臺灣博碩士論文加值系統

肺癌是目前世界上高致死率的癌症之ㄧ，由於肺癌具有高度轉移能力，使得治癒極為不易。過去的研究指出苦瓜(Momordica charantia)有包括抗發炎、降血糖和抗腫瘤等生物功能。為了探討苦瓜對於肺癌的影響，本研究以苦瓜的甲醇萃取液分別處理具有不同轉移能力的肺癌細胞株CL1-0、CL1-1、CL1-5及F4細胞，比較苦瓜對於這些肺癌細胞生長及轉移的抑制能力，並藉由蛋白質表現的分析，探討其對肺癌細胞的作用機制。研究結果顯示，苦瓜萃取物對不同轉移能力細胞的抑制活性，與苦瓜萃取的方式及處理濃度有關，對轉移能力較低的CL1-0細胞株具有較明顯的生長抑制活性。利用4-6-diamidine-2- pheny1indole (DAPI) / propidium iodide (PI)的染色結果，也觀察到苦瓜處理的CL1-0和CL1-5細胞，在細胞核濃染等與凋亡相關細胞結構上的改變，有相當程度的差異。此外，以明膠蛋白酵素電泳法(gelatin zymography assay)進行肺癌細胞外培養基的蛋白質分析，顯示苦瓜萃取物處理後的MMP-9活性有明顯降低的趨勢。而在以傷口癒合試驗(wound healing assay)及細胞穿透試驗(transwell assay)進行苦瓜萃取物對肺癌細胞作用的評估結果，也證實肺癌細胞的轉移能力可被苦瓜萃取物所抑制，尤其以CL1-0細胞對苦瓜萃取物的反應較為明顯。在以西方墨點法所進行的細胞蛋白質表現分析，藉由探討與細胞凋亡、轉移以及細胞增生相關的蛋白質表現，可知苦瓜萃取物可透過誘發細胞凋亡、減緩細胞轉移及調控細胞週期等途徑，抑制肺癌細胞的生長。研究結果也顯示苦瓜萃取物對肺癌細胞的作用與細胞的轉移能力有關，對於轉移能力較低的細胞，其抑制活性較為顯著。

Lung cancer is one of the highest death rate cancers. The highly invasive and metastatic ability of lung cancer cells results in the difficulties of treatments for lung cancer. The past research indicates that Momordica charantia has anti-inflammatory, anti-hyperglycemia, anti-tumor effects and other bioactivities such as anti-viral effect. We used CL1-0, CL1-1, CL1-5 and F4 cells with different metastatic activity to evaluate the effect of the methanol extract of the fruit of Momordica charantia on lung cancer cells anti-proliferative and anti-metastatic activity. The underlying mechanism was also studied by protein analysis. The result shows that the growth inhibitory potency of the extract of Momordica charantia on lung cancer cells with different metastatic ability is dose dependent. The anti-proliferative activity of Momordica charantia on CL1-0 cells, which have lower invasive ability than other CL cells, is more significant. We also observe the chromatin condensation and apoptosis-related morphological changes in both Momordica charantia treated CL-0 cells and CL1-5 cells by using DAPI/PI staining. According to the result, we found the MMP-9 activity was reduced after treating with Momordica charantia in the gelatin zymography assay. The results of cell migration and wound healing assay also indicate that the metastatic activity of lung cancer could be inhibited by the extract of Momordica charantia, especially in CL1-0 cells. The growth inhibition of lung cancer cells by Momordica charantia was mediated by induction of apoptosis, metastatic arrest and cell cycle regulation as assayed by Western blot analysis of some apoptosis, metastasis and cell proliferation related proteins. These results also show that the effect of Momordica charantia on lung cancer was related to the potency of cancer cells metastatic ability, higher inhibitory effects were observed in the cells with the lower metastatic ability.

中文摘要- 2
英文摘要 -3
前言 -4
材料與方法-9
結 果 -15
討 論 -19
參考文獻 -24
圖 表 -27

1.Raz, D. J.; He, B.; Rosell, R.; Jablons, D. M., Bronchioloalveolar carcinoma: a review. Clin Lung Cancer 2006, 7, (5), 313-22.
2.Doll, R.; Hill, A. B., Lung cancer and other causes of death in relation to smoking; a second report on the mortality of British doctors. Br Med J 1956, 2, (5001), 1071-81.
3.Travis, W. D.; Travis, L. B.; Devesa, S. S., Lung cancer. Cancer 1995, 75, (1 Suppl), 191-202.
4.Hamilton, W.; Peters, T. J.; Round, A.; Sharp, D., What are the clinical features of lung cancer before the diagnosis is made? A population based case-control study. Thorax 2005, 60, (12), 1059-65.
5.Clegg, A.; Scott, D. A.; Hewitson, P.; Sidhu, M.; Waugh, N., Clinical and cost effectiveness of paclitaxel, docetaxel, gemcitabine, and vinorelbine in non-small cell lung cancer: a systematic review. Thorax 2002, 57, (1), 20-8.
6.Saunders, M.; Dische, S.; Barrett, A.; Harvey, A.; Gibson, D.; Parmar, M., Continuous hyperfractionated accelerated radiotherapy (CHART) versus conventional radiotherapy in non-small-cell lung cancer: a randomised multicentre trial. CHART Steering Committee. Lancet 1997, 350, (9072), 161-5.
7.Gudbjartsson, T.; Smaradottir, A.; Skuladottir, H.; Grimsson, H. N.; Hardardottir, H.; Bjornsson, J.; Hannesson, P.; Haraldsdottir, S. O.; Jonsson, S., [Lung cancer--review]. Laeknabladid 2008, 94, (4), 297-311.
8.Proskuryakov, S. Y.; Konoplyannikov, A. G.; Gabai, V. L., Necrosis: a specific form of programmed cell death? Exp Cell Res 2003, 283, (1), 1-16.
9.Fan, T. J.; Han, L. H.; Cong, R. S.; Liang, J., Caspase family proteases and apoptosis. Acta Biochim Biophys Sin (Shanghai) 2005, 37, (11), 719-27.
10.Flores, E. R.; Tsai, K. Y.; Crowley, D.; Sengupta, S.; Yang, A.; McKeon, F.; Jacks, T., p63 and p73 are required for p53-dependent apoptosis in response to DNA damage. Nature 2002, 416, (6880), 560-4.
11.Salomoni, P.; Pandolfi, P. P., p53 De-ubiquitination: at the edge between life and death. Nat Cell Biol 2002, 4, (6), E152-3.
12.Lowe, S. W.; Cepero, E.; Evan, G., Intrinsic tumour suppression. Nature 2004, 432, (7015), 307-15.
13.Meiller, A.; Alvarez, S.; Drane, P.; Lallemand, C.; Blanchard, B.; Tovey, M.; May, E., p53-dependent stimulation of redox-related genes in the lymphoid organs of gamma-irradiated--mice identification of Haeme-oxygenase 1 as a direct p53 target gene. Nucleic Acids Res 2007, 35, (20), 6924-34.
14.Lockshin, R. A.; Zakeri, Z., Caspase-independent cell deaths. Curr Opin Cell Biol 2002, 14, (6), 727-33.
15.Diaz, G. D.; Li, Q.; Dashwood, R. H., Caspase-8 and apoptosis-inducing factor mediate a cytochrome c-independent pathway of apoptosis in human colon cancer cells induced by the dietary phytochemical chlorophyllin. Cancer Res 2003, 63, (6), 1254-61.
16.Sielecki, T. M.; Boylan, J. F.; Benfield, P. A.; Trainor, G. L., Cyclin-dependent kinase inhibitors: useful targets in cell cycle regulation. J Med Chem 2000, 43, (1), 1-18.
17.Harper, J. W.; Adams, P. D., Cyclin-dependent kinases. Chem Rev 2001, 101, (8), 2511-26.
18.Jones, S. M.; Kazlauskas, A., Growth factor-dependent signaling and cell cycle progression. Chem Rev 2001, 101, (8), 2413-23.
19.Lee, M. H.; Yang, H. Y., Negative regulators of cyclin-dependent kinases and their roles in cancers. Cell Mol Life Sci 2001, 58, (12-13), 1907-22.
20.Noda, H.; Maehara, Y.; Irie, K.; Kakeji, Y.; Yonemura, T.; Sugimachi, K., Increased proliferative activity caused by loss of p21(WAF1/CIP1) expression and its clinical significance in patients with early-stage gastric carcinoma. Cancer 2002, 94, (7), 2107-12.
21.Yoshida, B. A.; Sokoloff, M. M.; Welch, D. R.; Rinker-Schaeffer, C. W., Metastasis-suppressor genes: a review and perspective on an emerging field. J Natl Cancer Inst 2000, 92, (21), 1717-30.
22.Hotary, K. B.; Yana, I.; Sabeh, F.; Li, X. Y.; Holmbeck, K.; Birkedal-Hansen, H.; Allen, E. D.; Hiraoka, N.; Weiss, S. J., Matrix metalloproteinases (MMPs) regulate fibrin-invasive activity via MT1-MMP-dependent and -independent processes. J Exp Med 2002, 195, (3), 295-308.
23.Birkedal-Hansen, H., Proteolytic remodeling of extracellular matrix. Curr Opin Cell Biol 1995, 7, (5), 728-35.
24.Rollin, J.; Regina, S.; Vourc'h, P.; Iochmann, S.; Blechet, C.; Reverdiau, P.; Gruel, Y., Influence of MMP-2 and MMP-9 promoter polymorphisms on gene expression and clinical outcome of non-small cell lung cancer. Lung Cancer 2007, 56, (2), 273-80.
25.Pongracz, J. E.; Stockley, R. A., Wnt signalling in lung development and diseases. Respir Res 2006, 7, 15.
26.Mazieres, J.; He, B.; You, L.; Xu, Z.; Jablons, D. M., Wnt signaling in lung cancer. Cancer Lett 2005, 222, (1), 1-10.
27.You, L.; He, B.; Xu, Z.; Uematsu, K.; Mazieres, J.; Mikami, I.; Reguart, N.; Moody, T. W.; Kitajewski, J.; McCormick, F.; Jablons, D. M., Inhibition of Wnt-2-mediated signaling induces programmed cell death in non-small-cell lung cancer cells. Oncogene 2004, 23, (36), 6170-4.
28.Lyu, J.; Joo, C. K., Wnt-7a up-regulates matrix metalloproteinase-12 expression and promotes cell proliferation in corneal epithelial cells during wound healing. J Biol Chem 2005, 280, (22), 21653-60.
29.Rao, B. K.; Kesavulu, M. M.; Giri, R.; Appa Rao, C., Antidiabetic and hypolipidemic effects of Momordica cymbalaria Hook. fruit powder in alloxan-diabetic rats. J Ethnopharmacol 1999, 67, (1), 103-9.
30.Vijayalakshmi, B.; Suresh Kumar, G.; Salimath, P. V., Effect of bitter gourd and spent turmeric on glycoconjugate metabolism in streptozotocin-induced diabetic rats. J Diabetes Complications 2007.
31.Tan, M. J.; Ye, J. M.; Turner, N.; Hohnen-Behrens, C.; Ke, C. Q.; Tang, C. P.; Chen, T.; Weiss, H. C.; Gesing, E. R.; Rowland, A.; James, D. E.; Ye, Y., Antidiabetic activities of triterpenoids isolated from bitter melon associated with activation of the AMPK pathway. Chem Biol 2008, 15, (3), 263-73.
32.Khan, S. A., Bitter gourd (Momordica charantia): a potential mechanism in anti-carcinogenesis of colon. World J Gastroenterol 2007, 13, (11), 1761-2.
33.Jiratchariyakul, W.; Wiwat, C.; Vongsakul, M.; Somanabandhu, A.; Leelamanit, W.; Fujii, I.; Suwannaroj, N.; Ebizuka, Y., HIV inhibitor from Thai bitter gourd. Planta Med 2001, 67, (4), 350-3.
34.Lee-Huang, S.; Huang, P. L.; Bourinbaiar, A. S.; Chen, H. C.; Kung, H. F., Inhibition of the integrase of human immunodeficiency virus (HIV) type 1 by anti-HIV plant proteins MAP30 and GAP31. Proc Natl Acad Sci U S A 1995, 92, (19), 8818-22.
35.Sun, Y.; Huang, P. L.; Li, J. J.; Huang, Y. Q.; Zhang, L.; Lee-Huang, S., Anti-HIV agent MAP30 modulates the expression profile of viral and cellular genes for proliferation and apoptosis in AIDS-related lymphoma cells infected with Kaposi's sarcoma-associated virus. Biochem Biophys Res Commun 2001, 287, (4), 983-94.
36.Nerurkar, P. V.; Lee, Y. K.; Linden, E. H.; Lim, S.; Pearson, L.; Frank, J.; Nerurkar, V. R., Lipid lowering effects of Momordica charantia (Bitter Melon) in HIV-1-protease inhibitor-treated human hepatoma cells, HepG2. Br J Pharmacol 2006, 148, (8), 1156-64.
37.Yasui, Y.; Hosokawa, M.; Sahara, T.; Suzuki, R.; Ohgiya, S.; Kohno, H.; Tanaka, T.; Miyashita, K., Bitter gourd seed fatty acid rich in 9c,11t,13t-conjugated linolenic acid induces apoptosis and up-regulates the GADD45, p53 and PPARgamma in human colon cancer Caco-2 cells. Prostaglandins Leukot Essent Fatty Acids 2005, 73, (2), 113-9.
38.Chu, Y. W.; Yang, P. C.; Yang, S. C.; Shyu, Y. C.; Hendrix, M. J.; Wu, R.; Wu, C. W., Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol 1997, 17, (3), 353-60.
39.The Carmichael School of Medicine. Ir Med J 1987, 80, (8), 216.
40.Goda, N.; Dozier, S. J.; Johnson, R. S., HIF-1 in cell cycle regulation, apoptosis, and tumor progression. Antioxid Redox Signal 2003, 5, (4), 467-73.
41.Salgia, R.; Skarin, A. T., Molecular abnormalities in lung cancer. J Clin Oncol 1998, 16, (3), 1207-17.
42.Wang, C. H.; Chang, H. C.; Hung, W. C., p16 inhibits matrix metalloproteinase-2 expression via suppression of Sp1-mediated gene transcription. J Cell Physiol 2006, 208, (1), 246-52.
43.Shi, Y.; He, B.; Kuchenbecker, K. M.; You, L.; Xu, Z.; Mikami, I.; Yagui-Beltran, A.; Clement, G.; Lin, Y. C.; Okamoto, J.; Bravo, D. T.; Jablons, D. M., Inhibition of Wnt-2 and galectin-3 synergistically destabilizes beta-catenin and induces apoptosis in human colorectal cancer cells. Int J Cancer 2007, 121, (6), 1175-81.
44.Bui, T. D.; Zhang, L.; Rees, M. C.; Bicknell, R.; Harris, A. L., Expression and hormone regulation of Wnt2, 3, 4, 5a, 7a, 7b and 10b in normal human endometrium and endometrial carcinoma. Br J Cancer 1997, 75, (8), 1131-6.