Pancreatic cancer is one of the most devastating human cancers with the lowest survival rate among 24 commonly diagnosed cancers. It is the seventh and the sixth leading cause of cancer-related deaths in the world and Hong Kong respectively. The current pancreatic cancer treatment options, have limited efficacy and undesirable side effects. Because of the high mortality rate and unsatisfactory treatment outcome, it is necessary to develop new strategies for pancreatic cancer therapy. / In human, an abundant arginine reserve is known to be crucial for tumor cell proliferation. Arginine is a semi-essential amino acid because most of the somatic cells can re-synthesize it from other metabolites like citrulline in urea cycle. However, arginine auxotrophy is observed in certain tumors, such as hepatocarcinoma, melanoma and sarcoma, where restriction or depletion of arginine will lead to tumor death. Further studies have found that deficiency in either argininosuccinate synthetase 1 (ASS1) or ornithine transcarbamylase (OTC) expression contributes to arginine auxotrophy in these tumors. These findings implicated the potential of using arginine deprivation as a novel pancreatic cancer treatment strategy. / PEG-BCT-100 is a pegylated recombinant human arginase that metabolizes arginine into urea and ornithine. This study examined the preclinical anti-tumor efficacy of PEG-BCT-100 and the underlying mechanism in pancreatic cancer. Six pancreatic cancer cell lines AsPC-1, BxPC-3, CFPAC-1, Capan-2, MIA PaCa-2 and Panc10.05 were used as in vitro cell model. Cell growth was either completely stopped or dramatically reduced in arginine-free medium, suggesting pancreatic cancer cells were arginine auxotrophic. The protein and mRNA expression levels of the ASS1, OTC and argininosuccinate lyase (ASL), which are enzymes involved in arginine, were studied. The results showed that ASL was highly expressed in all cell lines, suggesting it is not an essential regulator in arginine auxotrophy in pancreatic cancer. On the other hand, ASS1 was only detected in BxPC-3 and CFPAC-1, while OTC was undetectable in all cell lines in both mRNA and protein levels. The effect of PEG-BCT-100 was illustrated via cell cycle progression, cell proliferation and viability. Single drug effect combining PEG-BCT-100 with other anti-tumor drugs, such as 5-FU and gemcitabine, was further explored. Synergistic effect of PEG-BCT-100 and gemcitabine under combination of PEG-BCT-100 and gemcitabine was observed in CFPAC-1 and MIA PaCa-2. Overexpression of OTC and ASS1 decreased the sensitivity of towards PEG-BCT-100 significantly. Taken together, OTC deficiency is a potential indicative marker for the sensitivity of arginine depletion treatment in pancreatic cancer. / 胰腺癌是最具毀滅性的人類癌症之一，在二十四種常見的癌症中，它有着最低的存活率。儘管不在發病率最高的十種癌症中，胰腺癌仍舊是世界第七大致死癌症，以及香港第六大致死癌症。手術治療，放射治療，以及化學藥物治療是現今常用的胰腺癌治療手段，但是這些療法不是限制繁多，就是收效甚微，並常常伴有強烈的副作用。由於胰腺癌具有很高的致死率以及缺乏有效的治療方法，所以新的治療策略亟待開發。 / 於人類而言，精氨酸是一種半必需氨基酸，因爲它可以通過尿素循環中的其他代謝產物，如鳥氨酸以及瓜氨酸，重新合成。然而，精氨酸缺陷出現在多種腫瘤中，像肝癌，黑色素瘤，以及血癌。限制或者減少精氨酸的供應會導致這些腫瘤死亡。除此之外，腫瘤細胞的快速生長也依賴於充足的精氨酸。進一步的研究表明，在這些腫瘤中，精氨琥珀酸合成酶1（ASS1）或者鳥氨酸氨甲醯基轉移酶（OTC）的任意一個缺乏都會導致精氨酸缺陷。本文將探討將剝奪精氨酸作爲一種新策略來治療胰腺癌的可行性。 / PEG-BCT-100又名金氨素，是一種聚乙二醇化重組人精氨酸酶，它可以催化精氨酸分解爲尿素和鳥氨酸。我們研究了PEG-BCT-100在胰腺癌細胞中的抗癌效果以及探討了與其相關的作用機理。在我們的研究中，AsPC-1, BxPC-3, CFPAC-1, Capan-2, MIA PaCa-2以及Panc10.05這六個細胞株用作體外的細胞模型。爲了評估PEG-BCT-100治療胰腺癌的可行性，我們首先調查了精氨酸對胰腺癌細胞的重要性。通過將這些胰腺癌細胞培養在有精氨酸供應和沒有精氨酸供應的完全培養基中，我們發現剝奪精氨酸能完全停止或者極大地減少了胰腺癌細胞的生長。這說明了這些胰腺癌細胞也都是精氨酸營養缺陷型的細胞。通過蛋白印跡和實時定量聚合酶鏈式反應實驗，我們進一步研究了精氨酸代謝相關基因在這些胰腺癌細胞中的表達水平。結果表明，精氨琥珀酸裂解酶（ASL）在全部的六條細胞系中都有被檢測到。ASS1只出現在BxPC-3和CFPAC-1中。然而在全部的細胞中，無論是蛋白質水平還是mRNA水平，OTC都沒有被檢測到。緊接着，我們研究了PEG-BCT-100在胰腺癌細胞活力，細胞增殖，細胞週期以及細胞凋亡等方面的影響。結果表明，PEG-BCT-100可以從多個方面抑制胰腺癌細胞。我們還嘗試探索了PEG-BCT-100與其他胰腺癌治療藥物在胰腺癌細胞中的聯合使用效果。然後發現PEG-BCT-100與吉西他滨（gemcitabine）聯合使用具有協同效果。最後，我們構建了四種不同表達類型的MIA PaCa-2細胞模型：（ASS1-/OTC-）, (ASS1-/OTC+), (ASS1+/OTC-)以及(ASS1+/OTC+)。接着我們測試了PEG-BCT-100在這些細胞模型中的效果。結果表明，同時在MIA PaCa-2細胞中表達ASS1和OTC可以明顯地提高其對PEG-BCT-100的抗性，單表達其中一個基因對PEG-BCT-100的抗性也有些許提高，但效果不如雙表達明顯。 / 總而言之，對於胰腺癌細胞而言，精氨酸是必不可少的。PEG-BCT-100有很明顯的胰腺癌效果。在胰腺癌中，OTC的表達情況可以作爲預估PEG-BCT-100治療效果的重要生物標誌。 / Deng, Haohao. / Thesis M.Phil. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 111-117). / Abstracts also in Chinese. / Title from PDF title page (viewed on 14, October, 2016). / Deng, Haohao. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

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

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