Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths worldwide with a five-year survival rate of only 9%. The mevalonate (MVA) pathway, which results in synthesis of cholesterol, has been demonstrated to be overexpressed in numerous cancers, leading to its involvement in various aspects of the disease, including proliferation, invasion, and metastasis. In recent years, the MVA pathway has been implicated in PDAC as well, however the underlying mechanisms behind its upregulation in this context, as well as its role in tumor progression, remain largely unknown. An initial analysis of The Cancer Genome Atlas (TCGA) datasets using the Gene Expression Profiling Interactive Analysis (GEPIA) tool revealed one mevalonic gene in particular, SQLE—encoding squalene epoxidase (SQLE)—to be significantly overexpressed in PDAC compared to other genes within the pathway. We find that patients with higher SQLE expression profiles have significantly reduced five-year survival rates that can be further correlated to tumor stage and grade, and we demonstrate that knockdown of SQLE, a branchpoint enzyme of the cholesterol pathway, reduces tumor proliferation in both two-dimensional and three-dimensional assays. We further characterize the SQLE promoter landscape by demonstrating an interesting dynamic of gene activation between the sterol regulatory element binding protein-2 (SREBP2), BRG1-specific SWI/SNF chromatin remodelers, and mutant p53. These results highlight SQLE as a therapeutic target in PDAC and provide a better understanding of its regulation in the context of this devastating disease.

In conclusion, the present study successfully demonstrate BJ as a potent anti-pancreatic cancer herb; BD is the main ingredient for its cytotoxic and apoptotic effects on the pancreatic cancer cells through activation of the redox-sensitive p38-MAPK signaling pathway and reduction of anti-apoptotic activity by inhibition of NF-kappaB activation in pancreatic cancer cells. The in vivo efficacy and low toxicity of BD render this chemical compound to be a potential for its further development into an anti-pancreatic cancer agent. / In recent decades, the application of Chinese herbal medicine has become an increasingly popular approach and alternative to treating cancer. Moreover, Chinese herbal medicine is the source for the discovery of novel anti-cancer drugs. For example, irinotecan and topotecan, the analogues of camptothecin which is isolated from the bark and stem of Camptotheca acuminate are found to be effective in ovarian, lung and colon cancers. Given that Chinese medicine is commonly used in the treatment of cancers, we postulate that Chinese herbs are a valuable source to possess anti-pancreatic cancer compounds. Accordingly, the aims of the present project are: (1) to screen Chinese medicinal herbs which has the most potent cytotoxic activity in pancreatic cancer cells in vitro; (2) to isolate and identify the effective compound in Brucea javanica (BJ) which mediates apoptosis in pancreatic cancer cell lines; (3) to study the mechanistic pathways involved in brucein D - (BD, a quassinoid found in abundance in BJ) mediated apoptosis in pancreatic cancer in vitro; and (4) to evaluate the efficacy of BD in pancreatic cancer using an xenograft animal model of pancreatic cancer. / In vivo study demonstrated that daily administration of BD through intravenous injection for ten days in nude mice bearing pancreatic cancer cells effectively reduced tumor growth in terms of tumor weight and size, while showing no significant toxicity in heart, liver and kidney tissues of the mice. / Nine Chinese medicinal herbs were selected for the screening experiment and, among them, BJ exhibited the most potent cytotoxic action on the three pancreatic adenocarcinoma cell lines, namely PANC-1, SW-1990 and CAPAN-1, with IC50 values of 2.5mug/ml, 5.1mug/ml and 1.5mug/ml, respectively. BD, one of the main chemical compounds found in BJ was found to possess strong apoptogenic effect in PANC-1 cells, as evidenced by DNA condensation and fragmentation, sub-G1 phase formation, proteolytic activation of caspase 3, 8 and 9, and attenuation of bcl-2 activity. Further mechanistic studies revealed that the apoptotic signals generated by BD were transduced from the cell membrane to nucleus via the mediation of p38-MAPK signaling pathway while the reactive oxygen species (ROS) was found to accumulate in BD-treated PANC-1 cells. The activation p38-MAPK phosphorylation was inhibited by pretreatment with an antioxidant. However, the inhibition of NF-kappaB activity and downregulation of anitapoptotic genes in BD-treated cells was independent of the ROS changes. / Pancreatic cancer is the forth and sixth leading cause of cancer deaths in the United States and Hong Kong, respectively. The morbidity of pancreatic cancer is almost equal to its mortality rate. Poor diagnosis and intrinsic resistance to chemotherapy are the major characteristics for pancreatic cancer. Therefore, new therapeutic strategy is urgently warranted to overcome the drug-resistance challenge in the management of pancreatic cancer. / Lau Sin Ting Cynthia. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 228-271). / 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.

胰腺癌是一種死亡率極高的癌症, 據統計在所有的種族和性別中能達到五年存活的胰腺癌患者僅有5.5%。在現有醫學的治療方法中，除了手術切除之外，化學療法依然是主要的應對之策。接受胰腺癌切除術後的無瘤患者的生存期中位數和以現代一線化療藥吉西他濱治療的胰腺癌患者的生存期分別為13.4和6.9月。因此, 臨床上迫切需求更有效治療胰腺癌的新藥物。 / 我們從中藥鴉膽子中分離出了10種不同的化學單體。經過在人類胰腺癌細胞Capan-2上進行的細胞毒性篩選後, 發現Brucein D （BD）擁有最強的胰腺癌細胞毒性作用。我們目前的體外與體內實驗的目標是對BD可能具備的抗胰腺癌活性進行深入評估, 並進一步揭示其作用機理。 / 體外實驗研究表明BD可以極大程度上抑制Capan-2細胞生長, 同時對於人類肝細胞WRL68和人類胰腺幹細胞PPC僅存在很輕微的毒性作用。BD的抑制細胞生長作用和喜樹堿相當, 但顯著強於一線抗胰腺癌藥吉西他濱作。實驗中我們發現在BD作用的Capan-2細胞的線粒體膜電位被減弱, 其減弱程度與BD的濃度存在一定的劑量依賴性。另外, 被BD處理後的Capan-2細胞中的Bcl-2表達減弱, 與此同時capase 9和caspase 3的表達呈顯著性加強。除此之外, BD可以導致基因DNA破碎, 增加Capan-2細胞處於細胞凋亡期的數量, 而且處於凋亡期細胞的數量與BD存在劑量依賴性。 / 我們建立起原位型胰腺癌裸鼠模型並利用其進行體內實驗研究。研究結果顯示, BD治療組裸鼠的存活率遠遠大於吉西他濱治療組。此外, 與磷酸鹽緩衝鹽水注射組比較, BD治療組可以極大程度的減輕腫瘤的重量和減小腫瘤的體積。與此同時, 血液生化分析結果表明BD可以明顯降低CA19-9在血液中的表達。螢光免疫檢驗法結果揭示BD能夠調低CA19-9和Ki-67在胰腺腫瘤組織中的表達。蛋白質印記分析的結果也顯示BD治療後可以增強胰腺腫瘤組織中caspase 3, 8, 9的表達, 而減弱IKKα和NF-κB p65的表達。另外, 通過ELISA分析後顯示, BD治療明顯降低了NF-κB p65在細胞質與細胞核中的表達, 其表達程度與BD的濃度成反比。 / 綜上所述, 我們目前的體外和體內研究表明, BD作為一種存在于天然中藥中的化學單體具有很好的抗胰腺癌的潛質, 值得進一步研究和開發, 使之成為臨床治療胰腺癌的一種安全有效的新藥物。 / Pancreatic adenocarcinoma has a high morbidity and mortality rate in cancers as it possesses only 5.5% of 5-year survival rate for all races and both sexes. The median disease-free survival following complete resection of the pancreatic tumor and adjuvant chemotherapy with the first-line chemotherapeutic agent gemcitabine is 13.4 and 6.9 months, respectively. There issued an urgent need for alternative effective agents to producing a better clinical outcome for the management of this deadly disease. / Previous studies in our research group have shown that the fruit of Brucea javanica L. exhibited potent anti-pancreatic cancer activity. In the current project, ten chemical compounds were isolated from this Chinese herb and screened for their cytotoxicity against cultured Capan-2 cells, a human pancreatic adenocarcinoma cell line. Among these compounds, Brucein D (BD) exhibited the most potent cytotoxic activity. Further in vitro and in vivo studies were conducted to evaluate the potential anti-pancreatic cancer activity of BD and elucidate its underlying mechanisms of action. / In the In vitro study, BD was found to significantly inhibit the growth of Capan-2 cells, while exerting only modest cytotoxicity on human hepatocyte WRL68 cells and human pancreatic progenitor PPC cells. The anti-proliferative effects of BD were comparable to those exhibited by camptothecin and gemcitabine. We found a dose-dependent decrease of the mitochondrial membrane potential in BD-treated Capan-2 cells. In addition, BD exposure was able to attenuate the expression of Bcl-2 and significantly accentuate the expression of both caspase 9 and caspase 3. Moreover, BD was capable of inducing the fragmentation of genomic DNA while increasing the percentage of Capan-2 cells in the apoptotic phase and the quantity of apoptosis cells was observed in a dose- dependent manner. / A mouse model of orthotopic pancreatic cancer was established for the in vivo experiments. The results demonstrated that the BD-treated groups had a higher survival rate than that the gemcitabine-treated groups. Moreover, it was found that BD treatments significantly reduced the tumor weight and volume when compared with those of PBS injected group. Meanwhile, blood biochemistry analyses showed that BD significantly decreased the expression of CA19-9 (a tumor mark). Immunofluorescence study also revealed that BD could down-regulate the expression of both CA19-9 and Ki-67 in pancreatic tumor tissues. Furthermore, Western blot analysis showed that BD treatments could accentuate the expression of caspases 3, 8, 9 and decreased the expression of IKKα and NF-κB p65 in total. Moreover, BD attenuated the expression of NF-κB p65 in both cytoplasmic and nuclear factions of the tumor tissues as detected by ELISA kit, and the expression rate was inversely proportional to the doses of BD used. / Taken these data together, our in vitro and in vivo studies have successfully demonstrated that BD, a naturally occurring chemical compound from Fructus Bruceae, is a promising anti-pancreatic cancer agent worthy of further development into pharmaceutical agent for pancreatic cancer. / 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. / Liu, Ling. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 217-253). / Abstracts also in Chinese. / ABSTRACT --- p.I / 摘要 --- p.IV / PUBLICATIONS --- p.VI / ACKNOWLEDGEMENTS --- p.X / TABLE OF CONTENTS --- p.XI / LIST OF FIGURES --- p.XIX / LIST OF TABLES --- p.XXIII / LIST OF ABBREVIATIONS --- p.XXV / Chapter CHAPTER 1 --- GENERAL INTRODUCTION / Chapter 1.1 --- Pancreas --- p.2 / Chapter 1.1.1 --- Gross anatomy --- p.2 / Chapter 1.1.2 --- Microscopic anatomy --- p.5 / Chapter 1.1.2.1 --- Acini cells --- p.7 / Chapter 1.1.2.2 --- Duct cells --- p.7 / Chapter 1.1.2.3 --- Stroma --- p.14 / Chapter 1.1.2.4 --- Islets cells --- p.15 / Chapter 1.1.3 --- Pancreatic diseases --- p.16 / Chapter 1.2 --- Pancreatic Cancer --- p.30 / Chapter 1.2.1 --- Epidemiology --- p.30 / Chapter 1.2.2 --- Risk factors --- p.32 / Chapter 1.2.3 --- Clinical symptoms, diagnosis and staging --- p.34 / Chapter 1.2.4 --- Types of pancreas tumor --- p.42 / Chapter 1.3 --- Treatment of Pancreatic Cancer --- p.47 / Chapter 1.3.1 --- Treatment for localized disease --- p.49 / Chapter 1.3.2 --- Treatment for locally advanced disease --- p.50 / Chapter 1.3.3 --- Treatment for metastatic disease --- p.52 / Chapter 1.4 --- Molecular Targets for Pancreatic Cancer Therapy --- p.56 / Chapter 1.4.1 --- Mechanisms of apoptosis --- p.56 / Chapter 1.4.2 --- Roles of mitochondrial pathway in apoptosis --- p.58 / Chapter 1.4.3 --- NF-κB activation on cancers --- p.59 / Chapter 1.4.4 --- CA19-9 as a therapeutic target for Pancreatic Cancer --- p.62 / Chapter 1.4.5 --- Ki-67 is associated with for cellular proliferation --- p.64 / Chapter 1.5 --- Applications of Chinese Medicine in Cancer Treatment --- p.66 / Chapter 1.5.1 --- Background of traditional Chinese medicine --- p.66 / Chapter 1.5.2 --- Chinese medicine herbs commonly used for cancer treatment --- p.67 / Chapter 1.6 --- Mouse Models of Pancreatic Cancer --- p.75 / Chapter 1.6.1 --- Anatomy of pancreas in mouse --- p.75 / Chapter 1.6.2 --- Pancreatic cancer models --- p.77 / Chapter 1.7 --- Hypothesis and Objectives of the Study --- p.83 / Chapter CHAPTER 2 --- ANTI-PANCREATIC CANCER EFFECTS OF TEN CHEMICAL COMPOUNDS DERIVED FROM FRUCTUS BRUCEAE ON CULTURED CAPAN-2 CELLS / Chapter 2.1 --- Introduction --- p.86 / Chapter 2.1.1 --- Brucea javanica L. Merr --- p.86 / Chapter 2.1.2 --- The fruit of Brucea javanica --- p.88 / Chapter 2.1.3 --- Used of Fructus Bruceae to treat cancers by Chinese medicine practitioners --- p.90 / Chapter 2.1.4 --- Biological activities of some chemical compounds from Brucea javanica --- p.90 / Chapter 2.1.5 --- Chemical structure of ten compounds isolated from Fructus Bruceae --- p.92 / Chapter 2.2 --- Materials and Methods --- p.96 / Chapter 2.2.1 --- Plant material --- p.96 / Chapter 2.2.2 --- Extratcion, fractionation, isolate and characterization --- p.96 / Chapter 2.2.3 --- General procedures on structural elucidation and phytochemical work --- p.100 / Chapter 2.2.4 --- Preparation of solutions of tern chemical compounds derived from Fructus Bruceae --- p.101 / Chapter 2.2.5 --- General cell culture methods --- p.101 / Chapter 2.2.6 --- Selection of appropriate seeding density of Capan-2 cells --- p.102 / Chapter 2.2.7 --- Cytotoxicity evaluation by SRB assay --- p.102 / Chapter 2.2.8 --- Statistical analyses --- p.103 / Chapter 2.3 --- Results --- p.105 / Chapter 2.3.1 --- Seletion of appropriate seeding density of Capan-2 cells --- p.105 / Chapter 2.3.2 --- IC₅₀ values of ten tested compounds and chemical structures --- p.107 / Chapter 2.4 --- Discussion --- p.111 / Chapter CHAPTER 3 --- INVOLVEMENT OF THE MITOCHONDRIAL PATHWAY IN BRUCEIN D-INDUCED APOPTOSIS IN CAPAN-2 CELLS / Chapter 3.1 --- Introduction --- p.116 / Chapter 3.2 --- Materials and Methods --- p.117 / Chapter 3.2.1 --- General cell culture --- p.117 / Chapter 3.2.2 --- Cytotoxicity assay --- p.119 / Chapter 3.2.3 --- Proliferation assay --- p.120 / Chapter 3.2.4 --- Hoechest fluorescence staining for morphological evaluation --- p.121 / Chapter 3.2.5 --- Cell cycle analysis by flow cytometry --- p.122 / Chapter 3.2.6 --- Quantitative analysis of apoptosis by Annexin V-PI staining assay --- p.122 / Chapter 3.2.7 --- Estimation of the changes of MMP on BD-treated Capan-2 cells --- p.123 / Chapter 3.2.8 --- Western blot analysis --- p.124 / Chapter 3.2.9 --- Statistical analyses --- p.125 / Chapter 3.3 --- Results --- p.126 / Chapter 3.3.1 --- BD significantly inhibited the proliferation of Capan-2 cells --- p.126 / Chapter 3.3.2 --- BD was less cytotoxic on cultured WRL68 and PPC cells than that of controls --- p.128 / Chapter 3.3.3 --- BD induced DNA condensation in Capan-2 cells --- p.131 / Chapter 3.3.4 --- BD induced an increase in the percentage of subG1 phase (apoptotic cells) --- p.133 / Chapter 3.3.5 --- BD dose-dependently induced cellular apoptosis to Capan-2 cells --- p.136 / Chapter 3.3.6 --- The MMP of Capan-2 cells were significantly attenuated by BD treatment --- p.139 / Chapter 3.3.7 --- BD increased the expression of apoptotic caspases in Capan-2 cells --- p.142 / Chapter 3.4 --- Discussion --- p.144 / Chapter CHAPTER 4 --- BRUCEIN D SUPPRESSES PANCREATIC TUMOR GROWTH IN AN ORTHOTOPIC MOUSE MODEL THROUGH THE CASPASE 3, 8, 9 AND NF-κB PATHWAYS / Chapter 4.1 --- Introduction --- p.150 / Chapter 4.2 --- Materials and Methods --- p.153 / Chapter 4.2.1 --- Ethics statement and animal holdings --- p.153 / Chapter 4.2.2 --- Cell culture --- p.153 / Chapter 4.2.3 --- Establishment of an orthotopic pancreatic cancer mouse model --- p.154 / Chapter 4.2.4 --- Treatment of orthotopic pancreatic cancer mice with BD --- p.155 / Chapter 4.2.5 --- Necropsy procedure and histological studies --- p.156 / Chapter 4.2.6 --- Hematoxylin-eosin staining --- p.156 / Chapter 4.2.7 --- Determination of CA19-9 and Ki-67 by immunofluorescence staining --- p.160 / Chapter 4.2.8 --- CA 19-9 expression in blood --- p.161 / Chapter 4.2.9 --- Western blot analysis of Caspase 3,8,9, IKKα and NF-κB p65 --- p.162 / Chapter 4.2.10 --- Extraction of the nucleus and cytoplasm from pancreatic tumor tissues --- p.163 / Chapter 4.2.11 --- Detection of the expression of NF-κB p65 in both cytoplasm and nuclear parts of pancreatic cancer cells --- p.165 / Chapter 4.2.12 --- Statistical analyses --- p.166 / Chapter 4.3 --- Results --- p.167 / Chapter 4.3.1 --- BD treatment enhanced the survival rate of tumor-bearing mice and significantly attenuated the tumor weight and volume --- p.167 / Chapter 4.3.2 --- Histological evaluation of the pancreas and pancreatic tumor after BD treatment --- p.175 / Chapter 4.3.3 --- BD significantly decreased the expression of CA19-9 in the blood samples of the experimental mice --- p.178 / Chapter 4.3.4 --- BD down regulated the expression of CA19-9 in pancreatic tumor tissues --- p.180 / Chapter 4.3.5 --- BD down regulated the expression of Ki-67 in pancreatic tumor tissues --- p.183 / Chapter 4.3.6 --- BD accentuated the expression of Caspase3, 8, 9 and decreased the expression of NF-κB p65 --- p.186 / Chapter 4.3.7 --- BD decreased the expression of NF-κB p65 in both cytoplasm and nucleus of pancreatic tumor cells --- p.189 / Chapter 4.4 --- Discussion --- p.191 / Chapter CHAPTER 5 --- GENERAL DISCUSSION, CONCLUSIONS AND FUTURE STUDIES / Chapter 5.1 --- General Discussion --- p.200 / Chapter 5.2 --- General Conclusions --- p.209 / Chapter 5.3 --- Limitation of Study --- p.211 / Chapter 5.4 --- Clinical Significance of Study Results --- p.212 / Chapter 5.5 --- Future Studies --- p.214 / Chapter 5.5.1 --- Investigation of the possible synergistic effect of combination of BD with gemcitabine on orthotopic pancreatic cancer mouse model --- p.214 / Chapter 5.5.2 --- Testing BD on different animal models --- p.215 / REFERENCES / References by Alphabetical Order --- p.217

微小RNA是内源性小非编码RNA，在肿瘤生成中扮演重要角色。Brucein D(BD)是一种B. javanica果实提取物，已被报道在胰腺癌中具有抗肿瘤作用。在此研究中，我们证明了BD在体内和体外均可抑制肝癌细胞生长。为了研究BD是否通过调节微小RNA来执行其抗肿瘤功能，我们进行了一个肿瘤微小RNA定量PCR阵列谱分析。此阵列包括95个已被报道与肿瘤有关的微小RNA。通过对比BD处理前后微小RNA谱的变化，我们发现微小RNA-95在BD处理后被显著下调了。其后促凋亡的CUGBP2被确定为微小RNA-95的下游靶基因。 / 胰腺癌是一种预后很差的恶性肿瘤，常常在确诊时已发生转移。为了找出在胰腺癌转移过程中发挥决定性作用的基因，我们进行了全基因组范围的RNA干扰筛选。一个包含针对全部人类基因的shRNA文库被导入胰腺癌细胞系capan-2.然后将这些细胞移植到裸鼠的胰腺中来建立一个原位胰腺癌小鼠模型。我们的假设是下调某个基因会促使低转移潜力的capan-2细胞转移到肝脏。通过从肝转移结节中回收shRNA模板，我们找到了几个推定的转移抑制基因。其中之一，SOX9，通过体内实验验证，证明下调SOX9基因的表达可促进胰腺癌转移。 / 化疗适用于进展期胰腺癌病人。然而他们对一线化疗药吉西他滨的反应并不乐观，这进一步使胰腺癌的预后变差。我们展开了一个全基因组范围的RNA干扰筛选来确定一些在化疗耐药过程中起关键作用的基因。携带上述shRNA文库的capan-2细胞被用于吉西他滨药物处理之下的筛选。通过微阵列分析，一些基因被筛选成为可影响癌细胞对药物敏感性的潜在的靶基因。通过进一步验证，LLGL1基因被确定为在调节癌细胞对化疗敏感性过程中起重要作用的基因。 / MicroRNAs (miRNAs) are endogenous small non-coding RNAs that have been shown to play important roles in tumorigenesis. Brucein D (BD), a chemical compound isolated from Brucea javanica fruit, has previously been reported to have anti-cancer effect in pancreatic cancer. In this study, we showed that BD also inhibited the growth of liver cancer cells both in vitro and in vivo. To investigate whether BD exerts its anti-cancer effect through regulation of miRNAs, we performed a cancer miRNA qPCR array profiling. From the profiling, miR-95 was found to be significantly down-regulated after BD treatment. Subsequently, a pro-apoptotic gene CUGBP2 was identified as a direct downstream target of miR-95. These findings suggested BD suppressed liver cancer cell growth through down-regulation of miR-95 and reinforcing CUGBP2. / Pancreatic cancer is an aggressive malignancy with extremely poor prognosis. It is usually diagnosed when metastases are already present. To identify genes that play critical roles in the processes of pancreatic cancer metastasis, a whole genome RNAi screening was performed. An shRNA library targeting all human genes was introduced into a human pancreatic cancer cell line capan-2. The infected cells were then transplanted into the pancreas of nude mice. Because capan-2 is of low metastatic potential, we hypothesized that knocking down of metastasis suppressor genes would facilitate capan-2 cells to spread to the liver. By retrieving shRNA templates from the liver metastatic nodules, several candidate genes were found. One of them, SOX9, has been validated as metastasis suppressor gene in vivo, implying that loss of expression of SOX9 promotes pancreatic cancer metastasis. / Chemotherapy is recommended for patients of pancreatic cancer in advanced stage. However, their response to the first-line chemotherapy drug gemcitabine is not satisfactory. A genome-wide RNAi screening was conducted to identify genes that were critical in chemotherapy resistance. Capan-2 cells containing the above shRNA library were applied for the screening under gemcitabine treatment. Through microarray analysis, a number of genes were screened as potential gemcitabine sensitivity genes. Validation experiments implied that the gene LLGL1 may play an important role in modulating pancreatic cancer cells’ sensitivity to gemcitabine. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xia, Tian. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 125-134). / Abstracts also in Chinese. / Chapter Abstract --- p.I / Chapter Acknowledgements --- p.V / Chapter Abbreviations --- p.VI / Chapter List of Figures --- p.XV / Chapter List of Tables --- p.XVI / Chapter Part I: --- Brucein D-modulated microRNA-95 expression inhibits hepatocellular carcinoma cell growth --- p.1 / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Hepatocellular carcinoma --- p.1 / Chapter 1.1.1 --- Definition and classification --- p.1 / Chapter 1.1.2 --- Epidemiology --- p.1 / Chapter 1.1.3 --- Etiology --- p.3 / Chapter 1.1.4 --- Molecular pathogenesis of HCC --- p.4 / Chapter 1.1.4.1 --- Genomic instability --- p.4 / Chapter 1.1.4.2 --- Deregulation of key signaling pathways --- p.5 / Chapter 1.1.4.3 --- Epigenetic changes of HCC --- p.6 / Chapter 1.1.4.4 --- Two models of HCC pathogenesis --- p.7 / Chapter 1.1.5 --- Therapeutic methods and prognosis of HCC --- p.8 / Chapter 1.2 --- Apoptosis --- p.9 / Chapter 1.2.1 --- Types of cell death --- p.9 / Chapter 1.2.2 --- Apoptosis --- p.10 / Chapter 1.2.3 --- Morphological features of apoptosis --- p.10 / Chapter 1.2.4 --- Molecular mechanisms of apoptosis --- p.11 / Chapter 1.2.5 --- Apoptosis and cancer --- p.13 / Chapter 1.2.5.1 --- Imbalance of pro-apoptotic proteins and anti-apoptotic proteins --- p.13 / Chapter 1.2.5.2 --- Impaired caspases activity --- p.14 / Chapter 1.2.5.3 --- Deregulated death receptor signaling --- p.15 / Chapter 1.2.6 --- Cancer therapy targeting apoptotic defects --- p.15 / Chapter 1.3 --- microRNA --- p.16 / Chapter 1.3.1 --- Overview --- p.16 / Chapter 1.3.2 --- Biogenesis and maturation of microRNA --- p.17 / Chapter 1.3.3 --- Gene silencing by microRNA --- p.18 / Chapter 1.3.4 --- MicroRNA and cancers --- p.19 / Chapter 1.3.5 --- MicroRNA’s involvement in HCC --- p.21 / Chapter 1.3.6 --- Involvement of miR-95 in cancer --- p.22 / Chapter 1.4 --- Brucein D --- p.22 / Chapter 1.5 --- Aims of study --- p.23 / Chapter 2 --- Materials and Methods --- p.25 / Chapter 2.1 --- Cell Culture --- p.25 / Chapter 2.1.1 --- Mammalian Cell Culture --- p.25 / Chapter 2.1.2 --- Preparation of cell stock --- p.25 / Chapter 2.1.3 --- Cell recovery from liquid nitrogen stock --- p.26 / Chapter 2.1.4 --- Preparation of drugs for treatments --- p.26 / Chapter 2.1.5 --- Drug treatment --- p.26 / Chapter 2.1.6 --- Transfection of siRNA --- p.27 / Chapter 2.1.7 --- MTT Assay --- p.28 / Chapter 2.1.8 --- Luciferase reporter assays --- p.28 / Chapter 2.1.9 --- Annexin V Assay --- p.29 / Chapter 2.2 --- In vivo mouse model --- p.29 / Chapter 2.3 --- Tunel Assay (Terminal deoxynucleotide transferase dUTP Nick End Labeling Assay) --- p.30 / Chapter 2.4 --- RNA manipulation --- p.31 / Chapter 2.4.1 --- RNA Isolation --- p.31 / Chapter 2.4.2 --- Synthesis of cDNA from miRNA --- p.32 / Chapter 2.4.3 --- Synthesis of cDNA from RNA and quantitative PCR --- p.33 / Chapter 2.4.4 --- miRNA qPCR array --- p.34 / Chapter 2.5 --- DNA manipulation --- p.34 / Chapter 2.5.1 --- Agarose gel electrophoresis and purification of DNA --- p.34 / Chapter 2.5.2 --- Restriction enzymes digestion --- p.35 / Chapter 2.5.3 --- Ligation of DNA fragments --- p.36 / Chapter 2.5.4 --- Polymerase chain reaction --- p.36 / Chapter 2.5.5 --- Preparation of competent E. coli cells --- p.37 / Chapter 2.5.6 --- Transformation of E. coli cells --- p.37 / Chapter 2.5.7 --- Small scale plasmid isolation from E. coli (mini-prep) --- p.38 / Chapter 3 --- Results --- p.39 / Chapter 3.1 --- Brucein D inhibited the growth of HCC cells both in vitro and in vivo --- p.39 / Chapter 3.2 --- BD induced apoptosis in HCC cells --- p.43 / Chapter 3.3 --- miR-95 is an target of BD to modulate cell growth --- p.46 / Chapter 3.4 --- Identification of CUGBP2 as a downstream target of miR-95 --- p.55 / Chapter 4 --- Discussion --- p.60 / Chapter Part II: --- Genome-wide RNAi screening identifies tumor metastasis suppressor genes and drug sensitivity genes in pancreatic cancer --- p.65 / Chapter 1 --- Introduction --- p.65 / Chapter 1.1 --- Pancreatic cancer --- p.65 / Chapter 1.1.1 --- Overview --- p.65 / Chapter 1.1.2 --- Pancreatic ductal adenocarcinoma (PDAC) --- p.67 / Chapter 1.1.3 --- Molecular basis of PDAC --- p.67 / Chapter 1.1.3.1 --- KRAS --- p.67 / Chapter 1.1.3.2 --- TP53 --- p.68 / Chapter 1.1.3.3 --- CDKN2A --- p.69 / Chapter 1.1.4 --- Gemcitabine treatment in PDAC --- p.69 / Chapter 1.2 --- Metastasis --- p.71 / Chapter 1.2.1 --- Overview --- p.71 / Chapter 1.2.2 --- The stepwise process of metastasis --- p.72 / Chapter 1.2.3 --- Metastasis of pancreatic cancer --- p.74 / Chapter 1.3 --- SOX9 --- p.75 / Chapter 1.4 --- Aims of study --- p.77 / Chapter 2 --- Materials and Method --- p.78 / Chapter 2.1 --- Cell culture --- p.78 / Chapter 2.1.1 --- Mammalian Cell Culture --- p.78 / Chapter 2.1.2 --- MTT Assay --- p.78 / Chapter 2.1.3 --- Colony formation assay --- p.79 / Chapter 2.1.4 --- Wound healing assay --- p.79 / Chapter 2.1.5 --- Transwell migration chamber assay --- p.80 / Chapter 2.1.6 --- Immunocytochemistry --- p.80 / Chapter 2.1.7 --- Transient transfection of siRNA --- p.81 / Chapter 2.2 --- Establishment of in-vivo and in-vitro models --- p.82 / Chapter 2.2.1 --- shRNA library introduction --- p.82 / Chapter 2.2.2 --- Establishment of the orthotopic pancreatic cancer mouse model --- p.82 / Chapter 2.2.3 --- Package of lentivirus expressing shRNA --- p.83 / Chapter 2.2.4 --- Generation of stable cell line expressing shRNA --- p.84 / Chapter 2.3 --- DNA manipulation --- p.84 / Chapter 2.3.1 --- Large scale plasmid isolation from E. coli (maxi-prep) --- p.84 / Chapter 2.4 --- Analysis of Protein --- p.85 / Chapter 2.4.1 --- Preparation of protein cell lysates --- p.85 / Chapter 2.4.2 --- Protein concentration determination --- p.86 / Chapter 2.4.3 --- SDS-PAGE --- p.86 / Chapter 2.4.4 --- Immunoblotting (Western blotting) --- p.87 / Chapter 2.5 --- RNA manipulations --- p.88 / Chapter 2.5.1 --- RNA Isolation --- p.88 / Chapter 2.5.2 --- Synthesis of cDNA from RNA and quantitative PCR --- p.89 / Chapter 2.6 --- Analysis of Clinical Samples --- p.90 / Chapter 2.6.1 --- Clinical specimens --- p.90 / Chapter 2.6.2 --- Immunohistochemistry --- p.90 / Chapter 3 --- Results --- p.92 / Chapter 3.1 --- Genome-wide RNAi screening identifies genes as metastasis suppressors in an orthotopic pancreatic cancer mouse model --- p.92 / Chapter 3.2 --- SOX9 is a metastasis suppressor gene in pancreatic cancer --- p.97 / Chapter 3.3 --- Investigation into cellular functions of SOX9 --- p.102 / Chapter 3.3.1 --- SOX9’s effect on cell growth --- p.102 / Chapter 3.3.2 --- SOX9’s effect on cell migration --- p.105 / Chapter 3.4 --- Implication of SOX9 in human pancreatic cancer samples --- p.109 / Chapter 3.5 --- Genome-wide RNAi screening for the identification of gemcitabine sensitivity genes --- p.113 / Chapter 4 --- Discussion --- p.120 / Chapter General conclusions --- p.125

Serum levels of several molecules associated to pancreatic adenocarcinoma (PDAC) pathophysiology are evaluated in this work, in order to determine their diagnostic value, distinguishing between PDAC patients and healthy controls (HC), different gastrointestinal tumours (GIT) and chronic pancreatitis (CP). Plasma mRNA levels in plasma of sialyltransferases (ST3Gal III and ST3Gal IV) could differentiate between HC and PDAC. Moreover, lower levels of ST3Gal III in early stages of PDAC compared to PDAC advanced stages were reported. The Glasgow Prognostic Score (GPS), inflammatory response quantification, differentiated between all the study groups. IGF-1 levels were lower in neoplasic groups of patient vs HC and CP. We assessed the diagnostic capacity of different markers alone or in combination and compared with that of CA 19.9. The best diagnostic capacity was found combining CEA, CA 19.9 and IGF-1 compared with CA 19.9 and it could be useful to distinguish PDAC from CP. / En este trabajo se analizan distintos parámetros séricos relacionados con la fisiopatología del adenocarcinoma pancreático (PDAC), para evaluar su uso como marcadores que permitan distinguir entre pacientes con PDAC y controles sanos (C), otras neoplasias del tracto gastrointestinal (ONEOS) y pancreatitis crónica (PC). La expresión en plasma de sialiltransferasas, ST3Gal III y ST3Gal IV diferencia entre controles y PDAC. Así mismo, los niveles de ST3Gal III permiten diferenciar en PDAC entre estadíos iniciales y metastáticos. El Glasgow Prognostic Score (GPS), medida de la respuesta inflamatoria, diferencia entre todos los grupos del estudio. Los valores de IGF-1 disminuyen en procesos neoplásicos vs C y PC.Se analiza la capacidad diagnóstica de los distintos parámetros individualmente y combinados entre sí respecto al CA19.9. La combinación de CA 19.9, CEA, IGF-1 aumenta la precisión diagnóstica frente al CA19.9 y podría ser útil para distinguir PDAC de PC.

L’adénocarcinome du pancréas qui représente la quatrième cause de cancer en termes d’incidence reste une tumeur mal comprise et l’ensemble des traitements insuffisamment efficaces. Depuis quelques années, la prise en charge des patients cancéreux s’est individualisée et tente de proposer des traitements à la carte en prenant en compte divers paramètres tumoraux. Pour le cancer du pancréas, la pertinence des hypothèses biologiques pour le pronostic et le traitement des patients, a été très peu évaluée si bien qu’a coté des facteurs cliniques et histologiques, il n’existe pas de marqueurs moléculaires clairement identifiés. <p>Dans la première partie de nos traveaux, nous avons évalué l’intérêt pronostique d’une série de biomarqueurs dont les chimiorécepteurs CXCR4 et CXCR7. CXCR4 est exprimé par les ACP et joue un rôle dans la migration des cellules cancéreuses au travers la matrice extracellulaire, l’adressage métastatique, l’angiogénèse et la vasculogénèse tumorale. CXCR7 est impliqué dans la prolifération cellulaire. Ces deux chimiorécepteurs partagent le même ligand, CXCL12, qui est synthétisé par les cellules stellaires pancréatiques activées et est retrouvé en quantité importante dans le stroma fibrotique tumoral. L’expression de CXCR4 est en partie dépendante du facteur de transcription HIF-1&61537; (hypoxia inducible factor). L’expression de CXCR4, CXCR7, HIF-1&61537; et de Ki-67 à été évaluée par immunohistochimie et confrontée aux facteurs pronostiques classiques (cliniques et anatomopatholologiques). En analyse multivariée, le niveau d’expression de CXCR4 était un facteur pronostique indépendant associé à la survie sans récidive et la survie globale. De plus le niveau d’expression de CXCR4 était associé à la présence de métastase ganglionnaire sur la pièce opératoire et au risque de récidive hépatique.<p>Dans un deuxième temps, les protéines impliquées dans la métabolisation de la gemcitabine ont été évaluées à partir d’une cohorte de patients traités par radiochimiothérapie dans le cadre de deux protocoles cliniques multicentriques de phase II. La gemcitabine pénètre dans la cellule à travers la membrane plasmique soit par diffusion facilitée par des transporteurs nucléosidiques spécifiques appelés hENT (human equilibrative nucleoside transporter), soit par des transporteurs actifs Na+-dépendants ou hCNT (human concentrative nucleoside transporter). Dans la cellule, elle est métabolisée pour générer ses dérivés cytotoxiques actifs. La gemcitabine va tout d’abord être phosphorylée en dérivé monophosphate par la déoxycytidine kinase (dCK). La dCK est considérée comme étant l’enzyme limitante du métabolisme de la gemcitabine. Deux autres étapes de phosphorylation vont suivre faisant tout d’abord intervenir l’uridylate kinase (UMK) puis une troisième kinase qui, elle, est ubiquitaire.<p>In vitro, dans des lignées cellulaires d’adénocarcinome pancréatique, la résistance intrinsèque à la gemcitabine est associée à une diminution ou à une perte d’expression de hENT1 et de la dCK. En plus de son activité cytotoxique directe, la gemcitabine est un puissant radiosensibilisateur. La capacité de cet analogue pyrimidique à potentialiser les effets de la radiothérapie est en partie liée à sa capacité de pénétration. L’expression de hENT1, de hCNT3 et de la dCK a été évaluée et corrélée à la survie sans récidive et la survie globale des patients. En analyse multifactorielle, hENT1 et la dCK sont des facteurs pronostiques indépendants pour la survie sans récidive et la survie globale alors que hCNT3 est un facteur indépendant pour la survie globale. De plus l’analyse combinée de hENT1 et de hCNT3 s’avèrait plus discriminante dans l’identification des patients à haut rique de récidive que l’analyse de chacun de ces facteurs pris isolémment.<p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Bakgrund: Sockersötade drycker är drycker till vilka sockerarter tillsatts. Dessa drycker konsumeras globalt och många gånger överskrids rekommendationerna om intag av tillsatt socker, vilket kan leda till negativa hälsoeffekter. Sockersötade drycker kan öka risken att drabbas av övervikt, fetma och typ 2-diabetes som i sin tur är riskfaktorer för att utveckla bukspottkörtelcancer. Det finns därför ett ökat intresse att undersöka den potentiellt ökade cancerrisken vid konsumtion av sockersötade drycker. Syfte: Syftet var att undersöka om intag av sockersötade drycker kan öka risken att drabbas av bukspottkörtelcancer. Metod: Arbetet är en litteraturstudie där vetenskapliga artiklar sökts med databasen PubMed. Sökorden var ”sugar-sweetened beverages” och ”pancreas cancer”. Av de artiklar som erhölls inkluderades observationsstudier och kliniska studier. Utifrån ytterligare inklusions- och exklusionskriterier valdes sju studier ut och granskades. Resultat: Resultat från tre av sju studier visar att det inte förekommer något samband mellan konsumtion av sockersötad dryck och risk för bukspottkörtelcancer medan resultat från tre studier påvisar samband. Resultat från en studie visar att det finns samband hos kvinnor men inte hos män. Slutsats: Hälften av studierna visar ett samband mellan konsumtion av sockersötad dryck och en ökad risk att drabbas av bukspottkörtelcancer. För att minska risken kan det således vara fördelaktigt att följa de rekommendationer som finns kring tillsatt socker. / Background: Sugar-sweetened beverages are beverages to which sugars are added. These beverages are consumed globally and many times the recommendations on added sugar are exceeded, which can lead to negative health effects. Sugar-sweetened beverages may increase the risk of overweight, obesity and type 2 diabetes which are risk factors for developing pancreatic cancer. Thus, there is an increased interest to study the potentially increased cancer risk associated with the consumption of sugar-sweetened beverages. Aim: The aim was to examine whether consumption of sugar-sweetened beverages can increase the risk of pancreatic cancer. Method: This work is a literature study using scientific articles searched with the database PubMed. The search terms were “sugar-sweetened beverages” and “pancreas cancer.” Of the articles obtained, observational studies and clinical studies were included. Based on inclusion and exclusion criteria, seven studies were selected and reviewed. Results: Results from three of seven studies show that there is no relationship between consumption of sugar-sweetened beverages and risk of pancreatic cancer, while results from three other studies show that there is a relationship. Results from one study show that there is a relationship in women but not in men. Conclusion: Half of the studies shows that there is a relationship between consumption of sugar-sweetened beverages and increase the risk of pancreatic cancer. To reduce the risk, it can be beneficial to follow the recommendations that exist regarding added sugar.

Pancreatic cancer is known to be one of the most life-threatening cancers characterized by aggressive local invasion and distant metastasis. The high basal level of autophagy in pancreatic cancer may be responsible for the low chemotherapeutic drug response rate and poor disease prognosis. However, the clinical application of autophagy inhibitors was unsatisfactory due to their toxicity and minimal single-agent anticancer efficacy. Hence, oncologists begin to consider the tumor microenvironment when exploring new drug targets. In the present study, the anti-tumorigenic mechanisms of two major phytochemicals derived from Chinese medicinal herbs had been investigated against pancreatic cancer development. Calycosin is a bioactive isoflavonoid of the medicinal plant Astragalus membranaceus. Our results have shown that calycosin inhibited the growth of various pancreatic cancer cells both in vitro and in vivo by inducing cell cycle arrest and apoptosis. Alternatively, calycosin also facilitated MIA PaCa-2 pancreatic cancer cell migration in vitro and increased the expression of epithelial-mesenchymal transition (EMT) biomarkers in vivo. Further mechanistic study suggests that induction of the Raf/MEK/ERK pathway and facilitated polarization of M2 tumor-associated macrophage in the tumor microenvironment both contribute to the pro-metastatic potential of calycosin in pancreatic cancer. These events appear to be associated with calycosin-evoked activation of TGF-β signaling, which may explain the paradoxical drug actions due to the dual roles of TGF-β as both tumor suppressor and tumor promoter in pancreatic cancer development under different conditions. Isoliquiritigenin (ISL) is a chalcone obtained from the medicinal plant Glycyrrhiza glabra, which can be a precursor for chemical conversion to form calycosin. Results have shown that ISL decreased the growth and EMT of pancreatic cancer cells in vitro, probably due to modulation of autophagy. ISL-induced inhibition of autophagy subsequently promoted reactive oxygen species (ROS) production, leading to induction of apoptosis in pancreatic cancer cells. Such phenomenon also contributed to the synergistic growth-inhibitory effect in combined treatment with the orthodox chemotherapeutic drug 5-fluorouracil. In addition, ISL-induced tumor growth inhibition in vivo was further demonstrated in a tumor xenograft mice model of pancreatic cancer. ISL promoted apoptosis and inhibited autophagy in the tumor tissues. Study on immune cells indicates that ISL could reduce the number of myeloid-derived suppressor cells (MDSCs) both in tumor tissue and in peripheral blood, while CD4+ and CD8+ T cells were increased correspondingly. In vitro test has revealed that ISL inhibited the polarization of M2 macrophage along with its inhibition of autophagy in M2 macrophage. These immunomodulating effects of ISL had reversed the pro-invasive role of M2 macrophage in pancreatic cancer.In conclusion, calycosin acts as a "double-edged sword" on the growth and metastasis of pancreatic cancer, which may be related to the dual roles of TGF-β and its influence on the tumor microenvironment. Alternatively, ISL consistently inhibited the growth and metastatic drive of pancreatic cancer through regulation of autophagy and reprogramming of the immune system. The differential modes of action of these compounds have provided new insights in the development of effective pancreatic cancer treatment adjuvants.

Background: Pancreatic cancer is one of the leading causes of cancer-related death. Using DNA gene expression analysis based on a custom made Affymetrix cancer array, we investigated the expression pattern of both primary and established pancreatic carcinoma cell lines. Methods: We analyzed the gene expression of 5 established pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2 and HPAF II) and 5 primary isolates, 1 of them derived from benign pancreatic duct cells. Results: Out of 1,540 genes which were expressed in at least 3 experiments, we found 122 genes upregulated and 18 downregulated in tumor cell lines compared to benign cells with a fold change > 3. Several of the upregulated genes (like Prefoldin 5, ADAM9 and E-cadherin) have been associated with pancreatic cancer before. The other differentially regulated genes, however, play a so far unknown role in the course of human pancreatic carcinoma. By means of immunohistochemistry we could show that thymosin [β-10 (TMSB10), upregulated in tumor cell lines, is expressed in human pancreatic carcinoma, but not in non-neoplastic pancreatic tissue, suggesting a role for TMSB10 in the carcinogenesis of pancreatic carcinoma. Conclusion: Using gene expression profiling of pancreatic cell lines we were able to identify genes differentially expressed in pancreatic adenocarcinoma, which might contribute to pancreatic cancer development. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Indiana University-Purdue University Indianapolis (IUPUI) / Fatty acid synthase (FASN) is over-expressed in many cancers, and novel inhibitors that target FASN may find use in the treatment of cancers. It has been shown that orlistat, an FDA approved drug for weight loss, inhibits the thioesterase (TE) of FASN, but can be hydrolyzed by TE. To understand the mechanisms of TE action and for designing better FASN inhibitors, I examined the mechanism of orlistat hydrolysis by TE using molecular dynamics simulations. I found that the hexyl tail of orlistat undergoes a conformational transition, destabilizing a hydrogen bond that forms between orlistat and the active site histidine. A water molecule can then hydrogen bond with histidine and become activated to hydrolyze orlistat. These findings suggest that rational design of inhibitors that block hexyl tail transition may lead to a more potent TE inhibitor. To search for novel inhibitors of TE, I performed virtual DOCK screening of FDA approved drugs followed by a fluorogenic assay using recombinant TE protein and found that proton pump inhibitors (PPIs) can competitively inhibit TE. PPIs, which are used for the treatment of gastroesophageal reflux and peptic ulcers, work to decrease gastric acid production by binding irreversibly with gastric hydrogen potassium ATPase in the stomach. Recently, PPIs have been reported to reduce drug resistance in cancer cells when used in combination with chemotherapeutics, although the mechanism of resistance reduction is unknown. Further investigation showed that PPIs are able to decrease FASN activity and cancer cell proliferation in a dose-dependent manner. These findings provide new evidence that FDA approved PPIs may synergistically suppress cancer cells by inhibiting TE of FASN and suggests that the use of PPIs in combinational therapies for the treatment of many types of cancer, including pancreatic cancer, warrants further investigation.