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研究生: 溫偉伶
Wei-Ling Wen
論文名稱: 組蛋白去乙醯酶抑制劑抑制肺癌細胞生長之機制探討
Growth Inhibition of Lung Cancer Cells by Histone Deacetylase Inhibitors
指導教授: 王憶卿
Wang, Yi-Ching
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2007
畢業學年度: 95
語文別: 中文
論文頁數: 100
中文關鍵詞: 組蛋白去乙醯酶組蛋白去乙醯酶抑制劑肺癌
英文關鍵詞: histon deacetylase, HDAC inhibitors, lung cancer
論文種類: 學術論文
相關次數: 點閱:194下載:14
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  • 目的:前人研究顯示,在一些固體或血液腫瘤中,組蛋白去乙醯酶 (Histone deacetylases, HDACs) 常不正常活化;故引發出在癌症的治療中將 HDAC 作為癌症治療標靶的想法。本研究即探討新穎HDAC抑制劑:HDAC-44 與 HTPB 是否可以有效的抑制肺癌細胞的生長,以及其抑制癌細胞生長的分子機制。材料與方法:利用 trypan blue exclusion 的方法檢測 HDAC-44 與 HTPB 單獨處理下,對正常細胞或肺癌細胞的細胞毒殺性;或是將 HDAC-44 與 cisplatin 共同處理肺癌細胞株,檢測其對肺癌細胞株是否有加成性的細胞毒殺性;使用流式細胞儀 (flow cytometry) 檢測 HDAC 抑制劑是否會影響細胞週期的分佈;利用 DNA 片斷化分析 (DNA ladder assay) 確認 HDAC 抑制劑是否誘導細胞凋亡 (cell apoptosis);以細胞免疫染色的方式 (immunocytochemical analysis) 分析 HDAC 抑制劑使否會改變細胞骨架的結構;且利用反轉錄聚合酶鏈鎖反應 (RT-PCR) 與西方點墨法 (Western blot analysis) 分析 HDAC 抑制劑是否會影響肺癌細胞株各種目標基因其mRNA與蛋白質表達,或是影響蛋白質的乙醯化程度;接著,利用細胞質免疫沉澱 (Chromatin immunoprecipitation, ChIP)、細胞質免疫沉澱晶片分析(ChIP-on-chip),大規模尋找 HDAC 抑制劑直接影響的新穎目標基因。結果:新穎的 HDAC 抑制劑可以有效的促使組織蛋白 H3 與 H4 與非組織蛋白 p53 的蛋白質乙醯化;此外其也可以有效誘導 p21WAF1/Cip1 與 Tissue inhibitor of metalloproteinase-3 (TIMP-3) 等基因的轉錄活化。新穎的 HDAC 抑制劑,HDAC-44 與 HTPB,可以有效的促使肺癌細胞株死亡 (HDAC 44對H1299的IC50為1.09 μM、HDAC 44對A549的IC50為0.64 μM、HDAC 44對CL1-1的IC50為0.67 μM、HTPB對H1299的IC50為2.99 μM、HTPB對A549的IC50為1.60 μM、HTPB對CL1-1的IC50為2.78 μM、SAHA對H1299的IC50為4.59 μM、SAHA對A549的IC50為1.89 μM、SAHA對CL1-1的IC50為2.86 μM),但是對正常細胞株則沒有明顯的細胞毒殺性。將低劑量 HDAC-44 與 cisplatin 共同處理肺癌細胞株,發現對肺癌細胞有加成性的毒殺效果。HDAC-44 與 HTPB 可導致肺癌細胞株的細胞週期停在 G2/M 期並且導致細胞凋亡的現象,如:DNA ladder 與抗細胞凋亡的 Bcl2 蛋白質表達量下降。將肺癌細胞處理 HDAC-44 後,會導致細胞骨架蛋白 a-tubulin 的不正常分佈並且抑制癌細胞的細胞質分裂。ChIP-on-chip分析的結果顯示,HDAC抑制劑可以專一性的在三種測試癌細胞將一些 CpG island 乙醯化,這些 CpG island 所屬之基因可做為未來 HDAC 抑制劑的抗癌機制之目標基因群。結論: HDAC-44 與 HTPB 極具有潛力成為新穎的抗肺癌藥物,而HDAC抑制劑對其他種類的癌症影響也很值得做進一步的研究。

    Purpose: Recent studies have shown that overexpression and/or increased activity of histone deacetylases (HDACs) are observed in solid and hematologic tumors. It makes the HDACs as the attractive novel therapeutic target for cancer treatment. Therefore, we evaluated whether novel HDAC inhibitors (HDACIs) such as HDAC-44 and HTPB can be anti-cancer drugs in lung cancer and investigated their molecular mechanisms on inhibition of cancer cell growth. Materials and Methods: The cytotoxicity of HDAC-44 and HTPB alone or in combination with cisplatin in normal and lung caner cells were determined by trypan blue exclusion. Changes in cell cycle distribution by HDACIs were examined by flow cytometry. HDACIs-induced cell apoptosis was tested by DNA ladder assay. Alteration of cytoskeleton structure of the treated cells was examined by immunocytochemical analysis. RT-PCR and Western blot analysis were used to observe whether HDACIs alter mRNA and protein expressions and protein acetylation. In addition, we used Chromatin immunoprecipitation (ChIP) and ChIP-on-chip to search for more target genes that are influenced by HDACIs. Results: Novel HDACIs facilitated acetylation of histone H3, H4, and p53 proteins. In addition, novel HDACIs induced p21WAF1/Cip1 and Tissue inhibitor of metalloproteinase-3 (TIMP-3) transcriptional activation. Novel HDACIs (HDAC-44 and HTPB) induced lung cancer cell death (The IC50 of HDAC 44 in H1299 is 1.09 μM, HDAC44 in A549 is 0.64 μM, HDAC44 in CL1-1 is 0.67 μM, HTPB in H1299 is 2.99 μM, HTPB in A549 is 1.60 μM、HTPB in CL1-1 is 2.78 μM, SAHA in H1299 is 4.59 μM, SAHA in A549 is 1.89 μM, SAHA in CL1-1 is 2.86 μM) without showing apparent cytotoxicity towards normal lung cells. Combined treatment of HDAC-44 and cisplatin showed a synergistically cytotoxic effect in lung cancer cells. HDAC-44 and HTPB induced G2/M arrest and cell apoptosis such as DNA ladder and anti-apoptotic Bcl-2 protein down-regulation in different lung cancer cell lines. HDAC-44 may lead to a-tubulin abnormal distribution and cancer cell cytokinesis inhibition. ChIP-on-chip analysis indicated that histones on a subset of CpG islands were commonly acetylated by HDACIs in A549, H1299, CL1-1 tested. The genes containing these CpG islands will be used as targets for further mechanistic studies of HDACIs. Conclusion: The current studies and our data suggest that HDAC-44 and HTPB could be potential therapeutic drugs in lung cancer. Their effects toward other types of cancer are worthy of further analysis.

    壹、中文摘要 -------------------------------------------- 1 貳、英文摘要 -------------------------------------------- 3 參、文獻總論 -------------------------------------------- 5 1. 肺癌 ------------------------------------------- 5 1.1. 肺癌之流行病學與概述 ----------------------------- 5 1.2. 肺癌的分期與治療方法 ----------------------------- 6 1.3. 傳統化學治療 (Chemotherapy) 藥物 ----------------- 8 1.4. 新興標靶治療 (targeted therapy)藥物 -------------- 10 2. 染色質修飾 (Chromatin modification) 與組蛋白 去乙醯酵素 (Histone deacetylases, HDACs) -------- 12 2.1. 染色質修飾 (Chromatin modification) ------------- 12 2.2. HDACs的分類與功能 ------------------------------- 14 2.3. HDACs與癌症的關連性 ----------------------------- 16 3. 組蛋白去乙醯酵素抑制劑 (HDAC inhibitors) 作為癌症治療藥物的現況 --------------------------- 18 3.1. HDAC inhibitors的分類與分子機制 ------------------- 18 3.2. 現今 HDAC inhibitors的缺點 ---------------------- 19 3.3. 新穎HDAC抑制劑HTPB與HDAC-42的設計概念 ----- 20 肆、研究目的 -------------------------------------------- 22 (1)了解新穎HDAC inhibitors影響細胞生物功能 ---------- 22 (2)了解新穎HDAC抑制劑是否影響基因體 泛染色體緊密度 (Genome-wild analysis) ----------- 23 伍、研究材料與方法 --------------------------------------- 24 一、研究材料 -------------------------------------------- 24 1. 肺癌細胞株 -------------------------------------- 24 2. 傳統常用臨床化療藥物 ----------------------------- 24 3. HDAC抑制劑 ------------------------------------- 24 3.1. HTPB與HDAC-44合成方式 -------------------------- 25 二、研究方法 -------------------------------------------- 28 1. 模擬分子對接 (molecular docking) ---------------- 28 2. 細胞培養 ---------------------------------------- 29 3. 細胞毒殺性之IC50檢測 ----------------------------- 30 3.1. HDAC抑制劑單獨處理 ------------------------------ 30 3.2. HDAC-44與cisplatin傳統抗癌藥物共同處理 --------- 30 3.3. Trypan Blue exclusion assay ------------------ 30 4. 細胞週期 (Cell cycle) 的檢測 -------------------- 31 5. DNA片斷化分析 (DNA ladder assay) ---------------- 32 6. 細胞免疫染色 (Immunocytochemical analysis, ICC) - 32 7. 西方點墨法分析 (Western blotting analysis) ------ 33 7.1. 蛋白質萃取 -------------------------------------- 33 7.2. 蛋白質定量 -------------------------------------- 34 7.3. 電泳轉漬 ---------------------------------------- 34 7.4. 免疫呈色 ---------------------------------------- 34 8. RNA萃取與反轉錄聚合酶鏈鎖反應 [Reverse- transcriptase polymerase chain reaction (RT-PCR)]------ 35 9. 即時反轉錄聚合酶鏈鎖反應 (Real-time RT-PCR) ------ 36 10. 進行HDAC抑制劑所影響的基因體廣泛分析 (Genome-wild analysis) ------------------------ 36 10.1. 染色質沈澱分析法 [Chromatin immuno- precipitation (ChIP) assay] ------------------- 36 10.2. 染色質沈澱晶片分析 (ChIP-on-chip assay) --------- 39 陸、結果 ------------------------------------------------ 44 一、新穎HDAC抑制劑,HDAC-44及HTPB,可與HDAC8 活性區結合--------------------------------------- 44 二、新穎HDAC抑制劑,HDAC-44與HTPB,皆可有效 促使組蛋白H3和H4以及非組蛋白p53的乙醯化 ------- 44 三、新穎HDAC抑制劑,HDAC-44與HTPB,皆可 有效促使p21WAF1/Cip1與Tissue inhibitor of metalloproteinase-3 (TIMP3) 的基因轉綠活化 ------ 46 四、藥物HDAC-44與HTPB可有效率的導致 肺癌細胞死亡 ----------------------------------- 47 五、結合HDAC抑制劑與傳統化療藥物cisplatin,可 加成性的抑制肺癌細胞的生長 ------------------------ 48 六、HDAC-44、HTPB以及SAHA等HDAC抑制劑, 皆可以有效促使細胞週期停止於G2/M期, 並且導致細胞凋亡 --------------------------------- 49 七、HDAC-44、HTPB與SAHA等HDAC抑制劑皆可 有效的促使肺癌細胞產生DNA ladder以及Bcl-2 表達下降等細胞凋亡的現象 -------------------------- 50 八、藉由細胞免疫染色再次證實,HDAC-44、HTPB 及SAHA可有效促使細胞週期停止於G2/M期、 染色質分裂抑制、細胞凋亡與細胞骨架不正常等現象--- 51 九、ChIP-on-chip data 顯示HDAC抑制劑可促使 三株肺癌細胞株共52個基因座之組蛋白乙醯化程度 上升,且共參予38條路徑---------------------------- 52 柒、討論 ------------------------------------------------ 53 捌、附圖 ------------------------------------------------ 60 玖、附表 ------------------------------------------------ 78 拾、參考文獻 -------------------------------------------- 81

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