臺灣博碩士論文加值系統

5,5-diphenyl-2-thiohydantoin-N10 (DPTH-N10)是癲癇藥物 phenytoin (5,5-diphenylhydantoin)的合成衍生化合物。先前的研究結果顯示，DPTH-N10可以抑制新生血管的生成。本研究的目的，則是探討DPTH-N10對癌細胞的直接抑制作用及其分子機制。[3H]-Thymidine incorporation的實驗結果顯示，DPTH-N10可以抑制大腸癌細胞株COLO-205的DNA合成作用，且此抑制作用隨著DPTH-N10的濃度增加而有加強的現象。西方墨點法（Western Blot Analysis）的實驗結果則顯示，以DPTH-N10 (10-30 ?嵱) 處理COLO-205細胞24小時，細胞週期的抑制蛋白p21有顯著增加；而CDK2、CDK4、與Cyclins A、D1及D3蛋白的表現量則無明顯變化。免疫沈澱法（Immuno-precipitation Assay）的實驗結果則顯示DPTH-N10能增加p21蛋白與CDK2的結合量。利用蛋白激酶活性試驗（Kinase Activity Assay）結果顯示DPTH-N10處理後，COLO-205細胞的CDK2激酶活性會有顯著的降低現象。綜合上述實驗結果顯示，DPTH-N10可藉由增加p21蛋白的表現量，增加CDK2與p21的結合，而降低CDK2的激酶活性，並進而抑制大腸癌細胞的細胞週期之進行。由於DPTH-N10可以直接抑制癌細胞的增生，又同時具有抑制腫瘤新生血管（tumor angiogenesis）的雙重作用，非常有機會發展成為新一代的抗癌藥物。

5,5-diphenyl-2-thiohydantoin-N10 (DPTH-N10) is a synthetic derivative compound of anti-epileptic medicine phenytoin (5,5-diphenylhydantoin). Previously, we have shown that DPTH-N10 exerts an anti-angiogenesis activity. The aim of this study is to study the anti-proliferation effect of DPTH-N10 in colon cancer cells and its molecular mechanisms underlying. [3H]-Thymidine incorporation analysis demonstrated that DPTH-N10 at a range of concentrations (10-30 ?嵱) dose-dependently inhibited DNA synthesis in human colon cancer cell line (COLO-205). Western blot analysis showed that the p21 protein, a cell cycle inhibitory protein, increased significantly after treatment of COLO-205 cells with DPTH-N10 for 24 hours. In contrast, the protein levels of CDK2, CDK4, and Cyclins A, D1 and D3 were not changed significantly. Immuno-precipitation assay revealed that the protein-protein association between p21 and CDK2 was increased after DPTH-N10 treatment. Moreover, treatment of the COLO-205 with DPTH-N10 caused a decrease of the CDK2 kinase activity. Taken together, in COLO-205, DPTH-N10 up-regulates the expression of p21 protein, which in turn increases the CDK2/P21 association and decreases the CDK2 kinase activity, and finally causes cell cycle arrest at the G0/G1 phase. Due to the dual effects, including anti-angiogenesis and a direct inhibition of cancer cell proliferation, DPTH-N10 has a great potential to be developed as a new medicine for cancer treatment.

誌謝……………………………………………………………3
目錄……………………………………………………………5
摘要……………………………………………………………8
Abstract…………………………………………………….10
壹、緒論..................................12
一、癌症對人類健康的衝擊與大腸癌.........................................12
二、DPTH-N10...............................14
三、細胞週期...............................14
四、研究目的...............................17
貳、 實驗材料及方法....................19
一、 常用藥品及試劑....................19
二、細胞培養 (cell culture)……………………23
三、3H-Thymidine Incorporation Assay.....24
四、細胞毒性測試(MTT assay)...................... 24
五、西方墨點法(Western Blot Analysis).............25
六、免疫沉澱法(Immunoprceipitation)...............28
七、蛋白激酶活性測定(kinase assay)................29
參、 實驗結果分析.............................31
一、 DPTH-N10抑制人類大腸癌細胞COLO-205的增殖. 31
二、 DPTH-N10對COLO-205細胞內細胞週期調控蛋白的影響................................................32
三、 DPTH-N10促進COLO-205細胞內p21與CDK 2的蛋白結合...............................................34
四、 DPTH-N10抑制COLO-205細胞的CDK2激酶活性.. 34
肆、 討論.....................................36
伍、 參考文獻.................................40
陸、 附圖.....................................44
圖一、Phenytoin (5,5-diphenylhydantoin,DPH)、 5,5-diphenyl-2-thiohydantoin(DPTH), 及 2(naphthalen-2-ylmethylsulfanyl)-5,5-diphenyl-1,5-dihydro-imidazol-4-one (DPTH-N10)的結構式................................................44
圖二、細胞週期................................................45
圖三、[3H]-thymidine incorporation的量隨著DPTH-N10的濃度上昇而減少。.......................................46
圖四、移除藥物DPTH-N10之後細胞數目會有回昇（reverse）的現象.............................................. 47
圖五、DPTH-N10在30 ?嵱的濃度內不會引發明顯的細胞死亡
................................................48
圖六、以DPTH-N10處理COLO-205細胞24小時，西方墨點法的結果。p21蛋白的表現量較對照組上昇.....................49
圖七、以DPTH-N10處理COLO-205細胞24小時，CDK2及CDK4的西方墨點法分析結果。................................. 50
圖八、以DPTH-N10處理COLO-205後，細胞內的cyclins A、E、D及D3的西方墨點法分析............................... 51
圖九、DPTH-N10會促進p21與CDK 2的結合............52
圖十、DPTH-N10 抑制CDK 2的激酶活性..............53

1Cheng, C. K., J. Wu, Y. Liu, T. S. Lee, S. J. Kang, M. T. Sheu, and W. S. Lee, 2008, Structure and anti-proliferation function of 5,5-diphenyl-2-thiohydantoin (DPTH) derivatives in vascular endothelial cells: Vascul Pharmacol, v. 48, p. 138-42.
2Coats, S., W. M. Flanagan, J. Nourse, and J. M. Roberts, 1996, Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle: Science, v. 272, p. 877-80.
3Fodde, R., J. Kuipers, C. Rosenberg, R. Smits, M. Kielman, C. Gaspar, J. H. van Es, C. Breukel, J. Wiegant, R. H. Giles, and H. Clevers, 2001, Mutations in the APC tumour suppressor gene cause chromosomal instability: Nat Cell Biol, v. 3, p. 433-8.
4Folkman, J., 1971, Tumor angiogenesis: therapeutic implications: N Engl J Med, v. 285, p. 1182-6.
5Hsu, Y. F., T. S. Lee, S. Y. Lin, S. P. Hsu, S. H. Juan, Y. H. Hsu, W. B. Zhong, and W. S. Lee, 2007, Involvement of Ras/Raf-1/ERK actions in the magnolol-induced upregulation of p21 and cell-cycle arrest in colon cancer cells: Mol Carcinog, v. 46, p. 275-83.
6Hunter, T., and J. Pines, 1994, Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age: Cell, v. 79, p. 573-82.
7Kelly, B. L., K. G. Wolfe, and J. M. Roberts, 1998, Identification of a substrate-targeting domain in cyclin E necessary for phosphorylation of the retinoblastoma protein: Proc Natl Acad Sci U S A, v. 95, p. 2535-40.
8Liu, Y., J. Wu, P. Y. Ho, L. C. Chen, C. T. Chen, Y. C. Liang, C. K. Cheng, and W. S. Lee, 2008, Anti-angiogenic action of 5,5-diphenyl-2-thiohydantoin-N10 (DPTH-N10): Cancer Lett, v. 271, p. 294-305.
9Lundberg, A. S., and R. A. Weinberg, 1999, Control of the cell cycle and apoptosis: Eur J Cancer, v. 35, p. 531-9.
10MacKinney, A. A., Jr., L. Knobeloch, and D. Norback, 1988, The effect of diphenylhydantoin and cortisol on the cell cycle: Proc Soc Exp Biol Med, v. 188, p. 173-6.
11O''Reilly, M. S., L. Holmgren, Y. Shing, C. Chen, R. A. Rosenthal, M. Moses, W. S. Lane, Y. Cao, E. H. Sage, and J. Folkman, 1994, Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma: Cell, v. 79, p. 315-28.
12Olsen, C. M., E. T. Meussen-Elholm, L. S. Roste, and E. Tauboll, 2004, Antiepileptic drugs inhibit cell growth in the human breast cancer cell line MCF7: Mol Cell Endocrinol, v. 213, p. 173-9.
13Pohl, A., W. Zhang, Y. Ning, P. C. Manegold, G. Lurje, and H. J. Lenz, 2008, Targeting metastatic colorectal cancer in 2008: a long way from 5-FU: Oncology (Williston Park), v. 22, p. 456-62; discussion 462-3, 467-8, 474 passim.
14Ries, L. A., P. A. Wingo, D. S. Miller, H. L. Howe, H. K. Weir, H. M. Rosenberg, S. W. Vernon, K. Cronin, and B. K. Edwards, 2000, The annual report to the nation on the status of cancer, 1973-1997, with a special section on colorectal cancer: Cancer, v. 88, p. 2398-424.
15Sherr, C. J., 1996, Cancer cell cycles: Science, v. 274, p. 1672-7.
16Shih, C. R., J. Wu, Y. Liu, Y. C. Liang, S. Y. Lin, M. T. Sheu, and W. S. Lee, 2004, Anti-proliferation effect of 5,5-diphenyl-2-thiohydantoin (DPTH) in human vascular endothelial cells: Biochem Pharmacol, v. 67, p. 67-75.
17Tittle, T. V., and B. A. Schaumann, 1992, Effect of antiepileptic drugs on growth of murine lymphoid tumor cells in single-cell culture: Epilepsia, v. 33, p. 729-35.
18Weinberg, R. A., 1995, The retinoblastoma protein and cell cycle control: Cell, v. 81, p. 323-30.
19Xiong, Y., G. J. Hannon, H. Zhang, D. Casso, R. Kobayashi, and D. Beach, 1993, p21 is a universal inhibitor of cyclin kinases: Nature, v. 366, p. 701-4.