臺灣博碩士論文加值系統

泌尿移形上皮細胞癌在泌尿系統中佔90%的發生率，目前臨床治療方式大多為經過顯微手術去除部分癌細胞之後使用卡介苗或是絲裂霉素清除殘留癌細胞。由於之前實驗室所使用皮下注射方式建立膀胱癌並沒有辦法完整模擬臨床的治療方式，因此我們建立了小鼠原位膀胱癌動物模型，並且現階段臨床使用的絲裂霉素或是卡介苗其副作用較高，我們在本研究即選擇了在中醫中有去瘀血療效的桂枝茯苓丸做為治療的藥物。我們在膀胱植入小鼠膀胱MB-49細胞株的隔天灌注180 mg/ml 濃度的桂枝茯苓丸，之後每隔一天灌注，總共治療十次，結果可以有效減小膀胱腫瘤約80%的大小。另外在細胞實驗中我們也證實了桂枝茯苓丸使用1.5 mg/ml 以上的高濃度會降低小鼠膀胱癌細胞株MB-49的存活率，接著在探討藥物對於癌細胞造成的死亡機制中看到了當藥物的濃度給得越高，代表自噬作用的蛋白LC3-II表現量越高，抗凋亡蛋白Bcl-2會減少，在細胞週期的實驗中看到當給予高於1.5 mg/ml 濃度的桂枝茯苓丸藥物，會使細胞DNA的碘化丙啶 (Propidium Iodide) 螢光訊號升高，表示細胞給予高濃度的藥物時可能會直接造成細胞的壞死。我們也測試了給予藥物後細胞培養基裡釋放出來的乳酸脫氫酶 (Lactate dehydrognase ; LDH) 濃度，也可以看到相符的結果; 綜合以上的實驗結果我們認為高濃度的桂枝茯苓丸的確可以有效地減輕膀胱癌腫瘤大小，並且藥物對癌細胞造成的影響為細胞產生壓力發生自噬作用之後走向壞死。

Urothelial carcinoma is the most common tumor type in the urinary system, it has high recurrent rate after surgery. After transurethral resection of bladder tumor, it will combine treatment with Bacillus Calmette-Guerin (BCG) or mitomycin C to prevent the recurrence. However, the BCG treatment caused severe side effects such as hepatitis, abscess or pneumonitis. Thus, in this study we want to use the traditional chinese medicine Guei-Chih-Fu-Ling-Wan (桂枝茯苓丸, GFW) to investigate whether it will improve the treatment of bladder tumor. First, we established the orthotopic bladder cancer model to investigate the efficiency of GFW treatment and compared the efficacy with BCG, and mitomycin C in vivo. We found that GFW could inhibit the tumor growth in mouse bladder, and the efficacy was similar with BCG and mitomycin C, but did not show sever side effect such as hematuria. Next, we investigated the inhibitory mechanisms of GFW toward urothelial carcinoma. In vitro, cell survival were gradually decreased after GFW treatment and with dose dependent under 1.5mg/ml, however, most cell died when GFW dose were greater than 1.5mg/ml. And, the underlying mechanism of cell death after GFW treatment is through autophagy and necrosis pathway at the low and high dosage of GFW, respectively. Summarized our above data, in vivo, we established the orthotopic bladder cancer model and found that GFW inhibited the mouse bladder tumor growth orthotopically and with minor side effect compared with BCG and mitomycin C. After treatment with GFW, the cell performed cytotoxcicty through autophagy in lower dose but cell necrosis at higher dose.

目錄
目錄……………………………………………………………1
圖表目錄………………………………………………………3
中文摘要………………………………………………………5
英文摘要………………………………………………………7
緒論……………………………………………………………8
1. 泌尿上皮細胞癌 (Urothelial carcinoma)………………….8
2. 卡介苗(Bacillus Calmette-Guérin, BCG)…………………10
3. 絲裂黴素(Mitomycin C)………………………………….11
4. 桂枝茯苓丸 (Guei-Chih-Fu-Ling-Wan, GFW)………….12
4-1 桂枝茯苓丸對於體內免疫系統的調控…………………………13
5. 計劃性細胞凋亡(Programed cell death，PCD)…………15
5-1 細胞凋亡路徑(Cell Apoptotic pathway)………………………...15
5-2 細胞自噬路徑(Cell Autophagic pathway)……………………….17
5-3 細胞壞死(Cell necrosis)………………………………………….18
研究目的…………………………………………………….20
材料與方法………………………………………………….22
1. 細胞株……………………………………………………22
2
2. 實驗動物…………………………………………………22
3. MB-49小鼠膀胱癌細胞培養……………………………22
4. 原位膀胱癌小鼠建立藥物治療………………………….23
5. 膀胱癌小鼠藥物治療…………………………………….23
6. 細胞存活率實驗………………………………………….25
7. 細胞蛋白質抽取(Protein extraction)……………………..25
8. 西方墨點法(Western blot)………………………………..26
9. 細胞週期實驗…………………………………………….28
10. 乳酸氫酶釋放量實驗……………………………………29
實驗結果……………………………………………………..30
討論…………………………………………………………..39
圖表…………………………………………………………...42
參考文獻……………………………………………………...58

參考文獻
1. Mitra, A.P., et al. Combination of molecular alterations and smoking
intensity predicts bladder cancer outcome. Cancer 119, 756-765 (2013).
2. Cheng, L., Montironi, R., Davidson, D.D. & Lopez-Beltran, A. Staging and
reporting of urothelial carcinoma of the urinary bladder. Modern pathology :
an official journal of the United States and Canadian Academy of Pathology,
Inc 22 Suppl 2, S70-95 (2009).
3. Torti, F.M., et al. Cardiotoxicity of epirubicin and doxorubicin: assessment by
endomyocardial biopsy. Cancer Res 46, 3722-3727 (1986).
4. Hudson, M.L.A., Brown, E.J., Ritchey, J.K. & Ratliff, T.L. Modulation of
Fibronectin-mediated Bacillus Calmette-Guérin Attachment to Murine
Bladder Mucosa by Drugs Influencing the Coagulation Pathways. Cancer
Research 51, 3726-3732 (1991).
5. Shintani, Y., et al. Intravesical instillation therapy with bacillus
Calmette-Guerin for superficial bladder cancer: study of the mechanism of
bacillus Calmette-Guerin immunotherapy. Int J Urol 14, 140-146 (2007).
6. BÖHle, A. & Brandau, S. Immune Mechanisms in Bacillus Calmette-Guerin
Immunotherapy for Superficial Bladder Cancer. The Journal of urology 170,
964-969 (2003).
7. Lamm, D.L., et al. Incidence and treatment of complications of bacillus
Calmette-Guerin intravesical therapy in superficial bladder cancer. The
Journal of urology 147, 596-600 (1992).
8. Witjes, J.A. Management of BCG failures in superficial bladder cancer: a
review. European urology 49, 790-797 (2006).
9. Maffezzini, M., et al. Systemic Absorption and Pharmacokinetics of
Single-dose Early Intravesical Mitomycin C After Transurethral Resection of
Non-muscle-invasive Bladder Cancer. Urology (2013).
10. Park, W.H., Joo, S.T., Park, K.K., Chang, Y.C. & Kim, C.H. Effects of the
Geiji-Bokryung-Hwan on carrageenan-induced inflammation in mice and
cyclooxygenase-2 in hepatoma cells of HepG2 and Hep3B.
Immunopharmacology and immunotoxicology 26, 103-112 (2004).
11. Yao, Z. & Shulan, Z. Inhibition effect of Guizhi-Fuling-decoction on the
invasion of human cervical cancer. Journal of Ethnopharmacology 120,
25-35 (2008).
12. Mizawa, M., Makino, T., Hikiami, H., Shimada, Y. & Shimizu, T. Effectiveness
of keishibukuryogan on chronic-stage lichenification associated with atopic
dermatitis. ISRN dermatology 2012, 158598 (2012).
59
13. Nozaki, K., et al. Keishibukuryogan (gui-zhi-fu-ling-wan), a Kampo formula,
decreases disease activity and soluble vascular adhesion molecule-1 in
patients with rheumatoid arthritis. Evidence-based complementary and
alternative medicine : eCAM 3, 359-364 (2006).
14. Yoshihisa, Y., et al. The traditional Japanese formula keishibukuryogan
inhibits the production of inflammatory cytokines by dermal endothelial
cells. Mediators Inflamm 2010, 804298 (2010).
15. Assunção Guimarães, C. & Linden, R. Programmed cell deaths. European
Journal of Biochemistry 271, 1638-1650 (2004).
16. Kuan, N.K. & Passaro, E., Jr. Apoptosis: programmed cell death. Archives of
surgery (Chicago, Ill. : 1960) 133, 773-775 (1998).
17. Elmore, S. Apoptosis: a review of programmed cell death. Toxicologic
pathology 35, 495-516 (2007).
18. Oltval, Z.N., Milliman, C.L. & Korsmeyer, S.J. Bcl-2 heterodimerizes in vivo
with a conserved homolog, Bax, that accelerates programed cell death. Cell
74, 609-619 (1993).
19. Reddien, P.W., Cameron, S. & Horvitz, H.R. Phagocytosis promotes
programmed cell death in C. elegans. Nature 412, 198-202 (2001).
20. Cory, S. & Adams, J.M. The Bcl2 family: regulators of the cellular
life-or-death switch. Nature reviews. Cancer 2, 647-656 (2002).
21. Joza, N., et al. Essential role of the mitochondrial apoptosis-inducing factor
in programmed cell death. Nature 410, 549-554 (2001).
22. Scorrano, L., et al. A distinct pathway remodels mitochondrial cristae and
mobilizes cytochrome c during apoptosis. Developmental cell 2, 55-67
(2002).
23. Zou, H., Li, Y., Liu, X. & Wang, X. An APAF-1.cytochrome c multimeric
complex is a functional apoptosome that activates procaspase-9. The
Journal of biological chemistry 274, 11549-11556 (1999).
24. Kroemer, G. & Reed, J.C. Mitochondrial control of cell death. Nature
medicine 6, 513-519 (2000).
25. Arends, M.J. & Wyllie, A.H. Apoptosis: mechanisms and roles in pathology.
International review of experimental pathology 32, 223-254 (1991).
26. Golstein, P., Ojcius, D.M. & Young, J.D. Cell death mechanisms and the
immune system. Immunological reviews 121, 29-65 (1991).
27. Kerr, J.F., Winterford, C.M. & Harmon, B.V. Apoptosis. Its significance in
cancer and cancer therapy. Cancer 73, 2013-2026 (1994).
28. Lee, J., Giordano, S. & Zhang, J. Autophagy, mitochondria and oxidative
stress: cross-talk and redox signalling. The Biochemical journal 441, 523-540
60
(2012).
29. Mizushima, N., Ohsumi, Y. & Yoshimori, T. Autophagosome formation in
mammalian cells. Cell structure and function 27, 421-429 (2002).
30. Levine, B., Mizushima, N. & Virgin, H.W. Autophagy in immunity and
inflammation. Nature 469, 323-335 (2011).
31. Cesen, M.H., Pegan, K., Spes, A. & Turk, B. Lysosomal pathways to cell death
and their therapeutic applications. Experimental cell research 318,
1245-1251 (2012).
32. Bandyopadhyay, U., Kaushik, S., Varticovski, L. & Cuervo, A.M. The
chaperone-mediated autophagy receptor organizes in dynamic protein
complexes at the lysosomal membrane. Molecular and cellular biology 28,
5747-5763 (2008).
33. Homma, K., Suzuki, K. & Sugawara, H. The Autophagy Database: an
all-inclusive information resource on autophagy that provides nourishment
for research. Nucleic acids research 39, D986-990 (2011).
34. Kanduc, D., et al. Cell death: apoptosis versus necrosis (review).
International journal of oncology 21, 165-170 (2002).
35. Chen, K.L., et al. Targeting cathepsin S induces tumor cell autophagy via the
EGFR-ERK signaling pathway. Cancer letters 317, 89-98 (2012).
36. Xie, X., White, E.P. & Mehnert, J.M. Coordinate autophagy and mTOR
pathway inhibition enhances cell death in melanoma. PloS one 8, e55096
(2013).
37. Mathew, R., et al. Autophagy suppresses tumorigenesis through elimination
of p62. Cell 137, 1062-1075 (2009).
38. Han, J., Zhong, C.Q. & Zhang, D.W. Programmed necrosis: backup to and
competitor with apoptosis in the immune system. Nature immunology 12,
1143-1149 (2011).
39. Wu, Q., Esuvaranathan, K. & Mahendran, R. Monitoring the Response of
Orthotopic Bladder Tumors to Granulocyte Macrophage Colony-Stimulating
Factor Therapy Using the Prostate-Specific Antigen Gene as a Reporter.
Clinical Cancer Research 10, 6977-6984 (2004).
40. Gunther, J.H., et al. Optimizing syngeneic orthotopic murine bladder cancer
(MB49). Cancer Res 59, 2834-2837 (1999).
41. Bradford, M.M. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Analytical biochemistry 72, 248-254 (1976).