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研究生:邱冠婕
研究生(外文):Kuan-Chieh Chiu
論文名稱:BH3類似藥物Obatoclax之抗大腸癌效應:ATF3 之角色探討
論文名稱(外文):Study on the role of activating transcription factor 3 (ATF3) in the anti-colorectal cancer activity of Obatoclax, a BH3 mimetic
指導教授:張嘉哲張嘉哲引用關係
指導教授(外文):Chia-Che Chang
口試委員:陳霈霓柯俊良
口試委員(外文):Pei-Ni ChenJiunn-Liang Ko
口試日期:2020-01-07
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學院碩士在職專班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:46
中文關鍵詞:大腸癌細胞凋亡
外文關鍵詞:(colorectal cancerApoptosisBCL-2ObatoclaxATF3
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大腸癌高居台灣十大癌症發生率第一位,是國人不可輕忽之病症。其產生與遺傳、飲食、不良生活型態及環境等因素有關,近年來不僅40歲以上之族群,連年輕患者皆有增多之趨勢。大腸癌病情初期較不易察覺,發現時多數已進入癌症晚期甚至有癌細胞轉移現象,因此如何預防及抗癌藥物之研發是國人非常關注的議題。近幾年文獻指出大腸癌初期之抗凋亡B-cell lymphoma 2 (BCL-2) 蛋白會不受控而大量表現,以抑凋亡BCL-2為治療標靶的小分子藥物應運而生,Obatoclax (又稱 GX15-070)是一種 BCL-2 homology 3 (BH3)類似物,藉由結合多種抗凋亡BCL-2家族蛋白(包括: BCL-2、BCL-xL、MCL-1)而抑制其抗凋亡功能;許多研究實驗指出Obatoclax在多種癌症中能有效抑制癌細胞生長或促癌細胞死亡。Activating transcription factor 3 (ATF3)屬於 Activating transcription factor (ATF)/cAMP-response element binding protein (CREB)家族的其中一員,在不同生理壓力下可被快速誘發並調控下游蛋白的轉錄,文獻指出ATF3在不同癌症當中可能扮演促癌細胞凋亡或是促癌細胞存活兩個截然不同的角色,因此ATF3在癌症治療當中是非常具有潛力的目標。本篇論文我們利用大腸癌細胞株HCT 116作為細胞模式,研究結果發現Obatoclax可以劑量效應方式調高cleaved poly ADP-ribose polymerase (PARP)並調降BCL-2,證實Obatoclax可誘發大腸癌細胞株之細胞凋亡。實驗也發現ATF3蛋白表現量會隨著Obatoclax濃度提高而增加; knockdown ATF3後可明顯降低Obatoclax對HCT 116細胞的毒殺性、同時降低促凋亡轉錄因子CCAAT/Enhancer-Binding Protein (C/EBP) Homologous Protein (CHOP)的表現量。我們也證實Obatoclax可促進ATF3蛋白的穩定性也增加。綜合上述結果,我們的研究證實在HCT 116細胞株中,Obatoclax藉由促進ATF3蛋白穩定而提升ATF3、進而調高CHOP以降低BCL-2表現量、最後誘發細胞凋亡。本研究對於Obatoclax調控ATF3之抑癌性提供了更新的資訊。
Colorectal cancer ranks first in Taiwan''s top ten cancers, and it is a disease that cannot be neglected by Taiwanese people. Its production is related to genetics, diet, poor lifestyle and environment. In recent years, not only the population over 40 years old, but also young patients with colorectal cancer have been increasing. The early stage of colorectal cancer is less noticeable. Most of them have entered the late stage of cancer and even cancer metastasis. Therefore, how to prevent and develop anticancer drugs is a topic of great concern to the Taiwanese people. Recently, the literatures indicate that antiapoptotic B-cell lymphoma 2 (BCL-2) proteins in the early stage of colorectal cancer are abundantly expressed. Small-molecule drugs targeting BCL-2 have emerged. Obatoclax (also known as GX15-070) is a BCL-2 homology 3 (BH3) analogue. It binds and thus inhibits a panel of antiapoptotic BCL-2 family proteins such as BCL-2, BCL-xL and MCL-1, and many studies have shown that Obatoclax can effectively inhibit cancer cell growth or promote cancer cell death in a variety of cancers. Activating transcription factor 3 (ATF3) is a member of the activating transcription factor (ATF)/cAMP-response element binding protein (CREB) family and can be rapidly induced and regulates transcription of downstream proteins under different physiological stresses. Previous studies indicate that ATF3 may play an important role in promoting cancer cell apoptosis or promoting cancer cell survival in different cancers. In this thesis, I aimed to study the role of ATF3 in the anti-colorectal cancer effect of Obatoclax using the colorectal cancer cell line HCT 116 as our cell model. The results showed that Obatoclax dos-dependently upregulates cleavage of poly ADP-ribose polymerase (PARP) and also downregulates BCL-2, which confirmed that Obatoclax can induce apoptosis of HCT 116 cells. I also found that Obatoclax upregulates ATF3 in a dose-dependent manner; notably, ATF3 knockdown led to decreased Obatoclax-induced cytotoxicity as well as downregulation of proapoptotic transcription factor CCAAT/Enhancer-Binding Protein (C/EBP) Homologous Protein (CHOP). Lastly, we revealed that the stability of ATF3 protein is enhanced by Obatoclax. Based on these findings, I conclude that in the context of HCT 116 cells, Obatoclax promotes stabilization of ATF3 protein to upregulate ATF3, which increases CHOP expression to downregulate BCL-2, therefore inducing apoptosis.
中文摘要 --------------------------------------------------i
Abstract -----------------------------------------------iii
前言(Introduction)----------------------------------------1
一、大腸癌(colorectal cancer)------------------------------1
二、大腸癌細胞株HCT116-------------------------------------7
三、細胞凋亡(Apoptosis)------------------------------------7
四、BCL-2家族(B-cell lymphoma 2 family)--------------------9
五、BH3 only protein-------------------------------------10
六、Obatoclax(BCL-2 homology 3 mimetic) -----------------11
七、ATF3 (Activating transcription factor 3)-------------11
八、CHOP(CCAAT/Enhancer-Binding Protein (C/EBP) Homologous Protein)-------------------------------------------------12
研究動機(Motive)------------------------------------------14
實驗材料及試劑配方(Materials) ----------------------------15
一、藥物(Drug) -------------------------------------------15
二、試劑(Buffer) -----------------------------------------15
實驗方法(Methods)-----------------------------------------17
一、細胞培養(Cell lines and cell culture) ----------------17
二、細胞存活測試(MTS assay) ------------------------------17
三、細胞總量蛋白萃取(Whole protein extraction) ------------17
四、蛋白濃度定量分析(Protein quantification) --------------18
五、西方墨點法(Western blot) -----------------------------19
六、蛋白質半衰期測定 (Cycloheximide chase assay) ----------21
七、病毒製備與病毒感染 (Retovirus and Lentivirus production and infection) ------------------------------------------21
實驗結果(Results)-----------------------------------------23
一、Obatoclax抑制人類大腸癌細胞株細胞存活率 ----------------23
二、Obatoclax在大腸癌細胞株中誘導細胞凋亡 ------------------23
三、Obatoclax在大腸癌細胞株中誘導ATF3蛋白表現量上升 --------24
四、 ATF3為大腸癌細胞株抗Obatoclax細胞毒性之關鍵要素 -------24
五、 ATF3可調升凋亡蛋白CHOP的表現量 -----------------------24
六、Obatocalx透過後轉譯層次調升ATF3 表現量 ----------------25
七、討論--------------------------------------------------26
(一)探討Obatoclax之抗癌潛力-------------------------------26
(二)Obatoclax處理人類大腸癌細胞株造成ATF3蛋白表現上升-------26
(三)Obatoclax處理人類大腸癌細胞株是否造成CHOP蛋白表現量上升-27
(四)ATF3 表現量增加是否為人類大腸癌細胞株對Obatoclax 抗大腸癌活性的關鍵機制?--------------------------------------------28
實驗結果圖表(Experiment result chart)--------------------29
圖一、Obatoclax 抑制人類大腸癌細胞株細胞存活率 -------------29
圖二、Obatoclax在大腸癌細胞株中誘導細胞凋亡---------------30
圖三、Obatoclax抑制抗細胞凋亡蛋白BCL-2表現量 ---------------31
圖四、Obatoclax在大腸癌細胞株中誘導ATF3蛋白表現量上升 ------32
圖五、Knockdown ATF3降低Obatoclax對大腸癌細胞株之細胞毒性---33
圖六、Knockdown ATF3後,CHOP表現量下降 --------------------34
圖七、Obatoclax 促進ATF3蛋白質穩定性增加 -----------------35
結論(Conclusion)-----------------------------------------36
參考文獻(References)--------------------------------------37
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28、圖(一)﹕華人健康網(文/圖片亞東醫院腫瘤科暨血液科謝佩穎醫師) https://tw.news.yahoo.com/%E5%A4%A7%E8%85%B8%E7% 99%8C%E5%8F%B0%E7%81%A3%E7%99%8C%E7%97%87%E7%99%BC%E7%94%9F%E7%8E%87%E7%AC%AC-%E4%BD%8D-%E5%B0%88%E5%AE%B6%E6%B7%BA%E8%AB%87%E8%85 %B8%E7%99%8C%E5%88%86%E6%9C%9F-%E6%B2%BB%E7%99 %82%E6%96% B9%E5%BC%8F-013000236.html
29、圖(二)﹕華人健康網 (文/圖片亞東醫院家庭醫學科何中誠醫師)
https://www.msn.com/zh-tw/health/topic/%E6%84%9B%E5%90% 83%E7%83%A4%E8%82%89%E3%80%81%E7%87%92%E7%83 %A4%EF%BC%9F%E6%90%9E%E6%87%82%E5%A4%A7%E8%85%B8%E7%99%8C%E6%88%90%E5%9B%A0%EF%BC%8C%E5%81%9A%E5%B0%8D%E9%80%99%E4%BA%9B%E4%BA%8B%E5%8A%A9%E9%A0%90%E9%98%B2/ar-AAGUWUG
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