臺灣博碩士論文加值系統

台灣十大死因中，癌症始終都排名在第一位，其中乳癌致死率也逐年增加。其中原因之一便是因為癌細胞會轉移，也因此癌細胞轉移相關機制在近幾年來越來越受到重視。上皮細胞間質轉化（Epithelial-mesenchymal transition，EMT）為癌細胞變成具有侵略性的一個過程，近年來已被視為腫瘤轉移的指標。另外，第一型轉型生長因子-β（Transforming growth factor beta 1，TGF-β1）也是引起EMT的重要因子之一。靈芝Ganoderma Lucidum（Reishi），在傳統草藥中被廣泛使用於治療許多疾病。靈芝多醣體是其中已知具有多項生物活性的成分之一。MDA-MB-231是一具有侵略性的人類乳癌細胞株。在我們的研究中，使用靈芝多醣體萃取物（Extract of Reishi polysaccharides, EORP）來處理MDA-MB-231，藉由E-cadherin、γ-catenin及N-cadherin等的EMT指標蛋白的改變，推測EORP會影響EMT。我們也發現第一型TGF-β受體（TGFβ-RI）及第二型TGF-β受體（TGFβ-RII）經過EORP處理後，其表現量皆有減少的趨勢。進一步確認，經過EORP處理後，TGFβ-RI和TGFβ-RII有泛素化（ubiquitination）的現象。另外，靈芝多醣體萃取物亦誘導細胞膜上TGFβ-RI和TGFβ-RII的脂質筏（lipid raft）表現量增加。綜合上述，EORP會藉由伴隨著脂質筏的泛素化，來減少TGFβ-RI和TGFβ-RII的表現量，進而改變MDA-MB-231 EMT的現象。

Ten leading cause of death in Taiwan, cancer is always at the first of placing, and breast cancer mortality has been rising. One reason of that because the cancer cell will metastasis, then cancer metastasis-related mechanisms be paid great attention more and more in recent years. Epithelial-mesenchymal transition (EMT) is a process that cancer cells become aggressive, and it has been marker of tumor metastasis in recent years. Besides, Transforming growth factor beta 1 (TGF-β1) is one of the important factors that cause EMT. Ganoderma Lucidum (Reishi) is treatment of many diseases in Chinese herbal medicine. Ganoderma lucidum polysaccharides is one of many constituents that have bioactivity which are known in Ganoderma Lucidum . MDA-MB-231 is an invasion human breast cancer cell line. In our study, we conjecture that EMT was affected by EORP because the EMT markers (E-cadherin, γ-catenin, N-cadherin and vimentin) change after we used EORP to treatment MDA-MB-231. We also found the production of TGFβ-RI and TGFβ-RII were down after a treatment of EORP. And the TGFβ-RI and TGFβ-RII was ubiquitination in MDA-MB-231 after a treatment of EORP. In addition, EORP also induced lipid rafts expression that with TGFβ-RI or TGFβ-RII expression. These results showed that EORP regulates EMT via the lipid rafts-dependent ubiquitination of TGFβ-R in MDA-MB-231 Breast Cancer Cells.

中文摘要...................................................4
Abstract..................................................5
實驗背景介紹................................................6
乳癌（Breast cancer）......................................6
上皮細胞間質轉化 （Epithelial-mesenchymal transition）.......6
轉化生長因子受體（TGF-β receptor）...........................7
脂質筏（Lipid raft）........................................8
TGF-β和脂質筏之間的關係 .....................................9
靈芝 ......................................................9
材料方法 ................................................. 11
製備EORP..................................................11
細胞株 ...................................................11
抗體和試劑 ................................................11
傷口癒合實驗（細胞遷移實驗）（Wound healing assay）...........12
細胞粹取物的製備 ...........................................12
西方墨點法（Westen blot） .................................13
免疫螢光染色（Immunofluorescence staining） ................13
免疫沉澱法（Immunoprecipitation assay, IP）及體外泛素化分析（In vitro Ubiquitination assay） .............................14
分餾脂質筏 ................................................15
結果 .....................................................16
EORP在人類乳癌細胞MDA-MB-231中會減少EMT的情況................16
EORP在人類乳癌細胞MDA-MB-231中會改變EMT相關指標蛋白蛋白表現量..16
EORP在人類乳癌細胞MDA-MB-231中會調降TGFβ-RI及TGFβ-RII的表現..17
EORP在人類乳癌細胞MDA-MB-231中影響了TGFβ-RI及TGFβ-RII的泛素化活性........................................................18
EORP在人類乳癌細胞MDA-MB-231中影響了TGFβ-RI及TGFβ-RII所形成的脂質筏的表現.................................................19
經由MG-132抑制蛋白脢後，EORP對於人類乳癌細胞MDA-MB-231的影響...19
在人類乳癌細胞MDA-MB-231中MβCD對於脂質筏標定蛋白的影響.........20
討論 .....................................................21
參考資料...................................................27
圖表......................................................31

1. K, P., - Breast cancer: origins and evolution. J Clin Invest, 2007. 117(11): p. 3155-63.
2. Kalluri, R. and R.A. Weinberg, The basics of epithelial-mesenchymal transition. J Clin Invest, 2009. 119(6): p. 1420-8.
3. Thiery, J.P., Epithelial–mesenchymal transitions in development and pathologies. Current Opinion in Cell Biology, 2003. 15(6): p. 740-746.
4. JP, T., - Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer, 2002. 2(6): p. 442-54.
5. JP, T. and S. JP, - Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol, 2006. 7(2): p. 131-42.
6. K, P. and W. RA, - Transitions between epithelial and mesenchymal states: acquisition of malignant. Nat Rev Cancer, 2009. 9(4): p. 265-73.
7. RB, D., et al., - Activation of growth factor secretion in tumorigenic states of breast cancer. Proc Natl Acad Sci U S A, 1987. 84(3): p. 837-41.
8. JM, Y., B. KL, and S. JS, - Development of TGF-beta signalling inhibitors for cancer therapy. Nat Rev Drug Discov, 2004. 3(12): p. 1011-22.
9. R, D., et al., - Synthesis of messenger RNAs for transforming growth factors alpha and beta and. Cancer Res, 1987. 47(3): p. 707-12.
10. R, D., A. RJ, and B. A, - TGF-beta signaling in tumor suppression and cancer progression. Nat Genet, 2001. 29(2): p. 117-29.
11. Massagué, J., S.W. Blain, and R.S. Lo, TGFβ Signaling in Growth Control, Cancer, and Heritable Disorders. Cell, 2000. 103(2): p. 295-309.
12. Zimmerman, C.M. and R.W. Padgett, Transforming growth factor β signaling mediators and modulators. Gene, 2000. 249(1–2): p. 17-30.
28
13. Miyazono, K., P. ten Dijke, and C.H. Heldin, TGF-beta signaling by Smad proteins. Adv Immunol, 2000. 75: p. 115-57.
14. Massague, J., TGF-beta signal transduction. Annu Rev Biochem, 1998. 67: p. 753-91.
15. Itoh, S. and P. ten Dijke, Negative regulation of TGF-β receptor/Smad signal transduction. Current Opinion in Cell Biology, 2007. 19(2): p. 176-184.
16. Barcellos-Hoff, M.H. and R.J. Akhurst, Transforming growth factor-beta in breast cancer: too much, too late. Breast Cancer Res, 2009. 11(1): p. 26.
17. Allen, J.A., R.A. Halverson-Tamboli, and M.M. Rasenick, Lipid raft microdomains and neurotransmitter signalling. Nat Rev Neurosci, 2007. 8(2): p. 128-140.
18. Nicolau, D.V., Jr., et al., Identifying optimal lipid raft characteristics required to promote nanoscale protein-protein interactions on the plasma membrane. Mol Cell Biol, 2006. 26(1): p. 313-23.
19. Simons, K. and D. Toomre, Lipid rafts and signal transduction. Nat Rev Mol Cell Biol, 2000. 1(1): p. 31-39.
20. Raghu, H., et al., Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells. BMC Cancer, 2010. 10: p. 647.
21. Shi, Y. and J. Massague, Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell, 2003. 113(6): p. 685-700.
22. RR, P., - Ganoderma - a therapeutic fungal biofactory. Phytochemistry, 2006. 67(18): p. 1985-2001.
23. ZB, L., - Cellular and molecular mechanisms of immuno-modulation by Ganoderma lucidum. J Pharmacol Sci, 2005. 99(2): p. 144-53.
24. ZB, L. and Z. HN, - Anti-tumor and immunoregulatory activities of Ganoderma lucidum and its possible. Acta Pharmacol Sin, 2004. 25(11): p. 1387-95.
25. D, S., et al., - Ganoderma lucidum suppresses motility of highly invasive breast and
29
prostate. Biochem Biophys Res Commun, 2002. 298(4): p. 603-12.
26. QZ, C. and L. ZB, - Antitumor and anti-angiogenic activity of Ganoderma lucidum polysaccharides. Acta Pharmacol Sin, 2004. 25(6): p. 833-8.
27. QZ, C. and L. ZB, - Ganoderma lucidum polysaccharides peptide inhibits the growth of vascular. Life Sci, 2006. 78(13): p. 1457-63.
28. Teng, B.S., et al., Hypoglycemic effect and mechanism of a proteoglycan from ganoderma lucidum on streptozotocin-induced type 2 diabetic rats. Eur Rev Med Pharmacol Sci, 2012. 16(2): p. 166-75.
29. Wang, S.H., et al., Ganoderma lucidum polysaccharides prevent platelet-derived growth factor-stimulated smooth muscle cell proliferation in vitro and neointimal hyperplasia in the endothelial-denuded artery in vivo. J Cell Physiol, 2012. 227(8): p. 3063-71.
30. Hsu, H.Y., et al., Extract of Reishi polysaccharides induces cytokine expression via TLR4-modulated protein kinase signaling pathways. J Immunol, 2004. 173(10): p. 5989-99.
31. Lai, C.Y., et al., Immunomodulatory and adjuvant activities of a polysaccharide extract of Ganoderma lucidum in vivo and in vitro. Vaccine, 2010. 28(31): p. 4945-54.
32. Lin, C.Y., et al., Ganoderma lucidum polysaccharides attenuate endotoxin-induced intercellular cell adhesion molecule-1 expression in cultured smooth muscle cells and in the neointima in mice. J Agric Food Chem, 2010. 58(17): p. 9563-71.
33. Thyagarajan, A., et al., Inhibition of oxidative stress-induced invasiveness of cancer cells by Ganoderma lucidum is mediated through the suppression of interleukin-8 secretion. Int J Mol Med, 2006. 18(4): p. 657-64.
34. Weng, C.J. and G.C. Yen, The in vitro and in vivo experimental evidences disclose the chemopreventive effects of Ganoderma lucidum on cancer invasion and metastasis. Clin Exp Metastasis, 2010. 27(5): p. 361-9.
35. Zuo, W. and Y.G. Chen, Specific activation of mitogen-activated protein kinase by transforming growth factor-beta receptors in lipid rafts is required for epithelial cell plasticity. Mol Biol Cell, 2009. 20(3): p. 1020-9.
30
36. Dumont, N., A.V. Bakin, and C.L. Arteaga, Autocrine transforming growth factor-beta signaling mediates Smad-independent motility in human cancer cells. J Biol Chem, 2003. 278(5): p. 3275-85.
37. Staubach, S. and F.G. Hanisch, Lipid rafts: signaling and sorting platforms of cells and their roles in cancer. Expert Rev Proteomics, 2011. 8(2): p. 263-77.
38. Finger, E.C., et al., Endocytosis of the type III transforming growth factor-beta (TGF-beta) receptor through the clathrin-independent/lipid raft pathway regulates TGF-beta signaling and receptor down-regulation. J Biol Chem, 2008. 283(50): p. 34808-18.
39. Chen, C.L., S.S. Huang, and J.S. Huang, Cholesterol modulates cellular TGF-beta responsiveness by altering TGF-beta binding to TGF-beta receptors. J Cell Physiol, 2008. 215(1): p. 223-33.
40. Rodal, S.K., et al., Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol Biol Cell, 1999. 10(4): p. 961-74.
41. Dai, Y., et al., CrkI and p130(Cas) complex regulates the migration and invasion of prostate cancer cells. Cell Biochem Funct, 2011. 29(8): p. 625-9.
42. Zhou, X., et al., Cucurbitacin B inhibits 12-O-tetradecanoylphorbol 13-acetate-induced invasion and migration of human hepatoma cells through inactivating mitogen-activated protein kinase and PI3K/Akt signal transduction pathways. Hepatol Res, 2012. 42(4): p. 401-11.
43. Barron, N., et al., Biochemical relapse following radical prostatectomy and miR-200a levels in prostate cancer. Prostate, 2012. 72(11): p. 1193-9.
44. Wade, M., et al., Functional analysis and consequences of Mdm2 E3 ligase inhibition in human tumor cells. Oncogene, 2012. 23(10): p. 625.