay. This extract also possessed the strongest inhibitory effects on 2, 2'-azobis-2-amidino-propane (AAPH)-induced haemolysis in human red blood cells, hydrogen peroxide-induced apoptosis in rat heart H9c2 cells and the highest relaxation effects on rat basilar artery. The antioxidant effects of Danshen water-extracts linearly correlated to their relaxation effects (r = 0.895 to 0.977). Through multiple linear regression analysis, danshensu was found to be the most significant marker in the antioxidant and vasodilation effects of Danshen water-extract, while tanshinone IIA as the marker on hydrogen peroxide-induced apoptosis in rat heart H9c2 cells. Danshensu is, therefore, a useful marker for the quality control of Danshen water-extracts in antioxidant and vasodilation, while tanshinone IIA for anti-apoptotic potential of water-extracts. / Tanshinones, the major lipid-soluble components isolated from Danshen, have been reported for their anti-cancer potential in various cell lines and tumor-bearing mice models. Their anti-cancer mechanisms are also well-studied, mainly through cell cycle arrest, caspase-dependent apoptotic pathways and mitogen activated protein kinase (MAPK) signaling pathways. Miltirone, another abietane type-diterpene quinone isolated from Danshen, has been reported for its anti-oxidative, anxiolytic and anti-cancer effects. This study evaluated the apoptotic effect of miltirone and the underlying mechanisms in a human hepatoma HepG2 cell line and its p-glycoprotein (P-gp)-overexpressed doxorubicin-resistant counterpart (R-HepG2). Miltirone showed similar cytotoxicity in HepG2 (EC₅₀ = 7.06 μM) and R-HepG2 (EC₅₀ = 12.0 μM), demonstrated synergistic effects (1.56 - 6.25 μM) with doxorubicin (DOX) on the growth inhibition of R-HepG2 (synergism: 0.3 < CI < 0.5 at 50 % inhibition). Flow cytometric analysis showed that miltirone decreased P-gp-mediated DOX efflux in R-HepG2, and molecular docking studies illustrated that this effect was through inhibition on the active site of P-gp. At non-necrotic concentrations (25 μM or below), miltirone activated caspase-dependent apoptotic pathways, and induced the generation of reactive oxygen species (ROS) and oxidative stress which triggered ROS-mediated MAPK signaling pathways, including p38 MAPK, stress-activated protein kinase / c-Jun N-terminal kinase (SAPK/JNK) and extracellular regulated kinase 1/2, in both HepG2 and R-HepG2 cells. It is therefore concluded that miltirone is a dual inhibitor on P-gp and cell proliferation in R-HepG2 cells, with potential for the treatment of human hepatocellular carcinoma (HCC). / In order to improve the successful rates in drug development, the in vivo metabolic parameters of new chemical entities (NCEs), such as protein bindings, clearance rate, pharmacokinetic parameters and metabolism-based drug-drug interactions, should be considered at the early stage of drug discovery. Previous studies have shown that major tanshinones isolated from Danshen inhibited the metabolism of model probe substrates of human and rat CYP450 enzymes, with potential in causing herb-drug interactions. The aim of this study was to study the effect of miltirone on the metabolism of model probe substrates of CYP1A2, 2C9, 2D6 and 3A4 in pooled human liver microsomes. Miltirone showed moderate inhibition on CYP1A2 (IC₅₀ = 1.73 μM) and CYP2C9 (IC₅₀ = 8.61 μM), and weak inhibition on CYP2D6 (IC₅₀ = 30.20 μM) and CYP3A4 (IC₅₀ = 33.88 μM). Enzyme kinetic studies showed that miltirone competitively inhibited CYP2C9 (Ki = 1.48 μM), and displayed mixed type inhibitions on CYP1A2, CYP2D6 and CYP3A4 with Ki values of 3.17 μM, 24.25 μM and 35.09 μM, respectively. Molecular docking study further confirmed the ligand-binding conformations of miltirone in the active sites of human CYP450 isoforms. These findings suggested that miltirone may have potential drug-drug interactions with CYP1A2- and CYP2C9-metabolized drugs, and to a lesser extent with CYP2D6- and CYP3A4-metabolized drugs. / In conclusion, this study investigated the effects of Danshen water-extracts produced by different extraction methods on the chemical compositions and pharmacological activities, and consequently confirmed the biomarkers for the quality control of Danshen water-extracts for different medicinal uses. This study also demonstrated the anti-cancer potential of miltirone for HCC in vitro and the metabolism-based drug-drug interactions for its drug-likeness, which may provide useful and promising data for in vivo anti-cancer study of miltirone and further pre-clinical studies. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhou, Xuelin / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 195-224). / 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 / 論文摘要 --- p.v / Publications based on the work in this thesis --- p.viii / Acknowledgements --- p.x / Abbreviations --- p.xii / Table of Contents --- p.xv / Chapter Chapter 1 --- General introduction --- p.1 / Chapter 1.1 --- Reactive oxygen species and carcinogenesis --- p.1 / Chapter 1.2 --- Reactive oxygen species and tumor progression & metastasis --- p.2 / Chapter 1.3 --- Antioxidant enzymes in chemoprevention and chemotherapy --- p.3 / Chapter 1.3.1 --- Glutathione and Glutathione reductase --- p.5 / Chapter 1.3.2 --- Glutathione Peroxidase --- p.5 / Chapter 1.3.3 --- Glutathione S-transferases --- p.6 / Chapter 1.3.4 --- NAD(P)H: quinone reductase 1 --- p.7 / Chapter 1.3.5 --- Heme oxygenase-1 --- p.8 / Chapter 1.3.6 --- Thioredoxin reductase --- p.9 / Chapter 1.3.7 --- Superoxide Dismutase --- p.10 / Chapter 1.3.8 --- Catalase --- p.11 / Chapter 1.4 --- Medicinal uses of Danshen --- p.12 / Chapter 1.5 --- Analysis of Danshen and its components --- p.14 / Chapter 1.6 --- Antioxidant effects of Danshen extract and its bioactive compounds in chemoprevention and chemotherapy-related disease --- p.19 / Chapter 1.7 --- Anti-cancer effects of tanshinones isolated from Danshen --- p.21 / Chapter 1.7.1 --- Tanshinone IIA --- p.22 / Chapter 1.7.2 --- Tanshinone I --- p.26 / Chapter 1.7.3 --- Cryptotanshinone --- p.27 / Chapter 1.7.4 --- Dihydrotanshinone --- p.27 / Chapter 1.8 --- Metabolism / disposition of Danshen and its major active ingredients --- p.28 / Chapter 1.9 --- Herb-drug interactions with Danshen --- p.31 / Chapter 1.10 --- Effects of Danshen (and its major active ingredients) on model probe substrates of CYP isoforms --- p.33 / Chapter 1.11 --- CYPs induction by Danshen and its active components --- p.38 / Chapter 1.12 --- Effects of Danshen / active ingredients on drug transporter proteins --- p.40 / Chapter 1.13 --- CYP450 inhibition screening for new chemical entity --- p.42 / Chapter 1.14 --- Molecular docking analysis --- p.44 / Chapter 1.15 --- The Aim of this study --- p.45 / Chapter Chapter 2 --- Quantitative and qualitative studies to evaluate the efficiency of different heat water-extractions --- p.48 / Chapter 2.1 --- Introduction --- p.48 / Chapter 2.2 --- Materials and methods --- p.51 / Chapter 2.2.1 --- Materials and apparatus --- p.51 / Chapter 2.2.2 --- Extraction procedures --- p.51 / Chapter 2.2.3 --- HPLC analysis --- p.54 / Chapter 2.2.4 --- DPPH assay and FRAP assay --- p.54 / Chapter 2.2.5 --- Inhibition of 2,2'-azobis-2-amidinopropane (AAPH)-induced haemolysis in human red blood cells --- p.55 / Chapter 2.2.6 --- Protective effects on hydrogen peroxide-induced apoptosis in rat heart H9c2 cells --- p.56 / Chapter 2.2.7 --- Vasodilation effects on rat basilar artery --- p.57 / Chapter 2.2.8 --- Statistical analysis --- p.58 / Chapter 2.3 --- Results and Discussion --- p.59 / Chapter 2.3.1 --- Chemical profiles analyzed by HPLC analysis --- p.59 / Chapter 2.3.2 --- DPPH assay and FRAP assay --- p.63 / Chapter 2.3.3 --- Inhibition of AAPH-induced haemolysis --- p.65 / Chapter 2.3.4 --- Protective effects on hydrogen peroxide-induced apoptosis --- p.69 / Chapter 2.3.5 --- Vasodilation effects on rat basilar artery --- p.71 / Chapter 2.3.6 --- Multiple linear regression analysis --- p.76 / Chapter Chapter 3 --- Effects of miltirone on cell proliferation in a hepatoma HepG2 cell line and its doxorubicin-resistant counterpart --- p.83 / Chapter 3.1 --- Introduction --- p.83 / Chapter 3.2 --- Materials and Methods --- p.87 / Chapter 3.2.1 --- Chemicals --- p.87 / Chapter 3.2.2 --- Cell culture --- p.87 / Chapter 3.2.3 --- Cell viability test --- p.88 / Chapter 3.2.4 --- Drug-efflux study by flow cytometry --- p.89 / Chapter 3.2.5 --- Molecular docking study and Ligand-based prediction --- p.90 / Chapter 3.2.6 --- Measurement of ROS generation by confocal microscopy and flow cytometry --- p.91 / Chapter 3.2.7 --- GSH and GSSG determination for oxidative stress --- p.93 / Chapter 3.2.8 --- Apoptosis-related proteins expression detected by Western blotting analysis --- p.94 / Chapter 3.2.9 --- Data analysis --- p.96 / Chapter 3.3 --- Results --- p.97 / Chapter 3.3.1 --- Cytotoxicity in hepatoma cells --- p.97 / Chapter 3.3.2 --- Drug-efflux study by flow cytometry --- p.104 / Chapter 3.3.3 --- Molecular docking study and Ligand-based prediction --- p.108 / Chapter 3.3.4 --- ROS generation --- p.113 / Chapter 3.3.5 --- Determination of GSH/GSSG ratio --- p.117 / Chapter 3.3.6 --- Caspase-dependent apoptosis. --- p.121 / Chapter 3.3.7 --- Phosphorylation of MAPKs --- p.126 / Chapter 3.4 --- Discussion --- p.134 / Chapter Chapter 4 --- Enzyme kinetic and molecular docking studies of miltirone on major human cytochrome P450 isozymes inhibitions --- p.139 / Chapter 4.1 --- Introduction --- p.139 / Chapter 4.2 --- Material and Methods --- p.141 / Chapter 4.2.1 --- Materials and Reagents --- p.141 / Chapter 4.2.2 --- Incubation conditions --- p.142 / Chapter 4.2.3 --- Samples preparation --- p.143 / Chapter 4.2.4 --- HPLC analysis --- p.143 / Chapter 4.2.5 --- CYP inhibition and enzymatic kinetic study --- p.144 / Chapter 4.2.6 --- Molecular docking analysis --- p.145 / Chapter 4.2.7 --- Data analysis --- p.146 / Chapter 4.3 --- Results --- p.148 / Chapter 4.3.1 --- CYP inhibition and enzymatic kinetic study --- p.148 / Chapter 4.3.2 --- Molecular docking study of miltirone --- p.167 / Chapter 4.4 --- Discussions --- p.184 / Chapter Chapter 5 --- General discussion --- p.188 / References --- p.195

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

Tang Wan-yee. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 192-212). / 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.

published_or_final_version / Chinese Medicine / Master / Master of Philosophy