臺灣博碩士論文加值系統

肺癌是癌症致死亡的主要死因。並且其五年的存活率相當低，約只有百分之十五。目前，對於癌症病患的治療主要是依其診斷的腫瘤種類及期別。但現今，治療仍是有其限制性。最主要原因是在被診斷出腫瘤時往往已有轉移現象而侷限了治療。上皮細胞間質轉化(Epithelial-mesenchymal transition (EMT))已被視為腫瘤發展過程中關鍵性的過程。而其中具多功能性的轉形生長因子TGF-β1更是一典型能引起上皮細胞間質轉質轉化的重要因子。靈芝Ganoderma Lucidum (Lingzhi or Reishi) 已被長久廣泛使用在傳統中草用藥上。靈芝成份中已知具有多項生物活性。而靈芝多醣體為其中之一。在我們的研究當中，我們使用靈芝多醣體萃取物來探討在肺癌細胞A549中TGF-β1所調節的細胞轉形(EMT)過程中所扮演角色。我們的結果發現，靈芝多醣體萃取物能夠改變TGF-β1所誘導的細胞型態改變。並且，上皮細胞標誌有表現量增加而同時間質細胞標誌有表現下降的情形。另外，在短時間3小時看到Smad2/3磷酸化蛋白表現受到抑制。此外，在短時間3小時作用之下，靈芝多醣體萃取物阻止了Smad2/3、Smad4蛋白由細胞質移往到細胞核中，並且使得Smad4蛋白表現量下降。再者，轉錄因子snail蛋白也在此時受到抑制。另外，由實驗結果顯示靈芝多醣體萃取物能夠抑制轉形生長因子TGF-β1以及第二型TGF-β受體蛋白表現。而在細胞功能上，我們發現靈芝多醣體能夠抑制TGF-β1所誘導細胞的爬行及侵入性能力。由研究結果顯示，靈芝多醣體萃取物能夠經由抑制Smad蛋白訊息傳遞及抑制轉錄因子snail的表現來進一步阻止了TGF-β1所調節的上皮細胞間質轉化於肺癌A549細胞。

Lung cancer is the leading cause of cancer death. And the five-year relative survival rate is only about 15%. There are many treatments for lung cancer patients. It depends on the diagnosed stage. Nowadays the treatment has restricted. The most important reason is metastasized when diagnosed. Epithelial-mesenchymal transition (EMT) has been regarded as the critical event in tumorigenesis and is typically induced by a multifunctional cytokine, transforming growth factor (TGF)-β1. Ganoderma lucidum (Ling-zhi or Reishi) has been used for a long time in tradition herbal medicine. It contains many bioactive components. Polysaccharide is one of the bioactive compounds. We used the extract of Reishi polysaccharides (EORP) in our research. To investigate the effects of EORP on TGF-β1-mediated EMT in lung cancer A549 cells. EORP reverses TGF-β1-induced EMT morphological changes in A549 cells, in addition, the epithelial markers are up-regulated; alternatively, mesenchymal markers are down-regulated. To further analyze the mechanism for altering EMT, we found that phosphorylation of Smad2/3 proteins is reduced when cells treated with EORP for 3 h, meanwhile, EORP inhibits TGF-β1-induced translocation of Smad2/3-protein from the cytoplasm to the nucleus. On the other hand, EORP treatment of cells for 3 h, it decreases Smad4 protein expression followed by reducing Smad4 translocation to nucleus. Furthermore, the transcription factor snail protein is inhibited after treated with EORP for 3 h. In addition, EORP decreases the levels of TGFβ1 and TGFβ receptor II. We also found that cells treated with EORP results in the inhibition of TGF-β1-induced cell migration and invasion. Our results show that EORP inhibits the TGF-β1-mediated epithelial-mesenchymal transition via inhibition of the Smad and snail protein expression in NSCLC A549 cells.

中文摘要 1
ABSTRACT 2
INTRODUCTION 4
MATERIALS AND METHODS 7
PREPARATION OF EORP 7
CELL LINE 7
WESTERN BLOT ANALYSIS 7
ANALYSIS OF CELL VIABILITY (MTT ASSAY) 8
IMMUNOFLUORESCENCE STAIN 8
NUCLEAR PROTEIN EXTRACTION 8
ELISA 9
MIGRATION AND INVASION ASSAY 9
REAGENTS AND ANTIBODIES 9
RESULTS 10
TGF-Β1-INDUCED EPITHELIAL-MESENCHYMAL-TRANSITION MORPHOLOGICAL CHANGES IN A549 IS REVERSED BY EORP 10
TGF-Β1 INDUCES HUMAN NON SMALL CELL LUNG CANCER A549 CELLS TO UNDERGO EPITHELIAL-MESENCHYMAL TRANSITION (EMT) 10
HUMAN NSCLC A549 CELLS UNDERGO EMT IN RESPONSE TO TGF-Β1 AND WERE REVERSED BY EORP 11
EORP REDUCE THE CELL PROLIFERATION IN A549 CELLS 11
EORP REDUCE TGFΒ1-INDUCED PHOSPHORYLATED SMAD2/3 PROTEIN LEVELS IN A549 CELLS 11
EFFECTS OF EORP ON SMAD2/3 AND SMAD4 PROTEINS EXPRESSION IN A549 CELLS 12
EFFECTS OF EORP ON PHOSPHORYLATED ERK AND AKT PROTEINS EXPRESSION INDUCED BY TGF-Β1 IN A549 CELLS 12
EFFECTS OF EORP ON SMAD PROTEINS EXPRESSION IN NUCLEUS OF A549 CELLS 12
EORP REGULATES EXPRESSION OF SNAIL IN TGF-Β1-INDUCED A549 CELLS 13
EORP AFFECTS EXPRESSIONS OF TGFΒ RECEPTOR II IN A549 CELLS 13
EORP INHIBIT THE PRODUCTION OF TGF-Β1 IN A549 CELLS 14
EORP INHIBITS TGF-Β1-INDUCED CELL MIGRATION AND INVASION IN A549 CELLS 14
DISCUSSION 15
REFERENCES 19
FIGURE LEGENDS 22

1. Cancer facts and figures. American Cancer Society, 2009.
2. Sharma, S.V., et al., Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer, 2007. 7(3): p. 169-81.
3. M. Zhanga, S.W.C., P.C.K. Cheungb and Q. Wanga, Antitumor polysaccharides from mushrooms : a review on their isolation process, structural characteristics and antitumor activity. Trends in Food Science & Technology, 2007. 18.
4. Siegel, P.M. and J. Massague, Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat Rev Cancer, 2003. 3(11): p. 807-21.
5. Wakefield, L.M. and A.B. Roberts, TGF-beta signaling: positive and negative effects on tumorigenesis. Curr Opin Genet Dev, 2002. 12(1): p. 22-9.
6. Dumont, N. and C.L. Arteaga, Targeting the TGF beta signaling network in human neoplasia. Cancer Cell, 2003. 3(6): p. 531-6.
7. Thiery, J.P., Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer, 2002. 2(6): p. 442-54.
8. Vega, S., et al., Snail blocks the cell cycle and confers resistance to cell death. Genes Dev, 2004. 18(10): p. 1131-43.
9. Chou, T.Y., et al., Clusterin silencing in human lung adenocarcinoma cells induces a mesenchymal-to-epithelial transition through modulating the ERK/Slug pathway. Cell Signal, 2009. 21(5): p. 704-11.
10. Yang, J. and R.A. Weinberg, Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell, 2008. 14(6): p. 818-29.
11. Huber, M.A., N. Kraut, and H. Beug, Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol, 2005. 17(5): p. 548-58.
12. Zavadil, J. and E.P. Bottinger, TGF-beta and epithelial-to-mesenchymal transitions. Oncogene, 2005. 24(37): p. 5764-74.
13. Gavert, N. and A. Ben-Ze'ev, Epithelial-mesenchymal transition and the invasive potential of tumors. Trends Mol Med, 2008. 14(5): p. 199-209.
14. Lin, Z.B. and H.N. Zhang, Anti-tumor and immunoregulatory activities of Ganoderma lucidum and its possible mechanisms. Acta Pharmacol Sin, 2004. 25(11): p. 1387-95.
15. Lin, Z.B., Cellular and molecular mechanisms of immuno-modulation by Ganoderma lucidum. J Pharmacol Sci, 2005. 99(2): p. 144-53.
16. Yuen, J.W. and M.D. Gohel, Anticancer effects of Ganoderma lucidum: a review of scientific evidence. Nutr Cancer, 2005. 53(1): p. 11-7.
17. Cao, Q.Z. and Z.B. Lin, Ganoderma lucidum polysaccharides peptide inhibits the growth of vascular endothelial cell and the induction of VEGF in human lung cancer cell. Life Sci, 2006. 78(13): p. 1457-63.
18. Sliva, D., et al., Ganoderma lucidum suppresses motility of highly invasive breast and prostate cancer cells. Biochem Biophys Res Commun, 2002. 298(4): p. 603-12.
19. Cao, Q.Z. and Z.B. Lin, Antitumor and anti-angiogenic activity of Ganoderma lucidum polysaccharides peptide. Acta Pharmacol Sin, 2004. 25(6): p. 833-8.
20. Cano, A., et al., The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol, 2000. 2(2): p. 76-83.
21. Jonathan M. Yingling, K.L.B.J.S.S., Development of TGF- signalling inhibitors for cancer therapy. Nature Reviews Drug Discovery 2004. 3.
22. Derynck, R., R.J. Akhurst, and A. Balmain, TGF-beta signaling in tumor suppression and cancer progression. Nat Genet, 2001. 29(2): p. 117-29.
23. Xu, J., S. Lamouille, and R. Derynck, TGF-beta-induced epithelial to mesenchymal transition. Cell Res, 2009. 19(2): p. 156-72.
24. Moustakas, A., S. Souchelnytskyi, and C.H. Heldin, Smad regulation in TGF-beta signal transduction. J Cell Sci, 2001. 114(Pt 24): p. 4359-69.
25. Valcourt, U., et al., TGF-beta and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition. Mol Biol Cell, 2005. 16(4): p. 1987-2002.
26. Kretzschmar, M., et al., A mechanism of repression of TGFbeta/ Smad signaling by oncogenic Ras. Genes Dev, 1999. 13(7): p. 804-16.
27. Shi, Y. and J. Massague, Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell, 2003. 113(6): p. 685-700.
28. Attisano, L. and J.L. Wrana, Smads as transcriptional co-modulators. Curr Opin Cell Biol, 2000. 12(2): p. 235-43.
29. Massague, J. and D. Wotton, Transcriptional control by the TGF-beta/Smad signaling system. EMBO J, 2000. 19(8): p. 1745-54.
30. Itoh, S., et al., Signaling of transforming growth factor-beta family members through Smad proteins. Eur J Biochem, 2000. 267(24): p. 6954-67.
31. Nieto, M.A., The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol, 2002. 3(3): p. 155-66.
32. Yang, J., S.A. Mani, and R.A. Weinberg, Exploring a new twist on tumor metastasis. Cancer Res, 2006. 66(9): p. 4549-52.
33. Dickson, R.B., et al., Activation of growth factor secretion in tumorigenic states of breast cancer induced by 17 beta-estradiol or v-Ha-ras oncogene. Proc Natl Acad Sci U S A, 1987. 84(3): p. 837-41.
34. de Boer, W.I., et al., Transforming growth factor beta1 and recruitment of macrophages and mast cells in airways in chronic obstructive pulmonary disease. Am J Respir Crit Care Med, 1998. 158(6): p. 1951-7.
35. Vanhee, D., et al., Mechanisms of fibrosis in coal workers' pneumoconiosis. Increased production of platelet-derived growth factor, insulin-like growth factor type I, and transforming growth factor beta and relationship to disease severity. Am J Respir Crit Care Med, 1994. 150(4): p. 1049-55.
36. Anand-Apte, B. and B. Zetter, Signaling mechanisms in growth factor-stimulated cell motility. Stem Cells, 1997. 15(4): p. 259-67.
37. Price, J.T. and E.W. Thompson, Mechanisms of tumour invasion and metastasis: emerging targets for therapy. Expert Opin Ther Targets, 2002. 6(2): p. 217-33.
38. Fenteany, G. and S. Zhu, Small-molecule inhibitors of actin dynamics and cell motility. Curr Top Med Chem, 2003. 3(6): p. 593-616.
39. Stetler-Stevenson, W.G., Type IV collagenases in tumor invasion and metastasis. Cancer Metastasis Rev, 1990. 9(4): p. 289-303.
40. Liotta, L.A., P.S. Steeg, and W.G. Stetler-Stevenson, Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell, 1991. 64(2): p. 327-36.
41. Zeng, Z.S., A.M. Cohen, and J.G. Guillem, Loss of basement membrane type IV collagen is associated with increased expression of metalloproteinases 2 and 9 (MMP-2 and MMP-9) during human colorectal tumorigenesis. Carcinogenesis, 1999. 20(5): p. 749-55.
42. Seon, B.K., et al., Long-lasting complete inhibition of human solid tumors in SCID mice by targeting endothelial cells of tumor vasculature with antihuman endoglin immunotoxin. Clin Cancer Res, 1997. 3(7): p. 1031-44.
43. Won, J., et al., Tumorigenicity of mouse thymoma is suppressed by soluble type II transforming growth factor beta receptor therapy. Cancer Res, 1999. 59(6): p. 1273-7.
44. Yamaguchi, Y., D.M. Mann, and E. Ruoslahti, Negative regulation of transforming growth factor-beta by the proteoglycan decorin. Nature, 1990. 346(6281): p. 281-4.