臺灣博碩士論文加值系統

本篇論文的主旨，在研究化學合成物質SDil-N10【2-(Naphthalen
-2-ylmethylsulfanyl)-5,5-diphenyl-1,5-dihydro-imidazol-4-one】 對於人類臍靜脈內皮細胞 (HUVEC) 的影響，並探討其中作用的機制。我們實驗結果顯示SDil-N10可抑制HUVEC細胞的生長，且其抑制作用與藥物濃度及處理時間呈正向相關。[3H] Thymidine incorporation的實驗結果顯示，SDil-N10可抑制HUVEC細胞的DNA合成作用，但對人類纖維母細胞Fibroblast的生長影響則相對小很多。西方墨點法 (Western blot analysis) 的實驗結果觀察到SDil-N10處理細胞24小時，其和細胞週期停滯有關的p21以及p27蛋白表現量高於對照組，同時Cyclin A蛋白的表現量有顯著下降，而p53、CDK2、CDK4、Cyclin E、Cyclin D1、Cyclin D3蛋白的表現量則無明顯變化。免疫沉澱法與蛋白激酶活性測定實驗結果發現以SDil-N10處理後，能增加p21與CDK2的結合量，並降低CDK2的激酶活性。實驗也發現SDil-N10能夠抑制vascular endothelial growth factor (VEGF) 所誘導之內皮細胞增生的現象。2D-Matrigel微小管腔形成以及Rat Aorta微小管腔形成(tube formation)實驗結果發現給予SDil-N10能夠抑制內皮細胞微小管腔形成的情況。我們認為SDil-N10會干擾內皮細胞的細胞週期進行，因而減少細胞增生，其作用主要透過抑制CDKs活性的途徑。因此，SDil-N10有機會可以成為抗血管增生的藥物。

The aim of this study was to examine the anti-proliferation effect of 2-(Naphthalen-2-ylmethylsulfanyl)-5,5-diphenyl-1,5-dihydro- imidazol-
4-one (SDil-N10), an analogue of antiepileptic drug phenytoin(5,5- diphenylhydantoin, DPT), on human umbilical vein endothelial cells (HUVEC) and its possible molecular mechanism underlying. SDil-N10 at a range of concentrations (10-50 uM) dose- and time-dependently inhibited DNA synthesis and decreased cell number in cultured HUVEC, but less effect in human fibroblasts. [3H] Thymidine incorporation assay demonstrated that treatment of HUVEC with SDil-N10 arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that the protein levels of p21 and p27 increased and cyclin A decreased after SDil-N10 treatment. In contrast, the protein levels of p53, cyclin D1, D3 and E, cyclin-dependent kinase (CDK2, and CDK4) in HUVEC were not changed significantly after SDil-N10 treatment. Immunoprecipitation showed that the formation of the CDK2-p21 complex, but not the CDK4-p21, CDK2-p27 and CDK4-p27 complex, was increased in the SDil-N10-treated HUVEC. Kinase assay further demonstrated that CDK2, but not CDK4, kinase activity was decreased in the SDil-N10-treated HUVEC. SDil-N10 also inhibited vascular endothelial growth factor (VEGF) induced endothelial cells proliferation. 2D-Matrigel and rat aorta tube formation assays further showed that SDil-N10 inhibited HUVEC tube formation. Taken together, these data suggest that SDil-N10 inhibits HUVEC proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 kinase activity, and finally interrupts the cell cycle. The findings from the present study suggest that SDil-N10 might have the potential to inhibit the occurrence of angiogenesis.

目 錄
誌謝……………………………………………………………………iv
目錄……………………………………………………………………vi
圖次……………………………………………………………………ix
中文摘要……………………………………………………………..xi
Abstract……………………………………………………………….xii
壹、緒論
一、前言…………………………………………………………….1
二、細胞週期 (cell cycle)………………………………………2
三、細胞程式凋亡 (apoptosis)……………………………………7
四、血管增生 (angiogenesis)……………………………………10
五、SDil-N10相關背景與研究方向………………………………...13
貳、實驗材料與方法
一、實驗材料………………………………………………………...14
二、實驗方法………………………………………………………...18
(一)、人類臍靜脈內皮細胞 (HUVEC)的初代培養 (Primary cell culture )與繼代之培養………………………………………......18
(二)、細胞計數 (Cell counting)…………………………………19
(三)、細胞毒性測試 (MTT assay)…………………………………19
(四)、DNA片段分析 (DNA fragment analysis)………………….20
(五)、西方墨點法 (Western blot)…………………………………21
(六)、3H-Thymidine Incorporation Assay………………………24
(七)、免疫沉澱法 (Immunoprecipitation)…………………………25
(八)、蛋白激酶活性測定 (Kinase assay)…………………………..25
(九)、2D-Matrigel Tube Formation Assay…………………………..26
(十)、Rat Aorta Tube Formation Assay…………………………….27
參、實驗結果分析……………………………………………………..29
一、SDil-N10 引起人類臍靜脈內皮細胞的生長的停滯…………...29
二、SDil-N10對於人類臍靜脈內皮細胞之存活率的影響………..30
三、SDil-N10對於人類臍靜脈內皮細胞G0/G1時期停滯之調控蛋白的影響……………………………………………………….32
四、給予Vascular endothelial growth factor (VEGF) 與SDil-N10對於人類臍靜脈內皮細胞的影響……………………………33
五、SDil-N10對人類大腸癌細胞COLO205的影響…………...34
六、SDil-N10在Tube Formation assay中對於人類臍靜脈內皮細胞生長抑制的影響………………………………………………34
七、SDil-N10對人類臍靜脈內皮細胞HUVEC其DNA fragmentation之影響………………………………………..36
肆、討論………………………………………………………………37
一、SDil-N10對於Cyclins與CDK s(cyclin dependent kinases) 調控細胞週期進行的機制……………………………………....37
二、SDil-N10在p21與p27對CDKs蛋白於細胞週期中
的調控…………………………………………………………39
三、SDil-N10調控細胞週期停滯在G0/G1時期的因素………..42
四、SDil-N10有潛力發展為抗癌藥物…………………………...42
伍、參考文獻…………………………………………………………45
陸、附圖………………………………………………………………55
圖 次
圖A 細胞週期Cell cycle調控之模式圖…………………………..55
圖B 產生細胞程式凋亡Apoptosis之pathway模式圖………….56
圖C 由腫瘤產生血管新生之示意圖……………………………....57
圖1 SDil-N10 之結構式………………………………………….58
圖2 SDil-N10有抑制細胞生長的現象…………………………..59
圖3 SDil-N10抑制HUVEC之存活率…………………………...60
圖4 SDil-N10抑制 HUVEC DNA之合成的情況………………61
圖5 SDil-N10對HUVEC DNA合成抑制作用之時間相關性.….62
圖6 Vascular endothelial growth factor (VEGF) 在SDil-N10對VEGF 引發HUVEC DNA合成的影響…………………….63
圖7 SDil-N10對HUVEC 之Cyclins表現的影響……………….64
圖8 SDil-N10對HUVEC 之CDKs表現的影響………………...65
圖9 SDil-N10對HUVEC上 之p21、 p27以及p53表現的影響…………….………………………………………………...66
圖10 SDil-N10對p21與p27結合於CDK2和CDK4上的影響……………….………………………………………….67
圖11 SDil-N10 抑制CDK2活性在HUVEC的表現………….68
圖12 SDil-N10抑制colo205 DNA之合成…………………….....69
圖13 SDil-N10抑制2D-matrigel tube formation……………….70
圖14 SDil-N10抑制rat aorta tube formation………………….71
圖15 SDil-N10處理HUVEC後引發細胞程式凋亡…………......72

1. Chen, C.Y., You, S.L., Lin, L.H., Hsu, W.L., Yang, Y.W. (2002) Cancer epidemiology and control in Taiwan: a brief review. Jpn J Clin Oncol 32:S66-S81
2. Winston, J.T.,Coats, S.R.,Wang, Y.Z., and Pledger, W.J. (1996) Regulation of the cell cycle machinery by oncogenic ras. Oncogene 12:127-134
3. Sherr, C.J. (1996) Cancer cell cycles. Science 274: 1672-1677
4. Akats, H., Cai, H., and Cooper, G.M. (1997) Ras links growth factor signaling to the cell cycle machinery via regulation of Cyclin D1 and the CDK inhibiter p27KIP1. Mol. Cell. Biol. 17: 3850-3857
5. Sielecki, T.M., Boylan, J.F., Benfield, P.A., and Trainor, G.L. (2000) Cyclin-dependent kinase inhibitors: Useful targets in cell cycle regulation. J.Med.Chem. 43:1-18
6. Hunter, T., Pines, J. (1994) Cyclins and cancer Ⅱ:Cyclin D and cdk inhibitors come of age. Cell 79:573-583
7. Liu, Z.J., Ueda, T., Myiazaki, T., Tanaka, N., Mine, S.,Tanaka, Y., Taniguchi, T., Yamamura, H., and Minami, Y. (1998) A critical role for Cyclin C in promotion of the hepatopoietic cell cycle by cooperation with c-Myc. Mol. Cell. Biol. 18:3445-3454
8. Albrechtsen, N., Dornreiter, I., Grosse, F., Kim, E., Wiesmuller, L., and Deppert, W. (1999) Maintenance of genomic integrity by p53:complementary roles for activated and non-activated p53. Oncogene 18: 7706-7717
9. Kelly, B., Wolfe, K.G., and Roberts, J.M. (1998) Identification of a substrate-targeting domain in Cyclin E for phosphrylation of the retinoblastoma protein. Proc. Natl. Acad. Sci. U. S. A. 95: 2535-2540
10. Weinberg, R.A. (1995) The retinoblastoma protein and cell cycle control. Cell 81:323-330
11. Lundberg, A.S., and Weinberg, R.A. (1999) Control of the cell cycle and apoptosis. Eur.J.cancer. 35:531-539
12. Conklin, D.S., Galaktionov, K., and Beach, D. (1995) 14-3-3 proteins associate with cdc25 phosphatase. Proc. Natl. Acad. Sci. U. S. A. 92: 7892-7896
13. Bery, L.D., and Gould, K.L. (1996) Regulation of cdc2 activity by phosphorylation at T14/Y15. Prog. Cell. Cycle. Ras. 2:99-105
14. Hartwell, L.H., and Weinart, T.A. (1989) Checkpoints: controls that ensure the order of cell cycle events. Science 246: 629-634
15. Reed, S.I. (1997) Control of the G1/S transition. Cancer Surv. 29:7-23
16. Xiong, Y., Hannon, G.J., Zhang, H., Casso, D., Kobayashi, R., and Beach, D. (1993) p21 is a universal inhibitor of cyclin kinasrs. Nature 366: 701-704
17. Blain, S.W., Montalvo, E., and Massague, J. (1997) Differential interaction of the cyclin A-cdk2 and cyclin D2-cdk4. J.Biol.Chem. 272: 25863-25872
18. Labaer, I.,Garrett, M.D., Stevenson, L.F., Slingerland, J.M., Sandhu, C., Chou, H.S., Fattaey, A., and Harlow, E. (1997) New functional activities for the p21 family of CDK inhibitors. Genes Dev. 11:847-862
19. Cheng, M., Olivier, P., Diehl, J.A., Fero, M., Roussel, M.F., Roberts, J.M., and Sherr, C.J., (1999) The p21cip1 and p27kip1 CDK “inhibitors” are essential activators of cyclin D-dependent kinases in muirne fibroblasts. EMBO J. 18: 1571-1583
20. Kerr, J.F., Wyllie, A.H.,Currie, A.R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26: 239-257
21. Nagata, S. (1997) Apoptosis by death factor. Cell 88: 355-365
22. Mignotte, B., Vayssiere, J.L., (1998) Mitochondria and apoptosis. Eur J Biochem. 252: 1-15
23. Earnshaw. W.C., Matrins, L.M., Kaufmann, S.H. (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Ann Rev Biochem. 68: 383-424
24. Nunez, G., Benedict, M.A., Hu, Y., Inohara, N. (1998) Caspases: the proteases of the apoptotic pathway. Oncogene 17: 3237-3245
25. Goldman, E. (1907) The growth of malignant disease in man and the lower animals with special reference to the vascular system. Lancet 2:1236-1240
26. Ida, A.G., Baker, N.H. & Warren, S.L. (1939) Vascularization of the Brown-Pearce rabbit epithelioma transplant as seen in the transparent ear chamber. Am. J. Radiol. 42: 891-899
27. Greenblatt, M. & Shubik, P. (1968) Tumor angiogenesis: transfilter diffusion studies in the hamster by the transparent chamber technique. J. Natl Cancer Inst. 41, 111-124
28. Ehrmann, R. L. & Knoth, M. (1968) Choriocarcinoma: transfilter stimulation of vasoproliferation in the hamster check pouch studied by light and electron microscopy. J. Natl Cancer Inst. 41, 1329-1341
29. Folkman, J. (Decker , Ontario, Canada, 2000) in Cancer Medicine (eds Holland, J. F. et al.) 135-152
30. Folkman, J. (1972) Anti-angiogenesis: new concept for therapy of solid tumors. Ann. Surg. 175: 409-416
31. Folkman, J. (1971) Tumor angiogenesis: therapeutic implications. N. Engl. J. Med. 256: 1182-1186
32. Risau, W., and Flamme, I. (1995) Vasculogenesis. Cell Dev. Biol. 11: 73-91
33. Risau, W. (1997) Mechanisms of angiogenesis. Nature. 286: 671-674
34. Gastl, G., Hermann, T., Steurer, M., Zmija, J., Gunsilius, E., Unger, C., and Kraft, A. (1997) Angiogenesis as a target for tumor treatment. Oncology. 54: 177-184
35. Hanahan, D., and Folkman, J. (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86: 353-364
36. Fukumura, D., Xavier, R., Sugiura, T., Chen, Y., Park, E-C., Lu, N., Selig, M., Nielsen, G., Taksir, T., Jain, R., and Seed, B. (1998) Tumor induction of VEGF promoter activity in stromal cells. Cell 94: 715-725
37. Fidler, I. J., and Ellis, L. M. (1994) The implications of angiogenesis for the biology and therapy of cancer metastasis. Cell 79: 185-188
38. Folkman, J. (1995) Angiogenesis in cancer, vascular, rheumatoid and other diease. Nat. Med. 1: 27-31
39. Singh, A. K., Sidhu, G.S., Deepa, T., and Maheshwari, P.K. (1996) Curcumin inhibits the proliferation and cell cycle progression of human umbilical vein endothelial cell. Cancer Letters 107: 109-115
40. Zeitler, H., Ko, Y., Glodny, B., and Totzke, G. Appenheimer, M., Sachinidis, A., and Vetter, H. (1997) Cell-cycle arrest in G0/G1 phase of growth factor-induced endothelial cell proliferation by various calcium channel blockers. Cancer Detection & Prevention. 21: 332-339
41. Reiser, F., Way, D., Bernas, M., Witte, M., and Witte, C. (1998) Inhibition of normal and experimental angiotumor endothelial cell proliferation and cell cycle progression by 2-methoxyestradiol. Proceedings of the Society for Experimental Biology & Medicine 219: 211-216
42. Hsieh, T.C., Juan, G., Darzynkiewicz, Z., and Wu, J.M. (1999) Resveratrol increases nitric oxide synthase, induces accumulation of p53 and p21 (WAF1/CIP1), and suppresses cultured bovine pulmonary artery endothelial cell proliferation by perturbing progression through S and G2. Cancer Research 59: 2596-2601
43. Lopez-Marure, R., Ventura, J.L. Sanchez, L., Montano, L.F., and Zentella, A. (2000) Ceramide mimics tumor necrosis factor-alpha in the induction of cell cycle arrest in endothelial cells. Induction of the tumor suppressou p53 with decrease in retino blastoma protein levels. European Journal of Biochemistry 267: 4325-4333
44. Zhang, Y., Griffith, E.C., Sage, J., Jacks, T., and Liu, J.O. (2000) Cell cycle inhibition by the anti-angiogenic agent TNP-470 is mediated by p53 and p21 WAF1/CIP1. Proc. Natl. Aced. Sci. USA. 97: 6427-6432
45. Dill, R.E., and Farmer, G.R. (1991) Phenytoin-induced DNA synthesis and inositol 1,4,5-trisphosphate formation in L-929. Experientia. 47: 728-730
46. Reed, S.I. (1997) Control of the G1/S transiton. Cancer Surveys 29: 7-23
47. Delavain, L., and La, Thangue, N.B. (1999) Control of E2F activity by p21 Waf1/Cip1. Oncogene 18: 5381-5392
48. Baldin, V., Lukas, J., Marcote, M.J., Pagano, M., and Draetts, G. (1993) Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes & Development 7: 812-821
49. Aktas, H., Cai, H., and Cooper, G.M. (1997) Ras links growth factor signaling to the cell cycle machinery via regulation of cyclin D1 and the Cdk inhibior p27 KIP1. Molecular & Cellular Biology 17: 3850-3857
50. Sherr, C.J. (1996) Cancer cell cycle. Science 274: 1672-1677
51. Kelly, B.L., Wolfe, K.G., and Roberts, J.M. (1998) Identification of a substrate-targeting domain in cyclin E necessary for phosphorylation of the retinoblastoma protein. Proc. Natl. Acad. Sci. USA. 95: 2535-2540
52. Xiong, Y., Hannon, G.J., Zhang, H., Casso, D., Kobayashi, R., and Beach, D. (1993) p21 is a universal inhibitor of cyclin kinases. Nature. 366: 701-704
53. Deng, C., Zhang, P., Harper, J.W., Elledge, S.J., and Leder, P. (1995) Mice lacking p21 CIP1/WAF1 undergo normal development, but are defective in G1 chckpoint control. Cell. 82: 675-684
54. Sambucetti, L.C., Fischer, D.D., Zabludoff, S., Kwon, P.O., Chamberlin, H., Trogani, N., Xu, H., and cohen, D. (1999) Histone deacetylase inhibition selectively alters the activity and expression of cell cycle proteins leading to specific chromatin acetylation and antiproliferative effects. J BioChem. 274: 34940-34947
55. Toyoshima, H., and Hunter, T. (1994) p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78: 67-74
56. Goukassian, D., Diez-Juan, A., Asahara, T., Schratzberger, P., Silver, M., Murayama, T., Isner, J.M, and Andres, V. (2001) Overexpression of p27(Kip1) by doxycyclin-regulated adenoviral vectors inhibits endothelial cell proliferation and migration and impairs angiogenesis. FASEB J. 15: 1877-1885
57. Coats, S., Flanagan, W.M., Nourse, J., and Roberts, J.M. (1996) Requirement of p27 kip1 for restriction point control of the fibroblast cell cycle. Science 272: 877-880
58. Pagano, M., Tam, S.W., Theodoras, A.M., Beer-Romero, P., Del, Sal, G., Chau, V. Yew, P.R., Draetta, G.F., and Rolfe, M. (1995) Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269: 682-685
59. Hirano, M., Hirano, K., Nishimura, J., and Kanaide, H. (2001) Transcriptional up-regulation of p27(Kip1) during contact-induced growth arrest in vascular endothelial cells. Experimental Cell Research 271:356-367
60. Blain, S.W., Montalvo, E., and Massague, J. (1997) Differential interaction of the cyclin-dependent kinase (Cdk) inhibitor p27 Kip1 with cyclin A-Cdk2 and cyclin D2-Cdk4. J BioChem. 272: 25863-25872
61. Rao, S., Lowe, M., Herliczek, T.W., and Keyomarsi, K. (1998) Lovastatin mediated G1 arrest in normal and tumor breast cell, is through inhibition of CDK2 activity and redistribution of p21 and p27, independent of p53. Oncogene 17: 2393-2402
62. Coats, S.,Flanagan, W.M., Nourse, J., and Robert, J.M. (1996) Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle. Science 272: 877-880