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研究生:張雅筑
研究生(外文):Ya Chu Chang
論文名稱:BH3類似物藥物Obatoclax之抗大腸癌效應機制探討:Cyclin D1之角色
論文名稱(外文):Biological Significance and Mechanistic Study on Obatoclax-induced Cyclin D1 Downregulation in Human Colon Cancer Cell Lines
指導教授:張嘉哲張嘉哲引用關係沈郁強沈郁強引用關係
指導教授(外文):Chia-Che ChangYuh-Chiang Shen
口試委員:羅至佑黃惠美
口試委員(外文):Chih-Yu LoHM Huang
口試日期:2016-05-27
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:64
中文關鍵詞:藥物大腸癌機制抗癌
外文關鍵詞:cyclin D1Colon cancerMechanistic
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大腸癌是目前全台癌症致死率第三名的癌症,且國人的大腸癌發生率為第一名。該癌症在罹患初期無顯著徵兆,若出現明顯症狀或是體重下降時,癌細胞常常已經發展至第二、三期以上而不易治療。大腸癌的產生除了跟年紀、飲食習慣高油鹽之外,跟家族性基因遺傳曾患有大腸息肉者也息息相關。文獻指出大腸癌初期之促存活的BCL-2蛋白會不受調控並大量表現,近幾年來,以BCL-2為治療標靶的小分子藥物因應而生,Obatoclax(又稱GX15-070)藉由其結構類似BH3蛋白特性,是一種抑制BCL-2家族蛋白的抑制劑,已在肺癌及血癌模式分別完成第一與第二期臨床試驗,也在許多研究實驗指出Obatoclax處理不同的癌細胞能造成細胞凋亡並有效抑制癌細胞生長,其他文獻還指出低濃度Obatoclax處理大腸癌細胞並不會造成癌細胞凋亡,但卻會抑制細胞的生長停滯。爰上,本計劃所以對於Obatoclax在大腸癌中的抗增殖暨其分子機制探討。因此本研究的目標即以人類大腸癌細胞株HCT116、HT-29及LoVo為細胞模式,探討Obatoclax是否具有抗大腸癌的特性以及使細胞週期停滯的能力。我們發現處理Obatoclax會使大腸癌細胞存活率明顯下降以及抑制群落生成能力,表示Obatoclax對大腸癌細胞具細胞毒性。而處理Obatoclax可造成G1期停滯的現象,表示Obatoclax會使癌細胞無法繼續複製並增生。cyclinD1由CCDN1基因所轉譯出來,在G1中期,cyclinD1與CDK4會結合,使下游kinase活化,但處理Obatoclax的大腸癌細胞會導致cyclinD1蛋白表現下降,藉由大量表現cyclinD1後發現Obatoclax對人類大腸癌細胞的抑制群落生成能力及細胞週期皆回復,由此得知cyclin D1確實與Obatoclax有著密切的相關性。深入探討Obatoclax如何調降cyclinD1蛋白,發現Obatoclax對Cyclin D1在轉譯與轉譯後修飾有明顯的調控,說明Obatoclax是透過蛋白質穩定度階層來調降cyclinD1表現。為了更進一步探討Obatoclax透過何種途徑調控蛋白質降解,結果顯示處理Obatoclax後在HT-29細胞中可能會透過磷酸化cyclin D1 Thr286與蛋白酶體路徑去降解cyclin D1蛋白,但在HCT116及LoVo細胞中卻觀察到是並非藉由磷酸化cyclin D1 Thr286去調控cyclin D1蛋白穩定性。總結,可知 Obatoclax可對多株大腸癌細胞中具有抗癌效果,誘導細胞週期G1期停滯並知道是藉由調控cyclinD1蛋白穩定; Obatoclax在不同株大腸癌細胞中有依賴及非依賴磷酸化cyclin D1 Thr286兩種方式活化蛋白酶體去降解cyclin D1蛋白。

Colorectal cancer is the most commonly diagnosed cancer around the world and the third cause of cancer mortality in Taiwan. Up-regulation of prosurvival BCL-2 family members has been shown to be correlated with early colorectal cancer formation. Obatoclax (a.k.a GX15-070) is a pan-BCL-2 inhibitor. Previous studies have pointed out the antiproliferative effect of obatoclax, but the mechanisms remain unclear. In this study, we used colorectal cancer cell lines HCT116, HT-29 and LoVo as the cellular model to study the antiproliferative action of obatoclax. We found that obatoclax causes cell cycle arrest at the G1 phase and also marked decrease in the clonogenic capacity in dose-dependently. Immunoblotting analyses indicated that a decline of cyclin D1 levels following obatoclax treatment. Importantly, overexpression of cyclin D1 conferred cells resistance to obatoclax-induced G1 arrest and suppression of clonogenicity, indicating that cyclin D1 decrease is a fundamental mechanism for obatoclax to induce antiproliferation. To further understand how obatoclax down-regulates cyclin D1, we found that the levels of the cyclin D1 mRNA and promoter activity were not significantly affected. In contrast, pre-treatment with the proteasome inhibitor MG132 markedly rescued cyclin D1 protein levels in obatoclax-treated cells. Additionally, cycloheximide chase analyses revealed that the half-life of cyclin D1 protein was evidently decreased following obatoclax stimulation, indicating that obatoclax reduces cyclin D1 expression mainly by destabilizing cyclin D1 protein. It is known that a major mechanism of cyclin D1 destabilization is through glycogen synthase kinase-3β (GSK3β)-mediated phosphorylation of Thr286 of cyclin D1 (p-Cyclin D1 (Thr286). However, we found that obatoclax induced ser473 phosphorylation and thus activation of AKT, which then causes inactivation of GSK3β as evidenced by the increase in the levels of p-GSK3β (Ser9), suggesting that GSK3β activity is not required for obatoclax-induced cyclin D1 destabilization. In summary, we herein provide the first evidence that obatoclax induced antiproliferation in human colorectal cancer cells by down-regulating cyclin D through cyclin D1 protein destabilization.

中文摘要 i
Abstract iii
前言 1
一、 大腸癌 1
二、 Obatoclax(BH3相似物) 4
三、 細胞週期 5
四、 Cyclin D1 6
五、 MAPK/ERK訊息傳遞路徑 6
六、 AKT訊息傳遞路徑 7
研究目的 9
實驗材料及試劑配方(Materials) 10
 藥物(Drug) 10
 試劑(Buffer) 10
 核酸操作藥劑配置: 11
 報導基因冷光酵素活性分析藥劑配置(Promega): 12
 質體建構(Plasmid construction) 12
 抗體配製(Antibodies) 13
實驗方法(Methods) 15
一、 細胞培養 (Cell lines and cell culture) 15
二、 細胞之冷凍保存與解凍 15
三、 細胞存活測試 (Cell viability assay) 16
四、 細胞總量蛋白萃取 (Whole protein extraction) 16
五、 蛋白濃度定量分析 (Protein quantification) 17
六、 西方墨點法 (Western blot) 17
七、 蛋白酶體抑制劑試驗(MG-132 Proteasome Inhibitor Assay) 19
八、 蛋白質半衰期測定(Cycloheximide chase assay) 19
九、 細胞週期分析(Cell cycle) 20
十、 核糖核酸萃取(RNA extraction) 20
十一、 反轉錄聚合連鎖反應 (reverse transcription PCR) 21
十二、 全定量反轉錄聚合連鎖反應 (Real Time RT-PCR) 22
十三、 大腸桿菌質體轉型作用 (Transformation) 22
十四、 病毒製備與病毒感染 (Retrovirus and Lentivirus production and infection) 23
十五、 細胞菌落形成能力試驗 (Colony formation assay) 24
十六、 報導基因冷光酵素活性分析 (Luciferase reporter assay) 24
十七、 細胞生長測定 (Determination of cell growth) 25
結果 26
一、 Obatoclax抑制人類大腸癌細胞株細胞存活率 26
二、 Obatoclax造成人類大腸癌細胞株細胞群落生成能力下降 26
三、 Obatoclax抑制人類大腸癌細胞株生長 26
四、 Obatoclax造成人類大腸癌細胞株細胞週期停滯於G1期 27
五、 Obatoclax造成人類大腸癌細胞株SKP2、cyclin D1蛋白表現下降則p27、p21蛋白表現上升 27
六、 Cyclin D1表現量下降在Obatoclax所誘導細胞週期停滯中扮演著重要的角色 28
七、 Obatoclax透過轉譯及後轉譯層次調控cyclin D1表現量 29
八、 Obatoclax對人類大腸癌調控cyclin D1蛋白質穩性的機制不盡相同 31
討論 32
一、 探討Obatoclax之抗癌潛力 32
二、 Obatoclax處理人類大腸癌細胞株造成cyclin D1蛋白表現下降 32
三、 Obatoclax由依賴及非依賴性磷酸化cyclin D1來調控蛋白質穩性 33
四、 其他可能的路徑 34
五、 結論 35
實驗結果圖表 36
圖一、Obatoclax抑制人類大腸癌細胞株細胞存活率 36
圖二(A)、Obatoclax造成人類大腸癌細胞株細胞群落生成能力下降 37
圖二(B)、Obatoclax造成人類大腸癌細胞株細胞群落生成能力下降 38
圖三、Obatoclax抑制人類大腸癌細胞株生長 39
圖四、Obatoclax造成人類大腸癌細胞株細胞週期停滯於G1期 40
圖五、Obatoclax造成人類大腸癌細胞株SKP2、Cyclin D1蛋白表現下降則p27、p21蛋白表現上升 41
圖六、Obatoclax對於過度表現SKP2引起的人類大腸癌細胞株HT-29細胞SKP2蛋白表現。 42
圖七、過度表現SKP2處理Obatoclax造成人類大腸癌細胞HT-29之細胞群落生成能力並無回復。 43
圖八、Obatoclax對於過度表現cyclin D1引起的人類大腸癌細胞株HCT116、HT-29及LoVo細胞之Cleaved PARP與cyclinD1蛋白表現。 44
圖九、過度表現Cyclin D1處理Obatoclax造成人類大腸癌細胞株停滯於G1期的情況回復 45
圖十(A)、過度表現Cyclin D1處理Obatoclax造成人類大腸癌細胞HCT116之細胞群落生成能力回復 46
圖十(B)、過度表現Cyclin D1處理Obatoclax造成人類大腸癌細胞HT-29之細胞群落生成能力回復 47
圖十(C)、過度表現Cyclin D1處理Obatoclax造成人類大腸癌細胞LoVo之細胞群落生成能力回復 48
圖十一、Obatoclax在人類大腸癌細胞株中在轉錄層次調控Cyclin D1基因表現量不盡相同 49
圖十二、Obatoclax在人類大腸癌細胞株中Cyclin D1 promoter活性沒有太大影響 50
圖十三(A)、Obatoclax使Cyclin D1的蛋白穩定度下降 51
圖十四(B)、Obatoclax使Cyclin D1的蛋白穩定度下降 52
圖十四、MG132處理後會導致大腸癌細胞株Cyclin D1蛋白表現量回升 53
圖十五、Obatoclax影響人類大腸癌細胞株Cyclin D1 、p-Cyclin D1、GSK3β、p-GSK3β、ERK及p-ERK蛋白表現 54
附錄 55
附表一、HCT116之G1期DNA含量 55
附表二、HT-29之G1期DNA含量 55
附表三、HCT116與過度表現Cyclin D1蛋白之G1期DNA含量 55
附表四、HT-29與過度表現Cyclin D1蛋白之G1期DNA含量 55
附圖一 56
附圖二 57
附圖三 58
附圖四 59
參考文獻 60



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