胰腺癌是一種致死率極高的惡性疾病,在全世界所有的癌症中死亡率排列第八, 在美國排列第四。 很多因素造成了胰腺癌較差的預後,其中包括: 早期檢出率極低; 較少胰腺癌患者的腫瘤適宜手術切除;高轉移率;以及對傳統放療和化療具有較高抗性等。 因此,發展新的治療藥物迫在眉睫。 / 近年來, 植物藥以及從這些植物藥裡分離出的天然化合物, 單獨使用或者與傳統化療藥物合併使用時, 都顯示出對不同類型的癌症具有較好療效。植物藥毛萼香茶菜(唇形科)含有豐富的具有抗癌活性的二萜類化合物。其中毛萼乙素(EriB) 是一個擁有最好抗癌活性的對映-貝殼杉烷型二萜化合物。 基於此背景, 本研究的目標為:利用胰腺癌體外體內模型, 研究EriB的抗胰腺癌活性以及誘導胰腺癌細胞凋亡的機理。 / 體外實驗中, EriB對四種胰腺癌細胞株都顯示了顯著的細胞毒活性,其活性與化療藥物喜樹堿類似。其中, EriB對胰腺癌細胞株CAPAN-2活性最強, 半數致死濃度IC₅₀為0.73 μM。細胞凋亡特徵:細胞核凝聚, 磷脂醯絲氨酸外翻, DNA梯狀條帶以及片斷化,在EriB誘導的胰腺癌細胞株CAPAN-2中出現。此外, EriB還造成癌細胞在細胞週期G2/M期的阻滯。機理研究發現, EriB是通過啟動絲裂原活化蛋白激酶(MAPK), caspase及 p53信號通路來誘導細胞凋亡和細胞週期阻滯的。抗凋亡蛋白與促凋亡蛋白比率(bcl-2/bak)的減少也可能對啟動細胞凋亡內途徑發揮一定作用。除此以外, EriB對癌細胞的細胞毒活性及致凋亡作用依賴于活性氧分子(ROS)的產生。在對細胞進行抗氧化劑預處理的實驗中發現, 只有含巰基基團的抗氧化劑能夠有效的阻斷EriB對癌細胞的活性。進一步實驗證明, EriB對細胞內兩個抗氧化系統: 谷胱甘肽系統及硫氧還蛋白系統的抑制作用導致了ROS在癌細胞中的積聚。同時,ROS的產生啟動了MAPK,熱休克蛋白70以及caspase信號通路,卻抑制了NFκB通路。 / 動物體內實驗證實, 每天對胰腺癌細胞移植瘤裸鼠進行腹腔注射EriB(2.5 毫克/千克),能有效的抑制腫瘤生長, 並且對心臟,肝臟和腎臟沒有引起顯著毒性。 對腫瘤組織的分析表明, 給藥組(EriB)比溶劑對照組出現更多的細胞凋亡, 並產生較多的ROS積聚。 / 綜上所述, 本項研究首次闡述了EriB具有顯著的體內外抗胰腺癌活性。機理研究證明, EriB抑制胰腺癌細胞內兩個含巰基基團的抗氧化系統, 從而導致ROS在細胞中積聚, 並啟動(或抑制)了包括MAPK, p53, caspase和NFB在內的信號通路, 最終導致癌細胞死亡。 此外, 動物體內研究證明EriB的抗腫瘤生長活性和低毒性, 令該化合物具有潛力進一步研究發展成為抗胰腺癌的新藥物。 / Pancreatic cancer is the fourth and eighth leading cause of cancer-related deaths in the U.S. and worldwide, respectively. Its poor prognosis is attributed to its late diagnosis, limitation to surgical resection, aggressive local invasion, and early metastases, as well as high resistance to chemotherapy and radiotherapy. Therefore, a search for an alternative to therapeutic agents is in desperate need. / In recent years, herbal medicines or natural compounds isolated from herbs either used alone or in combination with conventional anti-cancer agents have been shown to have beneficial effects on various cancers. In this context, the Chinese herb Isodon eriocalyx (Dunn.) Hara (family Lamiaceae) is a well-known source of anti-cancer diterpenoids, the most potent one being Eriocalyxin B (EriB, an ent-kauranoid). Therefore, the aims of the present study are to investigate the anti-tumor activities of EriB in human pancreatic adenocarcinoma cells and tumor-bearing mouse model, as well as the underlying mechanisms. / Our results showed that EriB exhibited significant cytotoxic effects on four pancreatic adenocarcinoma cell lines, with potencies being comparable to that of chemotherapeutic agent camptothecin. EriB had the most potent cytotoxicity in CAPAN-2 cells with IC₅₀ = 0.73 μM. The hallmark features of apoptosis, such as nuclear condensation, translocation of phosphatidylserine, DNA laddering, and DNA fragmentation were observed in EriB-treated CAPAN-2 cells. On the other hand, EriB also induced G2/M phase cell cycle arrest. Mechanistic studies revealed that EriB induced apoptosis and cell cycle arrest through the activation of MAPKs (p38, ERK1/2), caspase cascade, and p53/p21/cdk1-cyclinB1 signaling pathways. A decrease in the ratio of anti-apoptotic to pro-apoptotic proteins (bcl-2/bak) also contributed to the activation of intrinsic apoptotic pathway. Further investigation showed that EriB-induced cytotoxic and apoptotic effects were dependent on reactive oxygen species (ROS) production. Such demonstrated effects could be inhibited by pre-treatment with thiol-containing antioxidants. Furthermore, EriB induced ROS was mediated via the inhibition of two main antioxidant systems, namely glutathione and thioredoxin systems. EriB-mediated ROS activated multiple targets or signal pathways, including MAPK, heat shock protein (Hsp) 70, and caspase cascade, while inhibiting the NFκB pathway. / On the other hand, in vivo study demonstrated that daily intraperitoneal administration of EriB (2.5mg/kg/day) in human pancreatic tumor xenografts BALB/c nude mice significantly inhibited tumor growth, but without having toxicity in the heart, liver and kidney. In addition, EriB treatments induced in vivo cell apoptosis and superoxide production as observed in tumor tissues. / In conclusion, the present study reports for the first time that EriB has possessed anti-proliferative activities in pancreatic cancer cells. The anti-proliferative effects of EriB on CAPAN-2 cells could be attributable to the regulation of cellular apoptosis and cell cycle arrest. The inhibitory effects of EriB on two antioxidant systems result in the accumulation of ROS, which in turn activate MAPK, p53, Hsp70 and caspase cascade, while inhibiting NFB pathway and finally leading to pancreatic cancer cell death. Meanwhile, in vivo study further confirms the anti-tumor properties of EriB, suggesting that EriB could be considered as a potential chemotherapeutic agent for patients with 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. / Li, Lin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 207-230). / Abstracts also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Publications --- p.vi / Acknowledgements --- p.vii / Table of contents --- p.ix / List of figures --- p.xv / List of tables --- p.xix / List of abbreviations --- p.xx / Chapter Chapter1 --- General Introduction --- p.1 / Chapter 1.1 --- The pancreas --- p.2 / Chapter 1.1.1 --- Anatomy of the pancreas --- p.2 / Chapter 1.1.2 --- Histology of the pancreas --- p.4 / Chapter 1.1.3 --- Exocrine pancreas --- p.5 / Chapter 1.1.3.1 --- Structure of secretory acini, ducts and stroma in pancreas --- p.5 / Chapter 1.1.3.2 --- Functions of exocrine pancreas --- p.6 / Chapter 1.1.4 --- Endocrine pancreas --- p.9 / Chapter 1.1.4.1 --- Structure of islets cells --- p.10 / Chapter 1.1.4.2 --- Functions of endocrine pancreas --- p.10 / Chapter 1.1.5 --- Disorders of the pancreas --- p.11 / Chapter 1.2 --- Pancreatic cancer --- p.14 / Chapter 1.2.1 --- Epidemiology --- p.14 / Chapter 1.2.2 --- The risks and causes of pancreatic cancer --- p.15 / Chapter 1.2.3 --- Signs and symptoms of pancreatic cancer --- p.18 / Chapter 1.2.4 --- Types of pancreatic cancer --- p.19 / Chapter 1.2.5 --- Diagnosis of pancreatic cancer --- p.21 / Chapter 1.2.6 --- Staging of pancreatic cancer --- p.27 / Chapter 1.3 --- Treatments for pancreatic cancer --- p.29 / Chapter 1.3.1 --- Surgery --- p.29 / Chapter 1.3.2 --- Chemotherapy --- p.30 / Chapter 1.3.2.1 --- 5-fluorouracil (5-FU) --- p.32 / Chapter 1.3.2.2 --- Gemcitabine (Gem) --- p.33 / Chapter 1.3.2.3 --- Other cytotoxic agents --- p.34 / Chapter 1.3.3 --- Radiotherapy --- p.35 / Chapter 1.3.4 --- Target therapies --- p.37 / Chapter 1.3.4.1 --- Antiangiogenic therapy --- p.37 / Chapter 1.3.4.2 --- Epidermal growth factor receptor (EGFR) signaling inhibitors --- p.39 / Chapter 1.3.4.3 --- Hedgehog and Notch signaling pathways inhibitors --- p.41 / Chapter 1.3.5 --- Gene therapy --- p.42 / Chapter 1.3.6 --- Immunotherapy --- p.45 / Chapter 1.3.7 --- Combination therapies --- p.46 / Chapter 1.4 --- Molecular targets for pancreatic cancer chemotherapy --- p.49 / Chapter 1.4.1 --- Therapies-induced apoptosis --- p.49 / Chapter 1.4.1.1 --- Caspase cascade and bcl-2 Family --- p.49 / Chapter 1.4.1.2 --- Role of mitogen-activated protein kinases (MAPKs) in apoptosis --- p.50 / Chapter 1.4.2 --- Nuclear factor-κB activation in pancreatic cancer --- p.50 / Chapter 1.4.3 --- The PI3K and AKT pathway --- p.51 / Chapter 1.4.4 --- JAK/STAT pathway --- p.51 / Chapter 1.4.5 --- Other molecular targets --- p.52 / Chapter 1.5 --- Herbal medicine as an alternative treatment for cancer treatment --- p.53 / Chapter 1.5.1 --- Herbal medicines for different types of cancer treatment --- p.53 / Chapter 1.5.2 --- Herbal medicines for pancreatic cancer treatment --- p.59 / Chapter 1.6 --- Introduction of Isodon eriocalyx (Dunn.) Hara --- p.61 / Chapter 1.6.1 --- Background of Isodon genus and Isodon eriocalyx (Dunn.) Hara --- p.61 / Chapter 1.6.2 --- Diterpenoids from Isodon species and their activities --- p.62 / Chapter 1.6.3 --- The potential anti-cancer activity of Eriocalyxin B, a diterpenoid isolated from Isodon eriocalyx (Dunn.) Hara --- p.62 / Chapter 1.7 --- Aims and objectives of this study --- p.66 / Chapter Chapter 2 --- Eriocalyxin B induces apoptosis and cell cycle arrest in pancreatic adenocarcinoma cells through caspase- and p53-dependent pathways --- p.67 / Chapter 2.1 --- Introduction --- p.68 / Chapter 2.2 --- Materials and methods --- p.71 / Chapter 2.2.1 --- Preparation and quality control of Eriocalyxin B --- p.71 / Chapter 2.2.2 --- Materials --- p.72 / Chapter 2.2.3 --- Cell culture --- p.72 / Chapter 2.2.4 --- Preparation of human peripheral blood mononuclear cells (PBMC) --- p.73 / Chapter 2.2.5 --- Cytotoxicity assay --- p.75 / Chapter 2.2.6 --- Hoechst 33258 staining for morphological evaluation --- p.76 / Chapter 2.2.7 --- DNA fragmentation detection by DNA ladder --- p.76 / Chapter 2.2.8 --- Cell death detection ELISA --- p.77 / Chapter 2.2.9 --- Apoptosis detection by flow cytometry --- p.78 / Chapter 2.2.10 --- Cell cycle analysis by flow cytometry --- p.78 / Chapter 2.2.11 --- Western blot analysis --- p.79 / Chapter 2.2.12 --- Statistical analysis --- p.80 / Chapter 2.3 --- Results --- p.81 / Chapter 2.3.1 --- EriB induces cytotoxic effect in human pancreatic cancer cells --- p.81 / Chapter 2.3.2 --- EriB induces apoptosis in CAPAN-2 cells --- p.85 / Chapter 2.3.3 --- Activation of pro-apoptotic caspases in EriB-treated CAPAN-2 cells --- p.89 / Chapter 2.3.4 --- Modulation of bcl-2/bak ratio in EriB-treated CAPAN-2 cells --- p.92 / Chapter 2.3.5 --- EriB causes G2/M cell cycle arrest --- p.94 / Chapter 2.3.6 --- EriB modulates expression of G2/M cell cycle regulatory proteins through activation of the p53 pathway --- p.96 / Chapter 2.4 --- Discussion --- p.99 / Chapter Chapter 3 --- Eriocalyxin B induces apoptosis in pancreatic cancer CAPAN-2 cells via mediation of reactive oxygen species --- p.107 / Chapter 3.1 --- Introduction --- p.108 / Chapter 3.2 --- Materials and methods --- p.113 / Chapter 3.2.1 --- Materials --- p.113 / Chapter 3.2.2 --- Cell culture and MTT assay --- p.113 / Chapter 3.2.3 --- Apoptosis detection by flow cytometry --- p.114 / Chapter 3.2.4 --- Reactive oxygen species (ROS) detection by flow cytometry --- p.114 / Chapter 3.2.5 --- Glutathione assessment --- p.115 / Chapter 3.2.6 --- Glutathione peroxidase (GPx) activity detection --- p.116 / Chapter 3.2.7 --- Thioredoxin reductase (TrxR) activity detection --- p.116 / Chapter 3.2.8 --- Nuclear and cytosolic fractionation --- p.117 / Chapter 3.2.9 --- Western blot analysis --- p.117 / Chapter 3.2.10 --- Electrophoretic mobility shift assay --- p.119 / Chapter 3.2.11 --- Statistical analysis --- p.119 / Chapter 3.3 --- Results --- p.120 / Chapter 3.3.1 --- Thiol-containing antioxidants inhibits EriB-induced cytotoxic effects --- p.120 / Chapter 3.3.2 --- Thiol-containing antioxidants inhibits EriB-induced apoptotic effects --- p.122 / Chapter 3.3.3 --- Effects of EriB on hydrogen peroxide production --- p.125 / Chapter 3.3.4 --- EriB depletes glutathione level and suppresses GPx activity --- p.128 / Chapter 3.3.5 --- EriB inhibits thioredoxin system and activates ASK1 --- p.130 / Chapter 3.3.6 --- EriB increases Hsp70 and cleaved-PARP expression through ROS --- p.134 / Chapter 3.3.7 --- EriB inhibits NFkB pathway in CAPAN-2 cells --- p.137 / Chapter 3.4 --- Discussion --- p.142 / Chapter Chapter 4 --- In vivo study of the anti-tumor efficacy of Eriocalyxin B in human pancreatic tumor xenograft model --- p.149 / Chapter 4.1 --- Introduction --- p.150 / Chapter 4.2 --- Materials and methods --- p.154 / Chapter 4.2.1 --- Establishment of a subcutaneous pancreatic cancer xenograft model --- p.154 / Chapter 4.2.2 --- Evaluation of the effects of EriB on tumor growth --- p.155 / Chapter 4.2.2.1 --- Pilot study for EriB and camptothecin treatment --- p.155 / Chapter 4.2.2.2 --- Confirmation study of effective dose of EriB --- p.156 / Chapter 4.2.2.3 --- Dose-comparison study of CPT-11 --- p.156 / Chapter 4.2.2.4 --- Comparison study of EriB and CPT-11 treatments --- p..157 / Chapter 4.2.3 --- Measurement of plasma-specific enzyme levels --- p.157 / Chapter 4.2.4 --- Assays of terminal deoxytransferase-catalyzed DNA nick-end labeling (TUNEL) --- p..158 / Chapter 4.2.5 --- Histological evaluation --- p.159 / Chapter 4.2.6 --- Detection of superoxide by DHE staining --- p.159 / Chapter 4.2.7 --- Establishment of an orthotopic model (SW1990) of pancreatic cancer and detection of the plasma biomarker CA19-9 --- p.160 / Chapter 4.2.7.1 --- Detection of CA19-9 expression by immunofluorescent staining and western blot --- p.161 / Chapter 4.2.7.2 --- Establishment of an orthotopic pancreatic cancer xenograft model by SW1990 cells --- p.162 / Chapter 4.2.8 --- Statistical analysis --- p.164 / Chapter 4.3 --- Results --- p.165 / Chapter 4.3.1 --- EriB inhibits the growth of CAPAN-2 human pancreatic tumor xenografts --- p.165 / Chapter 4.3.2 --- EriB treatments induce cell apoptosis in tumor tissues --- p.173 / Chapter 4.3.3 --- Toxicity tests for EriB --- p.175 / Chapter 4.3.3.1 --- Plasma enzyme levels after EriB treatments --- p.175 / Chapter 4.3.3.2 --- No apparent alterations in histology of the heart, liver and kidney tissues --- p..176 / Chapter 4.3.4tEriB induces superoxide production in the tumor tissues --- p.178 / Chapter 4.3.5 --- Successful establishment of an orthotopic xenograft model --- p.180 / Chapter 4.4 --- Discussion --- p.184 / Chapter Chapter 5 --- General Discussion --- p.188 / Chapter 5.1 --- Discussion --- p.189 / Chapter 5.2 --- Conclusion --- p.204 / Chapter 5.3 --- Limitations of the study --- p.205 / Chapter 5.4 --- Future work --- p.206 / Chapter Chapter 6 --- References --- p.207

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.

The human pancreatic cancer cell line T3M4, is known to produce transforming growth factor-alpha (TGF-alpha); as well as overexpress the receptor for this ligand, epidermal growth factor (EGF) receptor. TGF-alpha messenger RNA (mRNA) levels were assayed using northern blot, after addition of epidermal growth factor or TGF-alpha. The level of TGF-alpha mRNA was found to increase 2-fold at 2 hours and then return to near basal levels at 10 hours, after treatment with either ligand. Both ligands were also equipotent in a 2 hour dose response assay, with half maximal stimulation seen at 1 nM and maximal stimulation reached at 4 nM. Furthermore, there appeared to be a 2-fold increase in TGF-alpha transcription as determined by nuclear runoff experiments. Induction of TGF-alpha mRNA coupled with the overexpression of the EGF receptor, may result in a potent autocrine cycle; which may be found in other cancers.

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.

Li, Chi Han Samson. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 158-175). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

In 2018, it is estimated that 55,440 Americans will be diagnosed with pancreatic cancer and this figure is expected to continue to rise with increased life expectancy. Despite some measurable progress over the past few decades, pancreatic cancer remains one of the most lethal malignancies with five-year survival rate of 8.7%. Novel therapies, and their timely translation to the clinic, are urgently needed. As part of an effort to identify and characterize novel therapeutic strategies for pancreatic ductal adenocarcinoma, we began a study of the role of Bmi1 in tumor maintenance and progression. While Bednar and colleagues showed that Bmi1 is critical for the development of pancreatic cancer, and that its pancreas-specific deletion impairs PanIN formation, we were interested in assessing its function in established tumors. During the course of this work, we acquired a novel compound, PTC596, developed by PTC Therapeutics as a post-translational inhibitor of BMI1. Treatment with PTC596 leads to hyperphosphorylated BMI1, and this modification is associated a loss of protein activity. We planned to study this compound, in vitro and in vivo, as a complement to genetic perturbations of Bmi1. Initial characterizations of the effects of PTC596 on human and murine-derived pancreatic cancer cell lines revealed a potent anti-proliferative effect, accompanied by BMI1 hyperphosphorylation, and followed by polyploidy and cell death after prolonged treatment. Further analysis showed a clear G2/M arrest and elevated levels of phospho-histone H3. Bmi1 is known to play a role the cell cycle, but its inhibition in pancreatic cancer cell lines has been shown to induce G1 arrest. We decided to further explore the mechanism of PTC596’s antiproliferative effects by carrying out RNA sequencing on Aspc1 cells treated with PTC596. We found that 8 of the ten most down-regulated genes were members of the tubulin family and began to study this compound’s effect on microtubules. Compelling results from a cell-free tubulin polymerization assay support inhibition of tubulin polymerization as the mechanism of action for PTC596. These data are further supported by evidence that PTC596 increases the fraction of free-tubulin in treated cells, as well as dramatically alters the cell’s microtubule network. Given our laboratory’s interest in identifying novel therapies for pancreatic cancer, and the fact that PTC596 has already begun clinical trials, we continued to characterize this compound in vivo. We found PTC596 to have properties favorable for in vivo administration. PTC596 is orally available, has a plasma half-life of approximately 22 hours following oral administration, and accumulates in tumor tissue where it has an expected pharmacodynamic effect. Furthermore, it is well tolerated in vivo in combination with gemcitabine. We carried out a four-arm intervention study in tumor-bearing KPC mice, examining PTC596 alone and in combination with gemcitabine. We found that PTC596 synergizes with gemcitabine to significantly reduce tumor growth rates and provide a 3-fold extension of survival as compared to vehicle. These findings are, to our knowledge, the first evidence of in vivo synergy between a microtubule-destabilizing agent and gemcitabine for the treatment of pancreatic cancer. Importantly, this study identifies an alternative mechanism for PTC596 and implicates its efficacy in a novel treatment regimen for pancreatic ductal adenocarcinoma.

<p>Background:Pancreas cancer is a most aggressive malignancy. More than 80% of patients diagnosed with pancreas cancer, exhibit such advanced disease, that curative surgery is impossible. Systemic chemotherapy prolongs survival to 5-9 months. High concentrations of chemotherapeutic agents in the abdominal cavity and in the lymphatics draining the area is achieved by intraperitoneal administration. Vasopressin decreases splanchnic blood flow, reducing the intraperitoneal uptake of drugs, thus raising the local and lymphatic dose intensity.</p><p>Aim: The aim of the study was to investigate the feasibility and tumour response of intraperitoneal 5-Fluorouracil (5-FU) treatment in non-resectable pancreas cancer, using vasopressin to improve the pharmacokinetic profile. Further, to study the effect of vasopressin on peritoneal blood flow, altered by intraperitoneal 5-FU or the presence of peritoneal carcinomatosis.</p><p>Methods: In the animal experiments, the 133Xe-clearance technique and as a comparison Laser doppler flow, were used to identify changes of peritoneal blood flow caused by vasopressin in unmanipulated animals and in animals with peritoneal carcinomatosis or animals given intraperitoneal 5-FU. In the clinical studies, 68 (39 women/29 men) patients, with a non-resectable ductal pancreas cancer and a Karnovsky Index ≥70 were included. Patients were treated with 750-1500 mg/m2 5-FU intraperitoneally through a Port-a-cath and Leucovorin 100 mg/m2 intravenously on two consecutive days every 21 days until progression. Seventeen patients, receiving 750 mg/m2 5-FU, were given concomitant vasopressin 0.1 IU/min during 180 minutes, alternatively day 1 or 2.</p><p>Results: In the animal experiments, vasopressin 0.07 IU/kg/min significantly reduced the 133Xe-clearance. Intraperitoneal 5-FU decreased the basal peritoneal blood flow and abrogated the vasopressin effect for 1-2 days. The presence of peritoneal carcinomatosis did not influence the basal peritoneal blood flow, nor the reduction of peritoneal blood flow caused by vasopressin. In the clinical studies, the treatment with intraperitoneal 5-FU was well tolerated, with no WHO Grade 3 or 4 toxicity with doses up to 1250 mg/m2. Thirty patients achieved at least stable disease at three months. The median survival time was 8.0 (range 0.8-54.1) months. There was a significant reduction of 5-FU Cmax on day 2, but no significant reduction of AUC, when vasopressin was given.</p><p>Conclusion: Peritoneal blood flow changes caused by vasopressin can be estimated with the 133Xe-clearance technique. Intraperitoneal 5-FU but not peritoneal carcinomatosis decreases the vasopressin induced 133Xe-clearance reduction, 1-2 days after administration. In patients with non-resectable pancreas cancer, intraperitoneal 5-FU up to 1250 mg/m2 for two days every third week can be given without WHO grade 3 and 4 toxicity. The treatment is well tolerated with few and minor side effects. Tumour responses were observed. Addition of vasopressin does not significantly enhance the pharmacokinetics of intraperitoneal 5-Flurorouracil, but adds toxicity.</p>

Background:Pancreas cancer is a most aggressive malignancy. More than 80% of patients diagnosed with pancreas cancer, exhibit such advanced disease, that curative surgery is impossible. Systemic chemotherapy prolongs survival to 5-9 months. High concentrations of chemotherapeutic agents in the abdominal cavity and in the lymphatics draining the area is achieved by intraperitoneal administration. Vasopressin decreases splanchnic blood flow, reducing the intraperitoneal uptake of drugs, thus raising the local and lymphatic dose intensity. Aim: The aim of the study was to investigate the feasibility and tumour response of intraperitoneal 5-Fluorouracil (5-FU) treatment in non-resectable pancreas cancer, using vasopressin to improve the pharmacokinetic profile. Further, to study the effect of vasopressin on peritoneal blood flow, altered by intraperitoneal 5-FU or the presence of peritoneal carcinomatosis. Methods: In the animal experiments, the 133Xe-clearance technique and as a comparison Laser doppler flow, were used to identify changes of peritoneal blood flow caused by vasopressin in unmanipulated animals and in animals with peritoneal carcinomatosis or animals given intraperitoneal 5-FU. In the clinical studies, 68 (39 women/29 men) patients, with a non-resectable ductal pancreas cancer and a Karnovsky Index ≥70 were included. Patients were treated with 750-1500 mg/m2 5-FU intraperitoneally through a Port-a-cath and Leucovorin 100 mg/m2 intravenously on two consecutive days every 21 days until progression. Seventeen patients, receiving 750 mg/m2 5-FU, were given concomitant vasopressin 0.1 IU/min during 180 minutes, alternatively day 1 or 2. Results: In the animal experiments, vasopressin 0.07 IU/kg/min significantly reduced the 133Xe-clearance. Intraperitoneal 5-FU decreased the basal peritoneal blood flow and abrogated the vasopressin effect for 1-2 days. The presence of peritoneal carcinomatosis did not influence the basal peritoneal blood flow, nor the reduction of peritoneal blood flow caused by vasopressin. In the clinical studies, the treatment with intraperitoneal 5-FU was well tolerated, with no WHO Grade 3 or 4 toxicity with doses up to 1250 mg/m2. Thirty patients achieved at least stable disease at three months. The median survival time was 8.0 (range 0.8-54.1) months. There was a significant reduction of 5-FU Cmax on day 2, but no significant reduction of AUC, when vasopressin was given. Conclusion: Peritoneal blood flow changes caused by vasopressin can be estimated with the 133Xe-clearance technique. Intraperitoneal 5-FU but not peritoneal carcinomatosis decreases the vasopressin induced 133Xe-clearance reduction, 1-2 days after administration. In patients with non-resectable pancreas cancer, intraperitoneal 5-FU up to 1250 mg/m2 for two days every third week can be given without WHO grade 3 and 4 toxicity. The treatment is well tolerated with few and minor side effects. Tumour responses were observed. Addition of vasopressin does not significantly enhance the pharmacokinetics of intraperitoneal 5-Flurorouracil, but adds toxicity.

Pancreatic ductal adenocarcinoma (PDAC) has the poorest five-year survival rate of any cancer. Currently, there are no effective diagnostics or chemotherapeutics. Surgical resection is the only curative therapy. However, most patients experience recurrence due largely to challenges in assessing tumor margin status in the operating room. Molecular probes that selectively highlight pancreatic cancer tissue, having the potential to improve PDAC margin assessment intraoperatively, are urgently needed. In this work, a series of red and near-infrared fluorescent probes is reported. Two were found to distribute to normal pancreas following systemic administration. One selectively accumulates in genetically modified mouse models of PDAC, providing cancer-specific fluorescence. In contrast to the small molecule probes reported previously, it possesses inherent affinity for PDAC cells and tissue, and thus does not require conjugation to targeting agents. Moreover, the probe exhibits intracellular accumulation and enables visualization of four levels of structure including the whole organ, tissue, individual cells and subcellular organelles. It can thus promote new strategies for precision image-guided surgery, pancreatic cancer detection, the monitoring of therapeutic outcomes and basic research.

Pancreatic cancer is the 10th most common cancer diagnosis and 4th most common cause of cancer mortality in the United States, highlighting a disparity between disease prevalence and outcome. Ineffective drug delivery to these tumors contributes to the poor prognosis for this disease, as intravenous drug delivery is hampered by poor vascularity within these tumors. Bacterial therapy, or the use of bacterial components to treat disease, is thought to be able to overcome such drug delivery challenges; through a combination of tumor homing and long-term colonization, bacteria can be utilized to produce anti-cancer molecules directly within the cores of tumors. As such, here, we interrogate the feasibility of bacterial cancer therapy for pancreatic ductal adenocarcinoma (PDAC). Before delving too deeply into bacterial therapy design, it was important to first address one major limitation in therapeutic screening models. As a therapeutic should be effective against the entirety of the tumor, without a specific emphasis on the malignant epithelia, we developed and characterized a novel protocol for culturing ex vivo (explant) murine PDAC tissue with a corresponding protocol for human PDAC tissue. We demonstrated that these tumor slice explants retain the complex cellular architecture and population complexity throughout culture, making them a useful resource for not only therapeutic screens, but also paracrine interactions, which are infeasible to explore with in vitro and in vivo models. Use of these murine and human PDAC explant models assisted in the selection of a potent, bacterial-derived cytotoxin, theta toxin, as a potential therapeutic candidate for PDAC, in both bacteria lysate and live bacteria contexts. Ultimately, we employed a strain of a probiotic bacteria, E. coli Nissle 1917, as a ‘living drug’ to selectively produce theta toxin within the confines of a PDAC tumor in a mouse model of pancreatic cancer. In in vivo studies, we demonstrated that live bacteria preferentially colonize tumor tissue following a single, direct, intratumoral injection into the primary PDAC tumor. We found that not only did the bacteria colonize the injected tumor, but also translocated to distant regions of metastasis and secondary tumors such as anogenital papillomas. However, the long-term efficacy of this strategy is in question, as bacterial colonization and therapeutic capability waned after several weeks. Despite the limited time scale of the bacterial colonization, treatment with a single dose of live, theta toxin-producing bacteria provided a nearly 3-fold improvement in overall survival compared to vehicle and standard of care chemotherapy (gemcitabine) treatment arms. Preliminary evidence suggests that this improvement is due to a combination of the direct cytotoxic effect of the theta toxin and an inherently immunostimulatory capacity of these bacteria, resulting in an influx of anti-tumor immune cells and an overall reduction in immunosuppression phenotype markers. These findings suggest that bacterial therapy could be a useful tool for the treatment of pancreatic cancer, not solely due to the direct cytotoxic effect on the tumor, but with the potential for a combination treatment with immunotherapies.