Yip, Ka Yan. / "August 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 87-113). / Abstracts in English and Chinese. / Acknowledgement --- p.III / Abstract --- p.V / 論文摘要 --- p.VI / Table of Contents --- p.VII / List of Figures --- p.IX / List of Abbreviations --- p.XI / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Hepatocellular carcinoma --- p.1 / Chapter 1.1.1 --- Overview --- p.1 / Chapter 1.1.2 --- Risk factors --- p.3 / Chapter 1.1.3 --- Treatment ofHCC --- p.12 / Chapter 1.2 --- Berberine - a compound derived from Traditional Chinese Medicine --- p.15 / Chapter 1.2.1 --- Traditional Chinese Medicine --- p.15 / Chapter 1.2.2 --- Berberine --- p.16 / Chapter 1.3 --- Cell cycle --- p.18 / Chapter 1.3.1 --- An Overview of cell cycle --- p.18 / Chapter 1.3.2 --- Cell cycle and carcinogenesis --- p.18 / Chapter 1.4 --- Molecular mechanism of apoptosis --- p.20 / Chapter 1.4.1 --- Overview of apoptosis --- p.20 / Chapter 1.4.2 --- Caspases cascade --- p.22 / Chapter 1.4.3 --- Bcl-2 family --- p.24 / Chapter 1.5 --- Apoptosis as a target of cancer therapy --- p.26 / Chapter 1.6 --- Aims of study --- p.27 / Chapter Chapter 2 --- Materials and Methods --- p.28 / Chapter 2.1 --- Cell culture and treatment --- p.28 / Chapter 2.1.1 --- Cell lines --- p.28 / Chapter 2.1.2 --- Berberine --- p.29 / Chapter 2.1.3 --- Chemicals and reagents --- p.29 / Chapter 2.1.4 --- Preparation of solutions --- p.29 / Chapter 2.1.5 --- Procedures --- p.31 / Chapter 2.2 --- Apoptosis detection by FITC Annexin V and PI co-staining --- p.33 / Chapter 2.2.1 --- Chemicals and reagents --- p.33 / Chapter 2.2.2 --- Procedures --- p.33 / Chapter 2.3 --- Gene expression in Berberine-induced apoptotic cells --- p.35 / Chapter 2.3.1 --- Chemicals and Reagents --- p.35 / Chapter 2.3.2 --- Procedures --- p.35 / Chapter 2.4 --- Protein expression in Berberine-induced apoptotic cells --- p.38 / Chapter 2.4.1 --- Chemicals and Reagents --- p.38 / Chapter 2.4.2 --- Preparation of solution --- p.39 / Chapter 2.4.3 --- Procedures --- p.41 / Chapter 2.5 --- Caspase cascade studies in berberine-induced apoptosis --- p.43 / Chapter 2.5.1 --- Chemicals and reagents --- p.43 / Chapter 2.5.2 --- Procedures --- p.43 / Chapter 2.6 --- Cell cycle study in berberine-induced apoptotic cells --- p.44 / Chapter 2.6.1 --- Chemicals and Reagents --- p.44 / Chapter 2.6.2 --- Preparation of solutions --- p.44 / Chapter 2.6.3 --- Procedures --- p.44 / Chapter Chapter 3 --- Results --- p.46 / Chapter 3.1 --- Berberine induces apoptosis in hepatocellular cells --- p.46 / Chapter 3.2 --- Gene expression in Berberine-induced apoptotic cells --- p.53 / Chapter 3.3 --- Caspase cascade studies in berberine-induced apoptosis --- p.58 / Chapter 3.4 --- Protein expression in Berberine-induced apoptotic cells --- p.62 / Chapter 3.5 --- Berberine caused G1 cell cycle arrest in HCC cell lines --- p.65 / Chapter Chapter 4 --- Discussion --- p.76 / References --- p.87

by Zhong Sheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (p. 186-203). / 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.

ArtinM é uma lectina ligante a carboidrato D-manose que se interage a receptores de células fagocíticas induzindo a produção de mediadores pró- inflamatórios relacionados à resposta imune antitumoral. Aflatoxinas são micotoxinas produzidas por fungos do gênero Aspergillus. A Aflatoxina B1 (AFB1) é a toxina sintetizada mais abundantemente e a que apresenta o maior poder toxigênico, sendo capaz de induzir carcinoma hepatocelular (CHC) em humanos. O objetivo deste estudo foi investigar o papel da lectina ArtinM na hepatocarcinogênese induzida pela AFB1 em ratos. Setenta e dois ratos recém-desmamados foram divididos em três grupos: Controle - animais tratados com veículo; AFB1 - animais intoxicados com AFB1; AFB1+ArtinM - animais tratados com AFB1 e ArtinM. Ratos Wistar foram intoxicados por gavagem com 400 ?g de AFB1 por quilograma de ração ingerida durante três meses, enquanto o grupo AFB1+ArtinM recebeu adicionalmente três doses da lectina por via subcutânea (50 ?g por quilograma de peso do animal por dose) nos 45, 60 e 75 após inicio do experimento. Animais foram eutanasiados 3 e 12 meses após início das gavagens. A expressão hepática de proteínas relacionadas à hepatocarcinogênese foi avaliada por técnicas de imunohistoquímica, Western blotting e PCR em tempo real nos animais eutanasiados após 3 meses de intoxicação. A incidência de lesões pré-neoplásicas e de tumores hepáticos foi mensurada 3 e 12 meses após início das gavagens, respectivamente. Os animais tratados com ArtinM apresentaram maior expressão hepática de proteínas supressoras tumorais além de redução do número de focos pré-neoplásicos e de tumores hepáticos em relação aos animais que receberam apenas a micotoxina. Conclui-se, portanto, que ArtinM possui efeito protetor durante o processo de hepatocarcinogênese induzida por AFB1. / ArtinM is a D-mannose carbohydrate-binding lectin that interacts with phagocytic cell receptors inducing the production of pro-inflammatory mediators related to the antitumor immune response. Aflatoxins are mycotoxins produced by fungi of the genus Aspergillus. Aflatoxin B1 (AFB1) is the toxin most abundantly synthesized and the one with the highest toxigenic power, being able to induce hepatocellular carcinoma (HCC) in humans. The aim of this study was to investigate the role of ArtinM lectin in AFB1-induced hepatocarcinogenesis in rats. Seventy-two newly weaned rats were divided into three groups: Control - vehicle-treated animals; AFB1 - animals poisoned with AFB1; AFB1 + ArtinM - animals treated with AFB1 and ArtinM. Wistar rats were gavage-poisoned with 400 ?g AFB1 per kilogram of ration fed for three months, while the AFB1 + ArtinM group received three subcutaneous doses of the lectin (50 ?g per kilogram of animal weight per dose) 45, 60 and 75 days after the start of the experiment. Animals were euthanized 3 and 12 months after initiation of treatments. Hepatic expression of hepatocarcinogenesis-related proteins was assessed by immunohistochemistry, Western blotting, and realtime PCR in euthanized animals after three months of intoxication. The incidence of pre-neoplastic lesions and liver tumors was measured 3 and 12 months after the start of treatments, respectively. Animals treated with ArtinM had fewer pre-neoplastic foci and hepatic tumors than the animals that receiving mycotoxin alone, as well as showing greater hepatic expression of tumor suppressor proteins. It is concluded, therefore, that ArtinM has a protective effect during the process of hepatocarcinogenesis induced by AFB1.

Diao, Shu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 72-73). / Abstracts in English and Chinese. / 摘要 --- p.i / Abstract --- p.ii / Acknowledgements --- p.iv / Contents --- p.viii / Chapter Chapter 1 --- Introduction and Background / Chapter 1.1 --- General introduction to miRNA --- p.1 / Chapter 1.1.1 --- The discovery and biogenesis of miRNA --- p.1 / Chapter 1.1.2 --- The function of miRNA --- p.3 / Chapter 1.2 --- The liver-specific miRNA : miR-122a --- p.5 / Chapter 1.2.1 --- Discovery and biogenesis of miR-122a --- p.5 / Chapter 1.2.2 --- "miR-122a, a liver specific miRNA" --- p.6 / Chapter 1.2.3 --- miR-122a and Hepatocellular carcinoma (HCC) --- p.6 / Chapter 1.2.4 --- Therapeutic opportunities and challenges of miR-122a --- p.7 / Chapter 1.3 --- Techniques and approaches to study miRNAs --- p.8 / Chapter 1.3.1 --- Discovery of novel miRNA --- p.8 / Chapter 1.3.2 --- Detection of miRNA --- p.9 / Chapter 1.3.3 --- Functional study of miRNAs --- p.10 / Chapter 1.3.4 --- Prediction and validation of miRNA targets --- p.10 / Chapter 1.4 --- References --- p.12 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Cell culture --- p.18 / Chapter 2.2 --- Cell transfection --- p.18 / Chapter 2.3 --- RNA extraction --- p.18 / Chapter 2.4 --- Plasmid extaction --- p.19 / Chapter 2.5 --- Competent cell preparation --- p.19 / Chapter 2.6 --- Bacterial Transformation --- p.20 / Chapter 2.7 --- Purification of DNA fragments from agarose gel --- p.21 / Chapter 2.8 --- Genomic DNA extranction from MIHA cell --- p.21 / Chapter 2.9 --- Real-time RT-PCR analysis --- p.21 / Chapter 2.10 --- Lenti-vector Construction for miRNA expression --- p.22 / Chapter 2.11 --- Lentivirus production --- p.22 / Chapter 2.12 --- Lentiviral vector titering --- p.23 / Chapter 2.13 --- Bradford protein assay --- p.23 / Chapter 2.14 --- Western Blot --- p.24 / Chapter 2.14.1 --- Sample preparation --- p.24 / Chapter 2.14.2 --- Gel electrophoresis --- p.24 / Chapter 2.14.3 --- Blocking --- p.25 / Chapter 2.14.4 --- Incubation with Primary and Secondary Antibodies --- p.25 / Chapter 2.14.5 --- Substrate Incubation --- p.26 / Chapter 2.14.6 --- Exposeto x-ray film --- p.26 / Chapter 2.15 --- MTT Cell Proliferation Assay --- p.26 / Chapter 2.16 --- Apoptosis analysis :DAPI Staining --- p.26 / Chapter 2.17 --- 2-D Protein Gel Electrophoresis and MS --- p.26 / Chapter 2.17.1 --- Materials --- p.27 / Chapter 2.17.2 --- Protein extraction --- p.27 / Chapter 2.17.3 --- 2-D Electrophoresis --- p.27 / Chapter 2.17.4 --- Gel staining and image analysis --- p.28 / Chapter 2.17.5 --- In-gel protein digestion with trypsin --- p.28 / Chapter 2.17.6 --- MALDI-TOF MS and database search --- p.28 / Chapter 2.18 --- Statistical Analysis --- p.29 / Chapter Chapter 3 --- Expression of HCC-associated miRNA in HCC cell lines / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Experimental Section --- p.31 / Chapter 3.2.1 --- Cell culture --- p.31 / Chapter 3.2.2 --- RNA extraction --- p.31 / Chapter 3.2.3 --- miRNA-specific quantitative Real-time PCR --- p.31 / Chapter 3.3 --- Results and Discussion --- p.32 / Chapter 3.3.1 --- Expression of miR-let7a in HCC cells --- p.32 / Chapter 3.3.2 --- Expression of miR-221 in HCC cells --- p.33 / Chapter 3.3.3 --- "Expression of miR-122a,a liver-specific miRNA in HCC cells" --- p.34 / Chapter 3.4 --- Conclusions --- p.35 / Chapter 3.5 --- References --- p.36 / Chapter Chapter 4 --- Ectopic overexpression of miR-122a in HCC cells / Chapter 4.1 --- Introduction --- p.38 / Chapter 4.2 --- Experimental Section --- p.38 / Chapter 4.2.1 --- Overexpression of miR-122a with mimics --- p.38 / Chapter 4.2.2 --- Lentivirus-mediated miR-122a expression --- p.40 / Chapter 4.2.3 --- RNA extraction --- p.44 / Chapter 4.2.4 --- Expression level of miR-122a after transduction --- p.44 / Chapter 4.2.5 --- Western Blot --- p.44 / Chapter 4.3 --- Result and discussion --- p.46 / Chapter 4.3.1 --- Expression level of miR-122a after transfection --- p.46 / Chapter 4.3.2 --- Lentivirus-mediated miR-122a expression --- p.47 / Chapter 4.4 --- Conclusions --- p.52 / Chapter 4.5 --- References --- p.54 / Chapter Chapter 5 --- Gain-of-function study of miR-122a in HCC cells / Chapter 5.1 --- Introduction --- p.55 / Chapter 5.2 --- Experimental Section --- p.56 / Chapter 5.2.1 --- Cell culture --- p.56 / Chapter 5.2.2 --- Cell transfection --- p.56 / Chapter 5.2.3 --- Lentiviral vector transduction --- p.56 / Chapter 5.2.4 --- MTT Cell Proliferation Assay --- p.56 / Chapter 5.2.5 --- Apoptosis analysis - DAPI Staining --- p.57 / Chapter 5.2.6 --- 2-D Protein Gel Electrophoresis and MS --- p.57 / Chapter 5.2.7 --- miRNA target prediction using bioinformatic approaches --- p.59 / Chapter 5.3 --- Result and discussion --- p.59 / Chapter 5.3.1 --- Phenotypic changes of HepG2 cells caused by ectopic overexpression of miR-122a --- p.59 / Chapter 5.3.2 --- miR-122a target prediction using bioinformatic approaches --- p.62 / Chapter 5.3.3 --- Experimental validation of miR-122a targets by proteomics approach --- p.67 / Chapter 5.4 --- Conclusion --- p.70 / Chapter 5.5 --- References --- p.71 / Appendix --- p.73

在世界範圍內，肝細胞癌(HCC)是其中一種惡性程度很高並且預後很差的疾病。和其他的癌症一樣，肝癌是一种基因疾病，基因異常的積累在肝癌的生成中扮演著重要的角色。近年來，第二代測序技術（NGS）的迅速發展顯現了前所未見的能力揭示癌癥基因組中分子的複雜性，這為癌癥的生物學，診斷和药物治療提供了一個嶄新的思路。 / 非酒精脂肪性肝炎（NASH）是一種與代謝有關的疾病，在發達國家地區例如美國，歐洲，日本和加拿大，這是其中一種越來越常見的HCC 的病因。在本論文的第一部份，三個NASH 相關的HCC 以及其配對的血DNA 進行了全基因組測序（WGS）。另外還有一個來源于這三個NASH 相關的HCC其中之一的細胞株也進行了全基因組測序。在全部樣品中，測序深度範圍介乎29.1X 到102.2X，序列覆蓋度均大於99%。結果顯示發現的新的單核苷酸變異（SNVs）數量介乎于6,898 至17,129，平均值是11,133。根據這些找到的SNV，隨機選出56 個體細胞SNV 進行Sanger 測序，其中92.9%可以被確認。基因突變譜顯示有頻繁的A:T>G:C 和C：G>A:T 體細胞突變，而C：G>T:A 則在CpG 位點頻繁出現。在眾多的非同義體細胞突變基因中，我們選擇了CTNNB1，PNLIP 和MLL2 這三個基因進行進一步研究，此三個基因都在多於一個病例中發現有突變。在額外的一組44 對NASH 相關HCC及50 對HBV 相關的HCC 的癌變組織和其臨近非腫瘤組織中，我們進一步對這三個基因和TP53 的編碼區域進行了測序，而TP53 是在HBV 相關HCC中被報導有高頻率突變的。在NASH 相關的HCC 中，這些基因都只在腫瘤組織中發現重複出現的錯義突變，在臨近的非腫瘤肝組織沒有發現有突變。在NASH 相關的HCC 中，CTNNB1 的突變率（36.4%）明顯高於在HBV相關的HCC 中的突變率（12.0%，P=0.007）。PNLIP 和MLL2 的突變只在NASH相關的HCC 中發現，其突變率分別為12.1%和7.1%，而在HBV 相關的HCC中，則沒有發現突變。然而，TP53 的突變率在NASH 相關的HCC 及HBV相關的HCC 中差別不明顯（P>0.1）。在功能性研究的實驗中，我们发现在HKCI-10（有PNLIP 突變D396N）細胞株中，PNILP 的活性比在正常肝細胞L02 細胞株（野生型PNLIP）中要低。在永生性肝細胞L02 細胞株中，CTNNB1的突變引起了TOPFLASH 活性的提高以及增加了細胞群落形成的能力。HKCI-10 是一條我們實驗室建立的NASH 相關的肝細胞癌細胞株，在HKCI-10細胞株中，抑制CTNNB1 表達引起了細胞生長和增殖的減少。另外，在DEN誘導肝癌的有代謝失衡的db/db 轉基因鼠中，發現了一個CTNNB1 的突變T41A。據報導，CTNNB1 發生的致癌突變會引起蛋白的穩定並且因此激活經典的Wnt/β-catenin 信號通路，從而引致特定基因的轉錄。對於CTNNB1中最常見的突變S45P(在發現的CTNNB1 突變中占31.3%)，我們做了ChIP-array 實驗，結果顯示，在HKCI-10（CTNNB1 有S45P 突變）中，CTNNB1比在Hep3B 中（野生型CTNNB1）有著更緊密的啟動子結合能力（P<0.001）。Gene ontology 分析結果表明，被S45P 富集的生物過程涉及有RNA 代謝調節，轉錄因子活性和凋亡。MYC，E2F1 和ZFX 被ChIP-PCR 證實是與CTNNB1突變子S45P 有著更緊密結合能力的轉錄因子。 / 在本論文的第二部份，爲了進一步闡明致癌性的CTNNB1 突變S45P在HCC 中的角色，我們研究了一個此前未在HCC 報導過的基因ZFX。ZFX是一個在脊椎動物中高度保守，屬於Zfy 家族的zinc finger 蛋白。有報導指出ZFX 對於胚胎和造血幹細胞的自我更新有著重要的作用。另外亦有文獻報導ZFX 在一系列人類癌癥病例中有過度表達，例如食道癌，胃癌，前列腺癌和神經膠質瘤，並且顯現出致癌基因的特性。在本研究中，qRT-PCR結果提示ZFX 在HCC 腫瘤中的表達明顯高於正常肝組織。ZFX 的表達在51.8%的HCC 腫瘤中明顯高於其配對的鄰近非腫瘤肝組織。功能性研究表明，在MTT 實驗中，細胞的生存力在ZFX 缺失的穩定克隆中明顯減弱（P<0.0001）。在細胞群落形成實驗中，ZFX 缺失的穩定克隆顯示出明顯減弱的群落生長能力（P<0.0001）。在單細胞克隆生成實驗中，ZFX 缺失的HCC穩定克隆展示出數量更少，體積更小的細胞群落。另外，ZFX 基因抑制或者cisplatin 單獨處理均顯示細胞生存力的抑制，其抑制效率分別是24.0%和30.9%。當ZFX 基因抑制合併cisplatin 處理時，細胞生存力的抑制效率顯著地提高至65.2%，這個結果提示ZFX 基因抑制和cisplatin 處理兩者有協同增效作用（P<0.0001）。ZFX 基因的缺失會引起兩個為人熟知的胚胎幹細胞（ESCs）標誌物SOX-2 和NANOG 表達的明顯降低。 / 綜上所述，通過進行全基因測序，本論文的研究結果為NASH 相關的HCC 分子層面上的異常提供了一個高解析度的視覺角度。在NASH 相關的HCC 中，一些可能對於肝細胞癌變有重要作用並且重複出現突變的基因被確定，例如CTNNB1 和PNLIP。CTNNB1 的突變體S45P 的其中一個下游目標基因ZFX，在HCC 中被證實有幹細胞和腫瘤啟動細胞特性。闡明在HCC發展過程中的分子改變以及機制，對於為肝癌病人探索新的治療手段有著重要意義。 / Hepatocellular Carcinoma (HCC) is one of the most malignant diseases worldwide with poor prognosis. Like other human cancers, HCC is a genetic disease, where accumulation of genetic aberration plays important role in the liver carcinogenesis. The rapid advances in Next Generation Sequencing (NGS) technology in recent years have allowed unprecedented ability to unravel the molecular complexity of the cancer genome, providing new insights into the cancer biology, diagnosis and therapeutic drug development. / Non-alcoholic steatohepatitis (NASH), which is related to metabolic disorder, is an increasing common etiological factor of HCC, especially in developed countries such as United States, Europe, Japan and Canada. In first part of this thesis, Whole Genome Sequencing (WGS) was performed on three NASH-associated HCCs and their matched lymphocytic DNA. A cell line developed from one of the three NASH-associated HCCs was also subjected to WGS. The sequencing depth ranged from 29.1X to 102.2X with the coverage >99% shown in all samples. Novel SNVs identified ranged from 6,898 to 17,129 with an average of 11,133. Based on the SNVs found, the validation rate was 92.9% in 56 randomly selected somatic SNVs by Sanger sequencing. Mutational spectrum showed frequent somatic substitution of A:T>G:C and C:G>A:T while C:G>T:A transition exhibited a predominant somatic mutation rate in CpG sites. Among the non-synonymous somatic mutated genes, we selected CTNNB1, PNLIP and MLL2 which were mutated in more than one tumor for further study. In additional cohort of 44 NASH-associated and 50 HBV-associated HCC tumors and adjacent non-tumoral tissues, further sequencing all the coding regions of these three genes and TP53, which has been reported to be highly mutated in HBV-associated HCCs, were carried out. In NASH-associated HCCs, all genes harboured recurrent missense mutations exclusive in tumor but none in adjacent ,non-tumoral liver. The prevalence of CTNNB1 mutations was significantly higher in NASH-associated HCCs (36.4%) when compared to HBV-associated HCCs (12.0%, P=0.007). Mutations of PNLIP and MLL2 were detected only in NASH-associated HCCs with rates of 12.1% and 7.1%, respectively, but none in HBV-associated HCCs. The mutation rate of TP53, however, did not differ much between NASH-associated and HBV-associated HCCs (P>0.1). In functional study, for PNLIP, a loss-of-function in PNLIP activity was found in HKCI-10 harbouring D396N mutation as compared to normal liver cell, L02 with wild-type PNLIP. We demonstrated that CTNNB1 mutants conferred elevated TOPFLASH activity and enhanced colony growth in an immortalized hepatocyte cell line L02. Knockdown of CTNNB1 in HKCI-10, which is a NASH-associated HCC in-house cell line, resulted in inhibition of cell growth and proliferation. Also, a CTNNB1 mutation (T41A) was found in DEN-induced liver cancer of db/db transgenic mouse with metabolic disorder. Since oncogenic mutations of CTNNB1 were reported to be contributed to the stabilization of the protein and hence activate the canonical Wnt/β-catenin signaling pathway, inducing transcription of specific gene sets. We performed ChIP-array focusing on the most common CTNNB1 S45P mutation (accounted for 31.3% of all detected CTNNB1 mutants) in NASH-associated HCCs, the result showed more intense promoter binding affinities in HKCI-10 with CTNNB1 S45P mutation than Hep3B with wild-type CTNNB1 (P<0.001). Gene ontology analysis revealed that S45P mutant enriched the process including RNA metabolic regulation, transcription factor activities and apoptosis. Furthermore, we found that CTNNB1 S45P mutant showed more profound binding affinity to the promoter regions of transcription factors MYC, E2F1 and ZFX by ChIP-PCR. / In the second part of the thesis, we aimed to further understand the role of oncogenic CTNNB1 S45P mutant in HCC. Zinc finger protein X-linked (ZFX) gene which is previously undescribed in HCC was studied. ZFX is a zinc finger protein of Zfy family that is highly conserved among vertebrates. It has been reported that ZFX is required for self-renewal of embryonic and hematopoietic stem cells. Also, ZFX is suggested to be overexpressed in a number of human cancers such as esophageal carcinoma, gastric cancer, prostate cancer and glioma, conferring oncogenic characteristics. In this study, qRT-PCR analysis showed a significant higher ZFX expression in HCC tumors compared to normal livers. 51.8% of HCC tumors showed significant up-regulations of ZFX when compared to paired adjacent non-tumoral livers. Functional studies showed significant reduction in in-vitro cell proliferation in HCC by MTT assay (P<0.0001) and colony formation ability by colony formation assay (P<0.0001) in ZFX-deficient stable clones. In single-cell clonogenic assay, ZFX-depleted HCC exhibited fewer and smaller colonies. In addition, while ZFX knockdown or cisplatin treatment alone showed an inhibitory effect on cell viability by 24.0% and 30.9%, respectively, the reduction efficiency of cell viability increased dramatically to 65.2% when combine ZFX-knockdown and cisplatin treatment, indicating a synergistic effect between them (P<0.0001). Also, significant reduced expressions of SOX-2 and NANOG, both well-known embryonic stem cells (ESCs) markers, were observed as a result of ZFX depletion. / In summary, findings from this thesis provided high-resolution insight into the molecular aberrations in NASH-associated HCCs by performing WGS. Recurrent mutated genes which may be of great importance in hepatocellular carcinogenesis induced by NASH were identified, such as CTNNB1 and PNLIP. One of the S45P CTNNB1 downstream targets ZFX was demonstrated to confer stemness and tumor-initiating cells (TICs) characteristics in HCC. The understanding of molecular changes and mechanisms underlying HCC development will thus facilitate the exploration of new therapeutic options in patients with this deadly disease. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Jiawei. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 166-190). / Abstracts also in Chinese.

ArtinM é uma lectina ligante a carboidrato D-manose que se interage a receptores de células fagocíticas induzindo a produção de mediadores pró- inflamatórios relacionados à resposta imune antitumoral. Aflatoxinas são micotoxinas produzidas por fungos do gênero Aspergillus. A Aflatoxina B1 (AFB1) é a toxina sintetizada mais abundantemente e a que apresenta o maior poder toxigênico, sendo capaz de induzir carcinoma hepatocelular (CHC) em humanos. O objetivo deste estudo foi investigar o papel da lectina ArtinM na hepatocarcinogênese induzida pela AFB1 em ratos. Setenta e dois ratos recém-desmamados foram divididos em três grupos: Controle - animais tratados com veículo; AFB1 - animais intoxicados com AFB1; AFB1+ArtinM - animais tratados com AFB1 e ArtinM. Ratos Wistar foram intoxicados por gavagem com 400 ?g de AFB1 por quilograma de ração ingerida durante três meses, enquanto o grupo AFB1+ArtinM recebeu adicionalmente três doses da lectina por via subcutânea (50 ?g por quilograma de peso do animal por dose) nos 45, 60 e 75 após inicio do experimento. Animais foram eutanasiados 3 e 12 meses após início das gavagens. A expressão hepática de proteínas relacionadas à hepatocarcinogênese foi avaliada por técnicas de imunohistoquímica, Western blotting e PCR em tempo real nos animais eutanasiados após 3 meses de intoxicação. A incidência de lesões pré-neoplásicas e de tumores hepáticos foi mensurada 3 e 12 meses após início das gavagens, respectivamente. Os animais tratados com ArtinM apresentaram maior expressão hepática de proteínas supressoras tumorais além de redução do número de focos pré-neoplásicos e de tumores hepáticos em relação aos animais que receberam apenas a micotoxina. Conclui-se, portanto, que ArtinM possui efeito protetor durante o processo de hepatocarcinogênese induzida por AFB1. / ArtinM is a D-mannose carbohydrate-binding lectin that interacts with phagocytic cell receptors inducing the production of pro-inflammatory mediators related to the antitumor immune response. Aflatoxins are mycotoxins produced by fungi of the genus Aspergillus. Aflatoxin B1 (AFB1) is the toxin most abundantly synthesized and the one with the highest toxigenic power, being able to induce hepatocellular carcinoma (HCC) in humans. The aim of this study was to investigate the role of ArtinM lectin in AFB1-induced hepatocarcinogenesis in rats. Seventy-two newly weaned rats were divided into three groups: Control - vehicle-treated animals; AFB1 - animals poisoned with AFB1; AFB1 + ArtinM - animals treated with AFB1 and ArtinM. Wistar rats were gavage-poisoned with 400 ?g AFB1 per kilogram of ration fed for three months, while the AFB1 + ArtinM group received three subcutaneous doses of the lectin (50 ?g per kilogram of animal weight per dose) 45, 60 and 75 days after the start of the experiment. Animals were euthanized 3 and 12 months after initiation of treatments. Hepatic expression of hepatocarcinogenesis-related proteins was assessed by immunohistochemistry, Western blotting, and realtime PCR in euthanized animals after three months of intoxication. The incidence of pre-neoplastic lesions and liver tumors was measured 3 and 12 months after the start of treatments, respectively. Animals treated with ArtinM had fewer pre-neoplastic foci and hepatic tumors than the animals that receiving mycotoxin alone, as well as showing greater hepatic expression of tumor suppressor proteins. It is concluded, therefore, that ArtinM has a protective effect during the process of hepatocarcinogenesis induced by AFB1.

Cancer is the second leading cause of death in the USA, following cardiovascular disease. Treating cancer using conventional therapies is associated with low response rates and high toxicity, because these therapies usually lack specific tumor accumulation. Loading anticancer drugs into intelligently designed polymeric nanoparticles (NPs) can serve in delivering these drugs specifically to the tumor site, thus boosting their efficacy and reducing any associated off target toxicity. Targeting NPs to the tumor site can occur through either passive or active means. In passive targeting, NPs of specific size and surface characteristics can exploit the tumor’s erratic vasculature and occluded lymphatic drainage to extravasate the systemic circulation and accumulate preferentially at the tumor site. Active targeting mandates grafting the surface of NPs with a ligand that specifically interacts with a protein expressed at higher levels at the tumor site, in comparison to elsewhere in the body. In the current research, we independently investigated the utilization of passive and active targeting strategies to treat aggressive forms of cancer. Initially, passively targeted poly(lactic-co-glycolic acid) (PLGA) NPs to treat aggressive forms of endometrial cancer (EC) were investigated. A novel combination of soluble paclitaxel (PTX), a first line chemotherapy for EC, and soluble BIBF1120 (BIBF, nintedanib), an antiangiogenic molecular inhibitor, was first tested against three EC cell lines bearing different p53 mutations. The results showed that only EC cells with loss of function (LOF) p53 were sensitive to the combination therapy, indicating the potential of this combination to engender synthetic lethality to PTX. Next, NPs loaded with PTX were investigated with respect to the impact of varying the polymer lactic acid to glycolic acid ratio and the surfactant type on the major physicochemical properties of the prepared nanoparticles, drug loading, cellular uptake, cytotoxicity, and drug release. The optimum formulation was then loaded with BIBF and the combination of independently loaded passively targeted NPs were further evaluated for in vivo activity against a xenograft model of LOF p53 EC. The combination of independently loaded NPs exhibited the highest reduction in tumor volume and prolonged survival when compared to soluble PTX, PTX NPs or untreated control. These data highlight this specific combination of NPs as a novel promising therapy for LOF p53 EC. In a second study, the use of actively targeted NPs to treat liver cancer was explored. In this study, a combination of small interfering RNA (siRNA) against astrocyte elevated gene-1 (AEG-1), and all-trans retinoic acid (ATRA) was investigated as a new therapy for hepatocellular carcinoma (HCC). AEG-1 is a highly expressed oncogene that is directly involved in HCC progression and aggressiveness, in addition to reducing the ability of retinoic acid to induce apoptosis in HCC cells. First, a new conjugate was synthesized that was capable of delivering siRNA selectively to HCC cells, using galactose as a targeting moiety. The conjugate was prepared by linking poly(amidoamine) (PAMAM) dendrimers, polyethylene glycol (PEG) and lactobionic acid (Gal, disaccharide containing galactose) (PAMAM-PEG-Gal). We confirmed the synthesis of the conjugate using 1H-NMR, Mass spectrometry and Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry. Next, nanoplexes of the synthesized conjugate, PAMAM-PEG-Gal, and AEG-1 siRNA were prepared. Nanoplexes were further characterized for their size, surface charge, morphology, and electrophoretic mobility to identify the optimum complexation ratio between PAMAM-PEG-Gal and the siRNA. Then, mice bearing orthotopic luciferase expressing HCC cells were treated with the optimum nanoplex formulation. Results showed that a combination of AEG-1 nanoplexes and ATRA results in a significant reduction in luciferase expression, reduced liver weight, lower AEG-1 mRNA levels and increased apoptosis, when compared to utilizing nanoplexes with silencing control (siCon), siCon+ATRA, or AEG-1 nanoplexes alone. The results indicate that the combination of liver-targeted AEG-1 nanoplexes and ATRA may be a potential treatment for aggressive HCC. These data place targeted NPs as a promising efficient delivery system for cancer treatment.

The goniothalamin (GTN) and related styryl-lactones were found to be cytotoxic and apoptotic to a variety of tumor cell lines including breast cancer MCF-7, cervical cancer HeLa and leukemia HL60. In HL60 cells, GTN triggers mitochondria dysfunction, caspase activation and eventually, apoptosis. In this study, we demonstrated that GTN was able to induce apoptosis in hepatocellular carcinoma derived cells, SK-Hep1 and Hep-3B cells via upregulation of the phorbol-12-myristate-13-induced 1 (PMAIP1) protein , alternation of mitochondrial membrane potentials (P < 0.05) via TP53-dependent and -independent transactivations. Treatment with GTN induced DNA damage, formation of reactive oxygen species (ROS, P < 0.05) and activated cleaved CASP8, CASP9, CASP3 and poly (ADP-ribose) polymerase 1 proteins. However, cleaved BH3 interacting domain (BID) death agonist protein was not identified, suggesting that an intrinsic cellular stress was produced after GTN treatments in both cell lines. A pan caspase inhibitor, z-VAD-FMK, suppressed GTN-induced apoptotic cell percentages (P < 0.05), demonstrating that GTN-induced apoptosis was mediated by the activation of the caspase cascade protein. The GTN induced ROS formation prior to DNA damage in SK-Hep1, yet in reverse order in Hep-3B cells. Moreover, GTN induced DNA damages through activation of the gamma H2A histone family member X (£^H2AFX, Ser139)/phospho-CHK1 checkpoint homolog (pCHEK1, Ser345)/phospho-CHK2 checkpoint homolog (pCHEK2, Thr68)/phosphos-tumor protein p53 (pTP53, Ser15), and £^H2AFX/pCHEK2 (Thr68), in SK-Hep1 and Hep-3B cells,respectively. Among five integral outer mitochondrial membrane proteins that blocks or induces apoptotic death, PMAIP1 protein and PMAIP1 mRNA levels were upregulated after GTN treatments for 4 to 6 h in both cell lines (P < 0.05). Transfection of shRNA interference plasmids targeting PMAIP1 gene downregulated PMAIP1 mRNA (P < 0.05) and PMAIP1 protein (P < 0.05) levels, as well as GTN-induced apoptotic cell percentages (P < 0.05) in both cell lines. In parallel, transfection of the shRNA interference plasmid targeting TP53 gene in SK-Hep1 cells, downregulated TP53 and PMAIP1 mRNA (P < 0.05) and TP53, pTP53, PMAIP1, cleaved PARP1 protein levels and apoptotic cell percentages (P < 0.05). Treatment with the TP53 inhibitor, PFT-£\ or transfection of a dominant negative TP53 plasmid, pTP53mt135, repressed TP53, pTP53 and PMAIP1 protein and/or TP53, PMAIP1 mRNA levels (P < 0.05), however, significantly augmented GTN-induced apoptotic cell percentages (P < 0.05). Cytosol/mitochondria fractionation identified that TP53, along with PMAIP1 proteins, were translocated to mitochondria after GTN treatment in time-dependent manners. Taken together, our studies demonstrated that GTN induced apoptosis via PMAIP1 via both TP53-dependent and -independent transactivation pathways. In TP53-positive SK-Hep1 cells, PMAIP1 and TP53 proteins conducted synergic effects.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world and Taiwan. The major factors involved in the molecular pathogenesis for the development of HCC had been explored in recent years. An extensive array of growth factors and their receptors had been identified and may act as positive and negative modulators in different stages of hepatocarcinogenesis. Current therapeutic approaches for HCC include surgical resection (include liver transplantation), trans-arterial embolization (TAE), alcohol injection, etc. However, the effect is limited due to most of the HCC patients present with advanced stages of the disease. Therefore, this underscores the need for the development of novel therapeutic strategies. It is pivotal to set up an orthotopic hepatoma model for the development of novel intervention strategies for HCC. Under the guidance of ultrasound, we are able to create hepatoma in the liver lobe of Sprague-Dawley (SD) rats by injection of Novikoff (N1-S1) hepatoma cells. In addition, sonographic technique was employed for the monitoring of tumor growth in this animal model in the following subprojects. The continuous, non-invasive measurement of orthotopic hepatoma development will be a valuable tool for the evaluation of effects of drugs for treatment of HCC. In Chapter 1, the study employed a relatively non-invasive approach to establish an orthotopic HCC model in immune-competent rats. This was done by ultrasound-guided implantation of cancer cells and the model was used to evaluate the therapeutic efficacy of short-term and low-dose epirubicin chemotherapy. Ultrasound-guided implantation of Novikoff hepatoma cells led to the formation of orthotopic HCC in 60.4% of the SD rats. Moreover, tumor sizes measured by ultrasound significantly correlated with those measured by calipers after sacrificing the animals (P < 0.00001). The rate of tumor induction by ultrasound-guided implantation was comparable to that of laparotomy (55/91, 60.4% vs. 39/52, 75%) and no significant difference in sizes of tumor was noted between the two groups. Moreover, there was a significant correlation in tumor size measurement by ultrasound and computerized tomography. In tumor-bearing rats, short-term and low-dose epirubicin chemotherapy caused a significant reduction in tumor growth, and was found to be associated with enhanced apoptosis and attenuated proliferation as well as a decrease in microvessel density in tumors. In chapter 2, we investigated the chemopreventive effects of celecoxib in the growth of orthotopic rat HCC and the possible signal pathways involved. The status of COX-2 expression in rat Novikoff HCC was consistent with that in human HCC. Both Western blot and PCR tests had proved that N1-S1 was a HCC model presenting with low COX-2 enzymes in tumor cells. Then, low doses of celecoxib was shown to effectively inhibited the proliferation and increased the apoptosis of N1-S1 cells in vitro, which were also safe to the normal hepatocytes. Moreover, chemoprevention by celecoxib inhibiting the HCC tumor growth was shown in rat orthotropic HCC model. Tumor incidence was not affected by the celecoxib prevention, but, tumor weight was found significantly suppressed by the drug. Possible mechanisms of chemoprevention by celecoxib seen in the animal model were thought to be related to the anti-angiogenic, anti-proliferative and anti-hCSC characters of the drug. In chapter 3, we tried to test the combined inhibitory effects of low doses of celecoxib and epirubicin on the growth of HCC. Combined low doses of epirubicin and celecoxib was effective in inhibiting the hepatic cancer stem cells, tumor angiogenesis, tumor cell proliferation, as well as promoting cancer apoptosis. These are compatible with the effects of the individual drugs on HCC growth shown in the previous two chapters. In general, combination therapy expressed more effectiveness in tumor suppression and less bone marrow suppression than the individual drugs used alone. Taken together, ultrasound-guided implantation of Novikoff hepatoma cells is an effective means of establishing orthotopic HCC in SD rats, which is suitable and convenient for therapeutic trial of anti-HCC treatment. In the current study, we had proved the efficacies of low doses of two drugs, epirubicin and celecoxib, acting individually, as well as the combined effects of them in treating HCC in this model.

Leukocyte cell-derived chemotaxin 2 (LECT2) is first isolated as a 16-kDa secreted protein from cultured fluid of phytohemagglutinin-activated human T-cell leukemia SKW-3 cells. Recently LECT2 has shown to be synthesized by human hepatocytes and stimulates the growth of chondrocytes. LECT2 is involved in chemotactic factor to neutrophils and may be associated with rheumatoid arthritis. Besides, LECT2 is evolutionarily conserved and acts as a repressor in the Wnt/£]-catenin signaling pathway. Wnt/£]-catenin signaling is implicated in liver carcinogenesis. However, the exact roles of LECT2 in liver carcinogenesis are not yet well characterized. This study is to investigate the extra roles of LECT2 in Wnt signaling. Our results showed that adenoviral administration of LECT2 over-expression suppress oncogenic processes such as migration, invasion, proliferation and colony formation, as well as alteration in cell cycle distributions. In animal model significantly suppress liver malignancies in orthotopic Novikoff hepatoma. In conclusion, we show that ad-LECT2 gene delivery attenuated cell carcinogenesis process via downregulated Wnt/£]-catenin signaling in vitro and suppressed tumor growth in vivo. Besides LECT2 over-expression represents a novel therapeutically factor for hepatocelluar carcinoma.