臺灣博碩士論文加值系統

膀胱癌是台灣目前最常見的泌尿系統的癌症，而目前最普遍的療法是先施予內視鏡切除手術，再給予膀胱灌注化學藥品或免疫治療法。然而，治療後病人的復發率很高，因此找尋新的膀胱灌注治療藥品是一項迫在眉睫的事。Combretastatin A-4 (CA-4)是一種已被認為具有抗癌活性的抗微小管聚合劑。在本篇研究中，我們利用人類膀胱癌細胞株以及老鼠膀胱癌模式來偵測CA-4的抗癌活性。結果顯示CA-4對人類膀胱癌細胞株細胞毒性的LC50低於4 nM。利用西方點墨法發現CA-4能夠有效抑制細胞內的tubulin聚合。接下來利用流式細胞儀分析CA-4對細胞週期影響，發現能使細胞週期明顯停滯在G2/M時期，且伴隨sub-G1的產生，以上結果顯示CA-4可能經由細胞凋亡模式導致細胞死亡。另外，CA-4能導致有絲分裂時期特異性蛋白的累積，包括cyclin B1以及MPM-2。進一步我們分析CA-4誘發細胞死亡的機制，結果發現CA-4會導致BFTC 905細胞的PARP及caspase-3活化，而TSGH 8301細胞並未出現此現象，但兩種細胞隨CA-4濃度增加皆能降低細胞內粒線體膜電位。爾後利用H33342染細胞發現CA-4會導致細胞出現多核型態，因此推測細胞死亡並非僅經由細胞凋亡路徑。除此之外，CA-4能抑制細胞轉移能力及抑制BFTC 905細胞磷酸化AKT表現，但對於TSGH 8301細胞反而有增強表現的效果，推測CA-4抑制細胞轉移能力並非只經由抑制磷酸化AKT相關訊息傳遞路徑。最後，原位癌動物模式發現CA-4能夠延緩膀胱癌病程。綜合以上結果發現，CA-4能藉由誘發有絲分裂風暴進而細胞凋亡導致細胞死亡，並且抑制癌細胞轉移，在原位癌動物模式中也能抑制腫瘤生長。顯示CA-4具有潛力能成為另一種膀胱灌注治療膀胱癌的藥物。

Bladder cancer is the most common urological cancer in Taiwan, and the most common therapy is treated with endoscopic resection and following intravesical immuno/chemotherapy. However, the recurrence rate is still high after that treatment, so it is necessary to find new drugs for intravesical chemotherapy. Combretastatin A-4 (CA-4), an antitubulin agent, was identified as an anti-cancer agent. In this study, we investigated the anti-cancer activity of CA-4 in human bladder cancer cells and murine orthotopic bladder tumor model. Cytotoxic LC50 of CA-4 in human bladder cancer cell lines was below 4 nM. Western blot analysis showed that CA-4 indeed inhibited tubulin polymerization in vivo. Analysis of cell-cycle distribution by flow cytometry, CA-4 induced obviously G2-M arrest with sub-G1 phenomenon. It suggests that CA-4 might induce apoptosis. On the other hand, CA-4 induced the accumulation of the mitotic markers MPM-2 and cyclin B1. In the analysis of apoptosis, it showed that CA-4 induced the activation of caspase-3, PARP cleavage of BFTC 905 cell, but TSGH 8301 cell was free from these apoptosis-related proteins activated. However, both cells’ mitochondria membrane potential decreased in a dose-dependent manner. After H33342 staining, we found that CA-4 induced multinuclei cells. It suggested that CA-4 induced cell death might not through apoptosis pathway mainly. In addition, CA-4 had significant in vitro effect on reducing cell migration and inhibiting the expression of AKT phosphorylation of BFTC 905 cell, but increasing the expression of AKT phosphorylation of TSGH 8301. It suggested that CA-4 inhibited cell migratory ability might not only through inhibiting phospho-AKT-related signal pathway. Moreover, the in vivo orthotopic study revealed CA-4 slowed down the development of bladder tumor. Collectively, these data demonstrate that CA-4 causes bladder cancer cells death through mitotic catastrophe and apoptosis following; inhibit cell migration in vitro and orthotopic tumor growth in vivo. It suggests that CA-4 intravesical therapy at least provides another strategy in treating bladder cancers.

目錄 I
圖表目錄 III
縮寫表 V
中文摘要 VII
英文摘要 IX
I 序論 1
I-1 膀胱癌 1
I-2 微小管 4
I-3 類二苯乙烯(stilbenoids) 7
I-4 Combretastatin A-4 8
II實驗材料 11
II-1 藥品試劑 11
II-2 常用溶液 14
III實驗方法 19
III-1 Combretastatin A-4合成方法 19
III-2 細胞培養 19
III-3 細胞毒性試驗 20
III-4 細胞內抑制tubulin聚合活性試驗 21
III-5 西方點墨法 21
III-6 流式細胞儀細胞週期分析 23
III-7 細胞週期M phase arrest與細胞凋亡相關蛋白分析 24
III-8 流式細胞儀粒線體膜電位測定 25
III-9 mitotic catastrophe 現象觀察 26
III-10 細胞轉移試驗 26
III-11 動物飼養 27
III-12 老鼠膀胱癌模式 27
III-13 統計分析 28
IV結果 29
IV-1 CA-4對人類膀胱癌細胞株的細胞毒性研究 29
IV-2 CA-4與trans CA-4 之 in vivo微管蛋白聚合試驗 29
IV-3 CA-4 對細胞週期的影響 30
IV-4 CA-4誘發人類膀胱癌細胞株之細胞凋亡相關現象研究 31
IV-5 CA-4誘發人類膀胱癌細胞mitotic catastrophe 32
IV-6 CA-4對癌細胞轉移以及p-AKT表現影響 33
IV-7以膀胱癌動物模式評估CA-4膀胱灌注治療效果 34
V 討論 37
VI 參考文獻 44

1. 行政院衛生民國96年主要死因統計.
2. Parkin, D.M., The global burden of urinary bladder cancer. Scand J Urol Nephrol Suppl, 2008(218): p. 12-20.
3. Jemal, A., et al., Cancer statistics, 2008. CA Cancer J Clin, 2008. 58(2): p. 71-96.
4. Murta-Nascimento, C., et al., Epidemiology of urinary bladder cancer: from tumor development to patient's death. World J Urol, 2007. 25(3): p. 285-95.
5. Lower, G.M., Jr., Concepts in causality: chemically induced human urinary bladder cancer. Cancer, 1982. 49(5): p. 1056-66.
6. 陶聲洋防癌基金會.
7. Spiess, P.E. and B. Czerniak, Dual-track pathway of bladder carcinogenesis: practical implications. Archives of Pathology & Laboratory Medicine, 2006. 130(6): p. 844-852.
8. Barocas, D.A. and P.E. Clark, Bladder cancer. Curr Opin Oncol, 2008. 20(3): p. 307-14.
9. Gee, J., A.L. Sabichi, and H.B. Grossman, Chemoprevention of superficial bladder cancer. Crit Rev Oncol Hematol, 2002. 43(3): p. 277-86.
10. Amling, C.L., Diagnosis and management of superficial bladder cancer. Curr Prob Cancer, 2001. 25(4): p. 219-278.
11. Margolis, R.L. and L. Wilson, Microtubule treadmilling: what goes around comes around. Bioessays, 1998. 20(10): p. 830-6.
12. Chang, P. and T. Stearns, Delta-tubulin and epsilon-tubulin: two new human centrosomal tubulins reveal new aspects of centrosome structure and function. Nat Cell Biol, 2000. 2(1): p. 30-5.
13. Nigg, E.A., Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol, 2001. 2(1): p. 21-32.
14. Zheng, Y., M.K. Jung, and B.R. Oakley, Gamma-tubulin is present in Drosophila melanogaster and Homo sapiens and is associated with the centrosome. Cell, 1991. 65(5): p. 817-23.
15. Amos, L.A., Focusing-in on microtubules. Curr Opin Struct Biol, 2000. 10(2): p. 236-41.
16. Dowling, M., et al., Mitotic spindle checkpoint inactivation by trichostatin a defines a mechanism for increasing cancer cell killing by microtubule-disrupting agents. Cancer Biol Ther, 2005. 4(2): p. 197-206.
17. Wang, L.G., et al., The effect of antimicrotubule agents on signal transduction pathways of apoptosis: a review. Cancer Chemoth Pharm, 1999. 44(5): p. 355-61.
18. McKiernan, J.M., et al., Phase I trial of intravesical docetaxel in the management of superficial bladder cancer refractory to standard intravesical therapy. J Clin Oncol, 2006. 24(19): p. 3075-3080.
19. Saloustros, E., D. Mavroudis, and V. Georgoulias, Paclitaxel and docetaxel in the treatment of breast cancer. Expert Opin Pharmacother, 2008. 9(15): p. 2603-16.
20. Jordan, A., et al., Tubulin as a target for anticancer drugs: agents which interact with the mitotic spindle. Med Res Rev, 1998. 18(4): p. 259-96.
21. Hamel, E. and C.M. Lin, Interactions of combretastatin, a new plant-derived antimitotic agent, with tubulin. Biochem Pharmacol, 1983. 32(24): p. 3864-7.
22. Wall, M.E., et al., Plant antitumor agents. 3. A convenient separation of tannins from other plant constituents. J Pharm Sci, 1969. 58(7): p. 839-41.
23. Lin, C.M., et al., Interactions of tubulin with potent natural and synthetic analogs of the antimitotic agent combretastatin: a structure-activity study. Mol Pharmacol, 1988. 34(2): p. 200-8.
24. Vincent, L., et al., Combretastatin A4 phosphate induces rapid regression of tumor neovessels and growth through interference with vascular endothelial-cadherin signaling. J Clin Invest, 2005. 115(11): p. 2992-3006.
25. Griggs, J., J.C. Metcalfe, and R. Hesketh, Targeting tumour vasculature: the development of combretastatin A4. Lancet Oncol, 2001. 2(2): p. 82-7.
26. Quan, H., Y. Xu, and L. Lou, p38 MAPK, but not ERK1/2, is critically involved in the cytotoxicity of the novel vascular disrupting agent combretastatin A4. Int J Cancer, 2008. 122(8): p. 1730-7.
27. Tozer, G.M., et al., Combretastatin A-4 phosphate as a tumor vascular-targeting agent: early effects in tumors and normal tissues. Cancer Res, 1999. 59(7): p. 1626-1634.
28. Kanthou, C., et al., The tubulin-binding agent combretastatin A-4-phosphate arrests endothelial cells in mitosis and induces mitotic cell death. Am J Pathol, 2004. 165(4): p. 1401-1411.
29. Stevenson, J.P., et al., Phase I trial of the antivascular agent combretastatin A4 phosphate on a 5-day schedule to patients with cancer: magnetic resonance imaging evidence for altered tumor blood flow. J Clin Oncol, 2003. 21(23): p. 4428-4438.
30. Nabha, S.M., et al., Combretastatin-A4 prodrug induces mitotic catastrophe in chronic lymphocytic leukemia cell line independent of caspase activation and poly(ADP-ribose) polymerase cleavage. Clin Cancer Res, 2002. 8(8): p. 2735-41.
31. de Bruin, E.C. and J.P. Medema, Apoptosis and non-apoptotic deaths in cancer development and treatment response. Cancer Treat Rev, 2008. 34(8): p. 737-49.
32. Vitale, I., et al., Combretastatin CA-4 and combretastatin derivative induce mitotic catastrophe dependent on spindle checkpoint and caspase-3 activation in non-small cell lung cancer cells. Apoptosis, 2007. 12(1): p. 155-166.
33. Lin, H.L., et al., Combretastatin A4-induced differential cytotoxicity and reduced metastatic ability by inhibition of AKT function in human gastric cancer cells. J Pharmacol Exp Ther, 2007. 323(1): p. 365-73.
34. Petit, I., et al., The microtubule-targeting agent CA4P regresses leukemic xenografts by disrupting interaction with vascular cells and mitochondrial-dependent cell death. Blood, 2008. 111(4): p. 1951-61.
35. Dumontet, C. and B.I. Sikic, Mechanisms of action of and resistance to antitubulin agents: microtubule dynamics, drug transport, and cell death. J Clin Oncol, 1999. 17(3): p. 1061-70.
36. Deng, L., et al., MDR and MRP gene families as cellular determinant factors for resistance to clinical anticancer agents. Cancer Treat Res, 2002. 112: p. 49-66.
37. Kuo, C.C., et al., BPR0L075, a novel synthetic indole compound with antimitotic activity in human cancer cells, exerts effective antitumoral activity in vivo. Cancer Res, 2004. 64(13): p. 4621-4628.
38. Chin, Y.R. and A. Toker, Function of Akt/PKB signaling to cell motility, invasion and the tumor stroma in cancer. Cell Signal, 2009. 21(4): p. 470-6.
39. Sheng, S., M. Qiao, and A.B. Pardee, Metastasis and AKT activation. J Cell Physiol, 2009. 218(3): p. 451-4.
40. Simoni, D., et al., Novel combretastatin analogues endowed with antitumor activity. J Med Chem, 2006. 49(11): p. 3143-52.
41. Bernhard, E.J., et al., Reducing the radiation-induced G2 delay causes HeLa cells to undergo apoptosis instead of mitotic death. Int J Radiat Biol, 1996. 69(5): p. 575-84.
42. Yoshida, M., et al., Biochemical differences between staurosporine-induced apoptosis and premature mitosis. Exp Cell Res, 1997. 232(2): p. 225-39.
43. Vakifahmetoglu, H., M. Olsson, and B. Zhivotovsky, Death through a tragedy: mitotic catastrophe. Cell Death Differ, 2008. 15(7): p. 1153-62.
44. Chade, D.C., et al., Immunomodulatory effects of recombinant BCG expressing pertussis toxin on TNF-alpha and IL-10 in a bladder cancer model. J Exp Clin Cancer Res, 2008. 27: p. 78.
45. Pettit, G.R., et al., Antineoplastic agents 322. synthesis of combretastatin A-4 prodrugs. Anticancer Drug Design, 1995. 10(4): p. 299-309.