臺灣博碩士論文加值系統

本實驗室先前證實蟛蜞菊學名的乙酸乙酯層粗萃物(EAW)具有抑制乳癌(Breast cancer)增生之影響，因此，本研究進一步探討EAW主成分蟛蜞菊內酯(Wedelolactone；WEL)和去甲基蟛蜞菊內酯 (Demethylwedelolactone；DWEL) 對乳癌細胞 (MDA-MB-231) 侵移及轉移影響的相關藥理及分子機制。首先，我們經由細胞毒性分析和細胞週期測定，發現蟛蜞菊主成份 (WEL and DWEL)可以有效抑制MDA-MB-231乳癌細胞生長，另外經由5-脂氧合酶活性測定、前列腺素含量測定、反轉錄聚合酶鏈鎖反應和免疫點墨法，結果顯示 WEL 和 DWEL 具有抑制發炎反應之潛能，另外，利用血清誘發乳癌細胞侵移的模式來研究 WEL 和 DWEL 對乳癌細胞 MDA-MB-231 侵移特性的影響，結果顯示 WEL 和 DWEL 可抑制乳癌細胞株發散、移動、侵犯…等現象，接著我們以免疫點墨法評估 WEL 和 DWEL 對血清所誘導活化與侵犯性生長有關訊息路徑的影響，結果顯示 WEL 和 DWEL 可以抑制血清誘導的 VEGF、ERK、Akt和IкB-α 磷酸化表現，但不影響 JNK、p38 的磷酸化。在癌細胞移轉的過程當中MMP (基質金屬蛋白酵素)扮演重要的角色，所以我們利用凝膠酶譜法和免疫點墨法分析癌細胞其 MMP-2、-9 和 uPA 表現狀況，結果發現，WEL 和 DWEL會抑制 MMP-2、-9 和 uPA 的活性及表現。此外，組蛋白乙醯化作用在調控基因轉錄上扮演重要的角色，經由組蛋白去乙醯化作用造成染色體不活化，涉及了許多抑癌基因的轉錄抑制，本研究發現 WEL 和 DWEL 可增加 H3 組蛋白乙醯化進而促進 RECK 蛋白的表現，最後我們利用動物模式，研究 DWEL 對乳癌細胞在裸鼠中肺部移生的影響，結果發現 DWEL 有意義減少肺部的大小及重量和肺部移生的現象。此外，我們第一次證實無論在體外或體內，DWEL 隨著處理劑量的增加皆可以降低 MMP-2，提升TIMP1、TIMP2 蛋白的表現量，綜合以上的結果，顯示WEL和 DWEL 的抗癌活性涉及了 IKK 和 PI3K/Akt 訊息路徑的影響及經由組蛋白乙醯化的修飾而增加 RECK 表現，進而抑制乳癌細胞的侵移及轉移。

Our laboratory has previously confirmed the ethyl acetate extract of Wedelia chinensis (EAW) can restrain the proliferation of breast cancer cells. The present study is further to explore the main components of EAW, wedelolactone (WEL) and demethylwedelolactone (DWEL), invasion and metastasis of breast cancer cells. First of all, we found WEL and DWEL exhibited anti-proliferation effect on MDA-MB-231 breast cancer. We also found that WEL and DEWL have anti-inflammatory potential by 5-lipoxygenase activity assay, PGE2 assay, RT-PCR and western blot analysis. While the effect of WEL and DWEL on the suppressing breast cancer invasion and metastasis is poorly understood. Therefore, we use Fetal bovine serum (FBS) as an invasive inducer to investigate the effect of WEL and DWEL on invasive growth of MDA-MB-231 human breast cancer cells. The results showed WEL and DWEL inhibited the cell scattering, motility and invasion. The effect of WEL and DWEL on FBS-induced signaling activation involving invasive growth was evaluated by immunoblotting analysis. It showed that WEL and DWEL blocks the inhibited VEGF, ERK, Akt and IкB-α phosphorylation expression but not JNK and JNK phosphorylation. Evidenced showed that metalloprotease (MMPs) play important role in breast cancer cells migration and invasion. We performed zymography assay and western to investigate whether WEL and DWEL inhibits MMP-2, -9 and uPA activity and expression. WEL and DWEL showed inhibitory effect on MMPs activity as well as on MMP-2 and MMP-9 expression. Furthermore, histone acetylation appeared to play an important role in transcriptional regulation. The study found WEL and DWEL enhanced histone H3 acetylation to promote RECK protein expression, which may associat with activation of MMPs. Finally, we use animal models to study DWEL on lung colonization of MDA-MB-231 cells in nude mice. The results showed DWEL significantly reduced the size and weigth of lung as well as lung colonization. By immune blotting analysis, DWEL displayed concentration-dependent in reduction of MMP-2 and increase of TIMP1, TIMP2. Taken together, the study showed the inhibition of IKK and PI3K/Akt signaling pathways by WEL and DWEL anti-invasion and activity of WEL and DWEL by modulating of histone acetylation increase RECK expression to inhibit breast cancer cell invasion and metastasis. Take together the result show anti-invasion and anti-metastsis activity of WEL and DWEL in breast cancer are associated with inhibition of signaling pathway and promotion of chromatin remodeling.

目錄
縮寫表 II
中文摘要 1
ABSTRACT 3
壹、 緒論 5
一、 背景介紹 5
1. 癌症(Cancer) 5
2. 乳癌 (Breast cancer) 7
二、 藥物介紹 9
三、 細胞訊息傳遞 11
1. 第二型環氧化酵素之表現與功能 11
2. MAP Kinases pathway 12
3. 細胞核蛋白 NF-кB 13
4. Matrix metalloproteinases ( MMPs ) 15
5. Tissue inhibitors of metalloproteinases (TIMPs) 17
貳、 研究動機與架構19
研究架構 20
参、 實驗材料和方法 21
一、 實驗化學藥劑 21
二、 抗體 23
三、 實驗器材 25
四、 常用儀器 26
肆、 實驗方法 27
伍、 實驗結果 38
柒、 討論 53
陸、 參考文獻 57
柒、 圖表與說明 63
捌、 附圖 116

1.Deryugina, E. I.Quigley, J. P., Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev, 2006. 25(1): p. 9-34.

2.Parkin, D. M.Bray, F.Ferlay, J.Pisani, P., Global cancer statistics, 2002. CA Cancer J Clin, 2005. 55(2): p. 74-108.

3.Harbeck, N., Never too late: reducing late breast cancer relapse risk. Curr Med Res Opin, 2008. 24(12): p. 3295-305.

4.Lin, S. C.Lin, C. C.Lin, Y. H.Shyuu, S. J., Hepatoprotective effects of Taiwan folk medicine: wedelia chinensis on three hepatotoxin-induced hepatotoxicity. Am J Chin Med, 1994. 22(2): p. 155-68.

5.Lin, F. M.Chen, L. R.Lin, E. H.Ke, F. C.Chen, H. Y.Tsai, M. J.Hsiao, P. W., Compounds from Wedelia chinensis synergistically suppress androgen activity and growth in prostate cancer cells. Carcinogenesis, 2007. 28(12): p. 2521-9.

6.Tsai, C. H.Lin, F. M.Yang, Y. C.Lee, M. T.Cha, T. L.Wu, G. J.Hsieh, S. C.Hsiao, P. W., Herbal extract of Wedelia chinensis attenuates androgen receptor activity and orthotopic growth of prostate cancer in nude mice. Clin Cancer Res, 2009. 15(17): p. 5435-44.

7.Ghosh, S.Gifford, A. M.Riviere, L. R.Tempst, P.Nolan, G. P.Baltimore, D., Cloning of the p50 DNA binding subunit of NF-kappa B: homology to rel and dorsal. Cell, 1990. 62(5): p. 1019-29.

8.Vane, J. R.Bakhle, Y. S.Botting, R. M., Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol, 1998. 38: p. 97-120.

9.Fosslien, E., Biochemistry of cyclooxygenase (COX)-2 inhibitors and molecular pathology of COX-2 in neoplasia. Crit Rev Clin Lab Sci, 2000. 37(5): p. 431-502.

10.Tanabe, T.Tohnai, N., Cyclooxygenase isozymes and their gene structures and expression. Prostaglandins Other Lipid Mediat, 2002. 68-69: p. 95-114.
11.Karin, M.Ben-Neriah, Y., Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol, 2000. 18: p. 621-63.

12.Lernbecher, T.Muller, U.Wirth, T., Distinct NF-kappa B/Rel transcription factors are responsible for tissue-specific and inducible gene activation. Nature, 1993. 365(6448): p. 767-70.

13.Albert S. Baldwin., Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB. Journal of Clinical Investigation 2001. 107(3): p. 241-6.

14.Sen, R.Baltimore, D., Inducibility of kappa immunoglobulin enhancer-binding protein Nf-kappa B by a posttranslational mechanism. Cell, 1986. 47(6): p. 921-8.

15.Kiernan, R.Bres, V.Ng, R. W.Coudart, M. P.El Messaoudi, S.Sardet, C.Jin, D. Y.Emiliani, S.Benkirane, M., Post-activation turn-off of NF-kappa B-dependent transcription is regulated by acetylation of p65. J Biol Chem, 2003. 278(4): p. 2758-66.

16.Matrisian, L.M., Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet, 1990. 6(4): p. 121-5.

17.Liotta, L. A.Stetler-Stevenson, W. G., Tumor invasion and metastasis: an imbalance of positive and negative regulation. Cancer Res, 1991. 51(18 Suppl): p. 5054s-5059s.

18.Woessner, J.F., Jr., Role of matrix proteases in processing enamel proteins. Connect Tissue Res, 1998. 39(1-3): p. 69-73; discussion 141-9.

19.Kugler, A., Matrix metalloproteinases and their inhibitors. Anticancer Res, 1999. 19(2C): p. 1589-92.

20.Gomez, D. E.Alonso, D. F.Yoshiji, H.Thorgeirsson, U. P., Tissue inhibitors of metalloproteinases: structure, regulation and biological functions. Eur J Cell Biol, 1997. 74(2): p. 111-22.

21.DeClerck, Y. A.Perez, N.Shimada, H.Boone, T. C.Langley, K. E.Taylor, S. M. Inhibition of invasion and metastasis in cells transfected with an inhibitor of metalloproteinases. Cancer Res, 1992. 52(3): p. 701-8.

22.Shapiro, S. D.Kobayashi, D. K.Welgus, H. G., Identification of TIMP-2 in human alveolar macrophages. Regulation of biosynthesis is opposite to that of metalloproteinases and TIMP-1. J Biol Chem, 1992. 267(20): p. 13890-4.

23.Yang, T. T.Hawkes, S. P., Role of the 21-kDa protein TIMP-3 in oncogenic transformation of cultured chicken embryo fibroblasts. Proc Natl Acad Sci U S A, 1992. 89(22): p. 10676-80.

24.Greene, J.Wang, M.Liu, Y. E.Raymond, L. A.Rosen, C.Shi, Y. E., Molecular cloning and characterization of human tissue inhibitor of metalloproteinase 4. J Biol Chem, 1996. 271(48): p. 30375-80.

25.Kobori, M.Yang, Z.Gong, D.Heissmeyer, V.Zhu, H.Jung, Y. K.Gakidis, M. A.Rao, A.Sekine, T.Ikegami, F.Yuan, C.Yuan, J., Wedelolactone suppresses LPS-induced caspase-11 expression by directly inhibiting the IKK complex. Cell Death Differ, 2004. 11(1): p. 123-30.

26.Lim, J. H.Ha, U. H.Woo, C. H.Xu, H.Li, J. D., CYLD is a crucial negative regulator of innate immune response in Escherichia coli pneumonia. Cell Microbiol, 2008. 10(11): p. 2247-56.

27.Mohan, S.Konopinski, R.Yan, B.Centonze, V. E.Natarajan, M., High glucose-induced IKK-Hsp-90 interaction contributes to endothelial dysfunction. Am J Physiol Cell Physiol, 2009. 296(1): p. C182-92.

28.Vender, J. R.Laird, M. D.Dhandapani, K. M., Inhibition of NFkappaB reduces cellular viability in GH3 pituitary adenoma cells. Neurosurgery, 2008. 62(5): p. 1122-7; discussion 1027-8.

29.Tanabe, K.Nishimura, K.Dohi, S.Kozawa, O., Mechanisms of interleukin-1beta-induced GDNF release from rat glioma cells. Brain Res, 2009. 1274: p. 11-20.

30.Howe, L. R.Dannenberg, A. J., A role for cyclooxygenase-2 inhibitors in the prevention and treatment of cancer. Semin Oncol, 2002. 29(3 Suppl 11): p. 111-9.

31.da Silva, A. J. M.Melo, P. A.Silva, N. M.Brito, F. V.Buarque, C. D.de Souza, D. V.Rodrigues, V. P.Pocas, E. S.Noel, F.Albuquerque, E. X.Costa, P. R., Synthesis and preliminary pharmacological evaluation of coumestans with different patterns of oxygenation. Bioorg Med Chem Lett, 2001. 11(3): p. 283-6.

32.Zweifel, B. S.Hardy, M. M.Anderson, G. D.Dufield, D. R.Pufahl, R. A.Masferrer, J. L., A rat air pouch model for evaluating the efficacy and selectivity of 5-lipoxygenase inhibitors. Eur J Pharmacol, 2008. 584(1): p. 166-74.

33.Ben-Av, P.Crofford, L. J.Wilder, R. L.Hla, T., T., Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: a potential mechanism for inflammatory angiogenesis. FEBS Lett, 1995. 372(1): p. 83-7.

34.Kundu, N.Yang, Q.Dorsey, R.Fulton, A. M., Increased cyclooxygenase-2 (cox-2) expression and activity in a murine model of metastatic breast cancer. Int J Cancer, 2001. 93(5): p. 681-6.

35.Sheng, H.Shao, J.Morrow, J. D.Beauchamp, R. D.DuBois, R. N., Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res, 1998. 58(2): p. 362-6.

36.Lee, W. J.Chen, W. K.Wang, C. J.Lin, W. L.Tseng, T. H., Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and beta 4 integrin function in MDA-MB-231 breast cancer cells. Toxicol Appl Pharmacol, 2008. 226(2): p. 178-91.

37.Abdel-Ghany, M. Cheng, H. C. Elble, R. C. Pauli, B. U., The breast cancer beta 4 integrin and endothelial human CLCA2 mediate lung metastasis. J Biol Chem, 2001. 276(27): p. 25438-46.

38.Kim, D.Kim, S.Koh, H.Yoon, S. O.Chung, A. S.Cho, K. S.Chung, J., Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production. FASEB J, 2001. 15(11): p. 1953-62.
39.Carriero, M. V.Longanesi-Cattani, I.Bifulco, K.Maglio, O.Lista, L.Barbieri, A.Votta, G.Masucci, M. T.Arra, C.Franco, R.De Rosa, M.Stoppelli, M. P.Pavone, V., Structure-based design of an urokinase-type plasminogen activator receptor-derived peptide inhibiting cell migration and lung metastasis. Mol Cancer Ther, 2009. 8(9): p. 2708-17.

40. Hofmann, G. E.Glatstein, I.Schatz, F.Heller, D.Deligdisch, L.
, Immunohistochemical localization of urokinase-type plasminogen activator and the plasminogen activator inhibitors 1 and 2 in early human implantation sites. Am J Obstet Gynecol, 1994. 170(2): p. 671-6.

41.Trusolino, L.Cavassa, S.Angelini, P.Ando, M.Bertotti, A.Comoglio, P. M.Boccaccio, C., HGF/scatter factor selectively promotes cell invasion by increasing integrin avidity. FASEB J, 2000. 14(11): p. 1629-40.

42.Pulukuri, S.M., Gorantla, B., and Rao, J.S., Inhibition of histone deacetylase activity promotes invasion of human cancer cells through activation of urokinase plasminogen activator. J Biol Chem, 2007. 282(49): p. 35594-603.

43.Strahl, B. D.Allis, C. D., The language of covalent histone modifications. Nature, 2000. 403(6765): p. 41-5.

44.Jeon, H. W.Lee, Y. M., Inhibition of histone deacetylase attenuates hypoxia-induced migration and invasion of cancer cells via the restoration of RECK expression. Mol Cancer Ther, 2010. 9(5): p. 1361-70.

45.Chang, C.F; Yang, L.Y; Chang, S.W.; Fang Y.T.; Lee, Y.J. , Total synthesis of demethylwedelolactone and wedelolactone by Cu-mediated/Pd(0)-catalysis and oxidative-cyclization. Tetrahedron, 2008. 64: p. 3661-3666.

46.Shah, M. A.Schwartz, G. K. Cell cycle-mediated drug resistance: an emerging concept in cancer therapy. Clin Cancer Res, 2001. 7(8): p. 2168-81.

47.Megha, T.Ferrari, F.Benvenuto, A.Bellan, C.Lalinga, A. V.Lazzi, S.Bartolommei, S.Cevenini, G.Leoncini, L.Tosi, P., p53 mutation in breast cancer. Correlation with cell kinetics and cell of origin. J Clin Pathol, 2002. 55(6): p. 461-6.

48.Sander, E. E.van Delft, S.ten Klooster, J. P.Reid, T.van der Kammen, R. A.Michiels, F.Collard, J. G., Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase. J Cell Biol, 1998. 143(5): p. 1385-98.

49.Ridley, A. J.Comoglio, P. M.Hall, A., Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells. Mol Cell Biol, 1995. 15(2): p. 1110-22.

50.Wang, Y.Lam, J. B.Lam, K. S.Liu, J.Lam, M. C.Hoo, R. L.Wu, D.Cooper, G. J.Xu, A., Adiponectin modulates the glycogen synthase kinase-3beta/beta-catenin signaling pathway and attenuates mammary tumorigenesis of MDA-MB-231 cells in nude mice. Cancer Res, 2006. 66(23): p. 11462-70.

51.Liu, L. T.Peng, J. P.Chang, H. C.Hung, W. C., RECK is a target of Epstein-Barr virus latent membrane protein 1. Oncogene, 2003. 22(51): p. 8263-70.