簡易檢索 / 詳目顯示

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:182下載:14
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 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

    Department of Health EY, Republic of China (Taiwan) STATISTICS OF CAUSES OF DEATH 2006.
    2. Collins LG, Haines C, Perkel R, Enck RE. Lung cancer: diagnosis and management. Am Fam Physician 2007;75:56-63.
    3. De Vuyst P, Dumortier P, Jacobovitz D, Emri S, Coplu L, Baris YI. Environmental asbestosis complicated by lung cancer. Chest 1994;105:1593-5.
    4. Lee CS, Cooper WA. Asbestos exposure and lung cancer. Pathology 2004;36:513-4.
    5. Samet JM. Environmental causes of lung cancer: what do we know in 2003? Chest 2004;125:80S-3S.
    6. Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest 1997;111:1710-7.
    7. Spira A, Ettinger DS. Multidisciplinary management of lung cancer. N Engl J Med 2004;350:379-92.
    8. Beckles MA, Spiro SG, Colice GL, Rudd RM. The physiologic evaluation of patients with lung cancer being considered for resectional surgery. Chest 2003;123:105S-14S.
    9. Network NCC. NCCN Clinical Practice Guidelines in Oncology. Non-Small Cell Lung Cancer. Version 1. 2007. 2007 [cited; Available from: http://www.nccn.org/professionals/physician_gls/PDF/nscl.pdf#search=%22abstract%20no%3ALBA7012%22
    10. Moufarij MA, Phillips DR, Cullinane C. Gemcitabine potentiates cisplatin cytotoxicity and inhibits repair of cisplatin-DNA damage in ovarian cancer cell lines. Mol Pharmacol 2003;63:862-9.
    11. Kim MS, Blake M, Baek JH, Kohlhagen G, Pommier Y, Carrier F. Inhibition of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA. Cancer Res 2003;63:7291-300.
    12. Wozniak K, Blasiak J. Recognition and repair of DNA-cisplatin adducts. Acta Biochim Pol 2002;49:583-96.
    13. Wang JC. Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 2002;3:430-40.
    14. Jordan MA. Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Curr Med Chem Anticancer Agents 2002;2:1-17.
    15. Chu JJ, Chen KD, Lin YL, Fei CY, Chiang AS, Chiang CD, Lai YK. Taxol induces concomitant hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. J Cell Biochem 1998;68:472-83.
    16. Von Hoff DD, Rozencweig M, Piccart M. The cardiotoxicity of anticancer agents. Semin Oncol 1982;9:23-33.
    17. Kavallaris M, Verrills NM, Hill BT. Anticancer therapy with novel tubulin-interacting drugs. Drug Resist Updat 2001;4:392-401.
    18. Huber RM, Stratakis DF. Molecular oncology--perspectives in lung cancer. Lung Cancer 2004;45 Suppl 2:S209-13.
    19. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70.
    20. Toschi L, Cappuzzo F. Understanding the new genetics of responsiveness to epidermal growth factor receptor tyrosine kinase inhibitors. Oncologist 2007;12:211-20.
    21. Wilson AJ, Byun DS, Popova N, Murray LB, L'Italien K, Sowa Y, Arango D, Velcich A, Augenlicht LH, Mariadason JM. Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer. J Biol Chem 2006;281:13548-58.
    22. Minucci S, Pelicci PG. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 2006;6:38-51.
    23. Acharya MR, Sparreboom A, Venitz J, Figg WD. Rational development of histone deacetylase inhibitors as anticancer agents: a review. Mol Pharmacol 2005;68:917-32.
    24. Marks PA, Miller T, Richon VM. Histone deacetylases. Curr Opin Pharmacol 2003;3:344-51.
    25. Hake SB, Xiao A, Allis CD. Linking the epigenetic 'language' of covalent histone modifications to cancer. Br J Cancer 2004;90:761-9.
    26. Lin HY, Chen CS, Lin SP, Weng JR, Chen CS. Targeting histone deacetylase in cancer therapy. Med Res Rev 2006;26:397-413.
    27. Gray SG, Ekstrom TJ. The human histone deacetylase family. Exp Cell Res 2001;262:75-83.
    28. de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van Kuilenburg AB. Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J 2003;370:737-49.
    29. Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A, Nixon A, Yoshida M, Wang XF, Yao TP. HDAC6 is a microtubule-associated deacetylase. Nature 2002;417:455-8.
    30. Kovacs JJ, Murphy PJ, Gaillard S, Zhao X, Wu JT, Nicchitta CV, Yoshida M, Toft DO, Pratt WB, Yao TP. HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor. Mol Cell 2005;18:601-7.
    31. Vaziri H, Dessain SK, Ng Eaton E, Imai SI, Frye RA, Pandita TK, Guarente L, Weinberg RA. hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell 2001;107:149-59.
    32. Lin RJ, Egan DA, Evans RM. Molecular genetics of acute promyelocytic leukemia. Trends Genet 1999;15:179-84.
    33. He LZ, Tolentino T, Grayson P, Zhong S, Warrell RP, Jr., Rifkind RA, Marks PA, Richon VM, Pandolfi PP. Histone deacetylase inhibitors induce remission in transgenic models of therapy-resistant acute promyelocytic leukemia. J Clin Invest 2001;108:1321-30.
    34. Jian Gu XZ, Remco A. Spanjaard, Tai C. Chen, John N. Flanagan, Michael, Boosalis SPP, and Douglas V. Faller. Histone Deacetylase-Inhibitors Sensitize Human Prostate Cancer Cell Lines to Growth Suppression and Apoptosis by Retinoids. Journal of Cancer Molecules 2006;2:25-36.
    35. Bereshchenko OR, Gu W, Dalla-Favera R. Acetylation inactivates the transcriptional repressor BCL6. Nat Genet 2002;32:606-13.
    36. Zhu P, Martin E, Mengwasser J, Schlag P, Janssen KP, Gottlicher M. Induction of HDAC2 expression upon loss of APC in colorectal tumorigenesis. Cancer Cell 2004;5:455-63.
    37. Toh Y, Ohga T, Endo K, Adachi E, Kusumoto H, Haraguchi M, Okamura T, Nicolson GL. Expression of the metastasis-associated MTA1 protein and its relationship to deacetylation of the histone H4 in esophageal squamous cell carcinomas. Int J Cancer 2004;110:362-7.
    38. Thiagalingam S, Cheng KH, Lee HJ, Mineva N, Thiagalingam A, Ponte JF. Histone deacetylases: unique players in shaping the epigenetic histone code. Ann N Y Acad Sci 2003;983:84-100.
    39. Drummond DC, Noble CO, Kirpotin DB, Guo Z, Scott GK, Benz CC. Clinical development of histone deacetylase inhibitors as anticancer agents. Annu Rev Pharmacol Toxicol 2005;45:495-528.
    40. Haggarty SJ, Koeller KM, Wong JC, Grozinger CM, Schreiber SL. Domain-selective small-molecule inhibitor of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation. Proc Natl Acad Sci U S A 2003;100:4389-94.
    41. Chen CS, Weng SC, Tseng PH, Lin HP, Chen CS. Histone acetylation-independent effect of histone deacetylase inhibitors on Akt through the reshuffling of protein phosphatase 1 complexes. J Biol Chem 2005;280:38879-87.
    42. Qiang Lu MS. POTENT SHORT-CHAIN FATTY ACID-BASED HISTONE DEACETYLASE INHIBITORS AS ANTI-TUMOR AGENTS: The Ohio State University, 2005.
    43. Lu Q, Wang DS, Chen CS, Hu YD, Chen CS. Structure-based optimization of phenylbutyrate-derived histone deacetylase inhibitors. J Med Chem 2005;48:5530-5.
    44. Lu Q, Yang YT, Chen CS, Davis M, Byrd JC, Etherton MR, Umar A, Chen CS. Zn2+-chelating motif-tethered short-chain fatty acids as a novel class of histone deacetylase inhibitors. J Med Chem 2004;47:467-74.
    45. Kulp SK, Chen CS, Wang DS, Chen CY, Chen CS. Antitumor effects of a novel phenylbutyrate-based histone deacetylase inhibitor, (S)-HDAC-42, in prostate cancer. Clin Cancer Res 2006;12:5199-206.
    46. Noh EJ, Jang ER, Jeong G, Lee YM, Min CK, Lee JS. Methyl CpG-binding domain protein 3 mediates cancer-selective cytotoxicity by histone deacetylase inhibitors via differential transcriptional reprogramming in lung cancer cells. Cancer Res 2005;65:11400-10.
    47. Xu WS, Perez G, Ngo L, Gui CY, Marks PA. Induction of polyploidy by histone deacetylase inhibitor: a pathway for antitumor effects. Cancer Res 2005;65:7832-9.
    48. Tsurutani J, Soda H, Oka M, Suenaga M, Doi S, Nakamura Y, Nakatomi K, Shiozawa K, Amada YY, Kamihira S, Kohno S. Antiproliferative effects of the histone deacetylase inhibitor FR901228 on small-cell lung cancer lines and drug-resistant sublines. Int J Cancer 2003;104:238-42.
    49. Richon VM, Sandhoff TW, Rifkind RA, Marks PA. Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation. Proc Natl Acad Sci U S A 2000;97:10014-9.
    50. Gagnon J, Shaker S, Primeau M, Hurtubise A, Momparler RL. Interaction of 5-aza-2'-deoxycytidine and depsipeptide on antineoplastic activity and activation of 14-3-3sigma, E-cadherin and tissue inhibitor of metalloproteinase 3 expression in human breast carcinoma cells. Anticancer Drugs 2003;14:193-202.
    51. Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 1999;21:103-7.
    52. Mino N, Takenaka K, Sonobe M, Miyahara R, Yanagihara K, Otake Y, Wada H, Tanaka F. Expression of tissue inhibitor of metalloproteinase-3 (TIMP-3) and its prognostic significance in resected non-small cell lung cancer. J Surg Oncol 2007;95:250-7.
    53. Cooper GM. The Cell - A Molecular Approach. 2nd ed.; 2000.
    54. Kufe DWP, Raphael E.; Weichselbaum, Ralph R.; Bast, Robert C., Jr.; Gansler, Ted S.; Holland, James F.; Frei III, Emil, editors. Cancer Medicine. 6th ed. Hamilton (Canada); 2003.

    QR CODE