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系統識別號 U0026-3001201311271500
論文名稱(中文) 探討精氨酸抽離療法用於治療人類膽道癌的可能性
論文名稱(英文) Potential Use of Arginine Deprivation Therapy in Human Biliary Tract Cancers
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 101
學期 1
出版年 102
研究生(中文) 陳芃名
研究生(英文) Peng-Ming Chen
學號 T16991054
學位類別 碩士
語文別 英文
論文頁數 39頁
口試委員 指導教授-陳立宗
口試委員-洪文俊
口試委員-李健逢
中文關鍵字 精胺丁二酸合成酵素  精氨酸脫亞氨酶  膽道癌  細胞週期 
英文關鍵字 Argininosuccinate synthetase  ADI-PEG20  cholangiocarcinoma  cell cycle arrest 
學科別分類
中文摘要 精胺酸(Arginine)是一種非必需胺基酸,在蛋白質的生合成過程中扮演重要的角色,並且參與腫瘤細胞生長和死亡路徑的調控,因此精胺酸在調控腫瘤細胞的營養代謝過程中扮演著十分重要的角色。精胺酸在細胞內透過尿素循環生合成,在此過程中精胺丁二酸合成酵素(Argininosuccinate synthease,ASS-1)為速率決定步驟的催化酵素。最近的研究發現源自不同器官的腫瘤細胞均可能缺乏ASS-1的表現,例如皮膚癌、肝癌、前列腺癌,而這些ASS-1癌細胞若處在缺乏精胺酸的環境,例如使用源自mycoplasma的精氨酸脫亞氨酶(Arginine deiminase)催化精胺酸轉化成瓜胺酸則細胞生長將受到抑制,精氨酸脫亞氨酶經聚乙二醇化修飾後(ADI-PEG20)可將低其免疫性及敏感性,目前此藥物已進入臨床試驗。近年來,膽道癌的發生率有逐年增加的趨勢,且因肝內膽管之症狀不明顯故其診斷不易,被診斷時都已相對晚期無法以手術切除治療,但不幸的是膽道癌細胞相對具有化療藥物的抗藥性,所以找到一個好的治療方式是必須的。因此我們想探討精胺酸去除療法應用在膽道癌上的可能性。然而我們初步發現近百分之四十肝內膽管癌的組織檢體中缺乏ASS-1的表現,因此本研究擬由細胞模型來探討將精胺酸去除療法是否可適用於膽道癌之治療。本研究使用ADI-PEG 20來將精胺酸去除,為了在細胞模式中研究ADI-PEG20用於治療膽道癌的可能性,首先檢查了實驗室有的膽道癌細胞株其ASS-1的表現,我們可以看到不論在蛋白質或者是核醣核酸的表現量在SNU1196細胞株的ASS表現量高,然而SNU1079的ASS幾乎沒有表現,其餘三個的ASS-1表現介於其中。更進一步的對於這些細胞株處理ADI-PEG20來看藥物對細胞存活率的影響,發現在處理藥物之後ASS-1 表現量越低的細胞其細胞的存活率越低。因此我們用缺乏ASS-1表現的SNU1079以及有ASS-1表現的SNU1196來做進一步的研究。在ASS-1缺乏的狀況下,ADI-PEG20的給予是會使得細胞的生長停止在DNA合成期,但若有ASS表現的細胞則ADI-PEG20的給予對細胞週期沒有影響。在分子層面我們也看到了處理ADI-PEG20後,細胞週期蛋白的表現量(cyclinA2, cyclinB1, cyclinD1, cyclinE1)隨著處理藥物的時間以及劑量而減少。我們也發現ADI-PEG20的處理會影響ASS缺乏的細胞株的細胞聚落形成,但抑制細胞聚落的形成可能是藉由抑制細胞生長或者是促進細胞死亡,在我們的實驗結果看來,ADI-PEG20並不會促進細胞凋亡。此外ADI-PEG20會引起內質網壓力,這可能是造成細胞週期蛋白表現下降的原因。由本論文可得知,ADI-PEG20可透過抑制細胞的生長達到對膽道癌細胞的影響,而內質網壓力可能在其中扮演重要的角色。
英文摘要 Arginine is a non-essential amino acid in human beings and involved in protein synthesis and tumor metabolism. Argininosuccinate synthetase (ASS) is the rate-limiting enzyme involved in the two-step synthesis of arginine from citrulline. It has been demonstrated that ASS expression is deficient in various tumor types, that makes arginine deprivation therapy as a potential antitumor strategy in ASS deficient cancers. Biliary tract cancer (BTC) is a relatively uncommon, heterogeneous epithelial cell malignancy involving the intra-/extra-hepatic bile ducts and gall bladder, and its incidence is apparently rising worldwide over the past decades. Systemic chemotherapy in patients with advanced BTC is ineffective and its administration frequently jeopardized by the presence of obstructive jaundice and associated cholangitis. Developing an alternative treatment strategy is mandatory to improve the dismal outcome of patients with advanced BTC. Recently, our collaborative laboratory showed that 40~50% of BTC expressed lower level of ASS by immunohistochemical study, which highlighted the potential of arginine deprivation therapy (such as pegylated arginine deiminase, ADI-PEG20) in a subset of advanced BTC patients. In current study, we investigated the applicability of arginine deprivation therapy in human intrahepatic cholangiocarcinoma cells in vitro. First, we determine the expression level of ASS and the growth inhibition effect of ADI-PEG20 in five human intrahepatic cholangiocarcinomas cell lines. Our results show the sensitivities of intrahepatic cholangiocarcinomas to ADI-PEG20 are strongly correlated with their expression level of ASS. Furthermore, ADI-PEG20 induces cell cycle arrest at S phase and inhibits colony formation of the ASS-deficient SNU1079 cells but not ASS–expressing SNU1196 cells. In addition, ADI-PEG20 inhibits the mRNA and protein expression of cyclins(cyclinD1, cyclinB1, cyclinE and cyclinA2), but not through the activation of cyclin-dependent kinase inhibitor proteins. We also analyze whether ADI-PEG20 induces apoptosis in ASS-deficient SNU1079 cells and the data show that ADI-PEG20 does not enhance apoptosis. In conclusion, our study demonstrates that ADI-PEG20 inhibits the cell growth and colony formation in cholangiocarcinoma cell lines. In addition, we find ADI-PEG20 induces the ER stress response and it might be the mechanism which inhibits the cell cycle progression with ADI-PEG20 treatment in cholangiocarcinoma.
論文目次 中文摘要 I
Abstract III
誌謝 V
Contents VII
I. Introduction 1
The role of arginine in cancers 1
The application of arginine deiminase in cancer therapy 1
cholangiocarcinoma 2
Therapeutic option and mutation in cholangiocarcinoma 3
II. Specific Aims 5
III. Material and Methods 6
IV. Results 12
Sensitivity to ADI-PEG20 correlates with ASS expression 12
Epigenetic silencing of ASS in cholangiocarcinoma cell lines 13
ADI-PEG20 inhibits the colony formation in ASS1-deficient SNU1079 cell lines 13
ADI-PEG20 induces the cell cycle S arrest of ASS-deficient SNU1079 cell line 14
Effects of ADI-PEG 20 on distribution of cell-cycle and the expression of apoptosis regulated proteins 15
V. Discussion 16
VI. Reference 19
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