台灣十大癌症死亡率中，肺癌是排名第一的，早期容易發生轉移且預後極差，造成癌症轉移有一項機制為上皮間質轉化(Epithelial-Mesenchymal Transition，EMT)。白藜蘆醇(resveratrol)是在葡萄中一個天然的抗氧化物，許多研究指出resveratrol具有廣泛的藥理特性及能作為抗癌藥物的潛力，同時具有抑制EMT的效果，但resveratrol的衍生物是否有相同的機制尚未清楚，所以我想瞭解resveratrol之低聚芪類化合物(ε- Viniferin或α- Viniferin)對於癌症上皮-間質轉換之機制。
文獻指出肺癌細胞株A549添加TGF-β1可誘導產生EMT，所以我們利用resveratrol及其相似化合物進行處理，再合併添加TGF-β轉錄因子(Transforming growth factor-β 1,TGF-β1)促使正常的上皮細胞進行EMT，從而改變癌細胞侵襲及移動能力。
首先以Wound healing assay來檢測TGF-β1對於肺癌細胞A549的轉移效果，另外，添加TGF-β1併用ε- Viniferin或α- Viniferin的條件下，結果顯示其具有抑制細胞轉移的作用。接著利用免疫螢光染色法分析上皮間質轉化蛋白的表現位置，其中Vimentin位於細胞膜，之後添加ε-Viniferin或α-Viniferin能抑制TGF-β1誘導的Vimentin蛋白表現量；再經由明膠試驗檢測基質金屬蛋白酶的表現量，發現ε-Viniferin或α-Viniferin能抑制TGF-β1所誘導的MMP2和MMP9表現量，並以西方墨點法驗證，肺癌細胞株A549經TGF-β1併用ε-Viniferin或α-Viniferin，能使Snail、Vimentin及ZEB1蛋白表現量下降，也會抑制p-SMAD2及p-SMAD3蛋白表現量。綜合上述結果推論resveratrol其聚合物ε- Viniferin或α-Viniferin對上皮間質轉化有良好的抑制效果。
關鍵詞：轉化生長因子β1、白藜蘆醇寡聚體、上皮細胞間質轉化、肺癌

Among the top ten cancer deaths in Taiwan, lung cancer ranks first. It is likely to metastasis and have poor prognosis in the primary stage of cancer. Epithelial-mesenchymal transition (EMT) have important roles in promoting caner metastasis. Furthermore, resveratrol is a sort of natural antioxidant in grapes, and many studies have been pointed out that resveratrol is efficiency for pharmacological properties, potential as an anticancer drug, and effective of inhibiting EMT, but whether the resveratrol derivative has the same mechanism as the resveratrol, which is still unclear, yet as a result I would like to realize the mechanism of the epithelial-mesenchymal transition in cancer treated the oligomeric steroidal compound of resveratrol (ε-Viniferin or α-Viniferin).
Previous studies have shown that transforming growth factor-β (TGF-β) can induces metastasis and EMT in human A549 lung cancer cells. Therefore, we used resveratrol and its similar compounds for treatment, and then added TGF-β to promote normal epithelial cells to mesenchymal phenotype, thereby changing the invasion and migration of cancer cells. First, we used the wound healing test to detect the migration ability of lung cancer cells A549. In addition, the addition of TGF-β1 and the use of ε-Viniferin or α-Viniferin showed that they inhibited cell migration. Then, immunofluorescence staining was used to analyze the expression of EMT-related protein. Vimentin was located in the cell membrane and induced EMT. After adding ε-Viniferin or α-Viniferin inhibited TGF-β1-induced vimentin protein expression.
Next, we detected the expression of matrix metalloproteinase by gelatin zymography test, and found that ε-Viniferin or α-Viniferin could inhibit TGF-β1 -induced MMP2 and MMP9 expression, and verified by Western blot method. TGF-β combined with ε-Viniferin or α-Viniferin reduced the expression of Snail, Vimentin and ZEB1 proteins, and also inhibited the expression of p-SMAD2 and p-SMAD3 in lung cancer cell line A549. Based on the above results, it is inferred that resveratrol polymer ε-Viniferin or α-Viniferin has a good inhibitory effect on epithelial-mesenchymal transition. Keyword: TGF-β1, resveratrol oligomers, epithelial-mesenchymal transition, lung cancer

[1] 國家衛生研究院。小細胞肺癌臨床指引。取自https://reurl.cc/G3Lz3
[2] 衛生福利部全球網站中文版(2017)。取自https://reurl.cc/YARM4
[3] Travis, W. D., Brambilla, E., Noguchi, M., Nicholson, A. G., Geisinger, K. R., Yatabe, Y., … Yankelewitz, D. (2011). International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer, 6(2), 244–285. doi:10.1097/JTO.0b013e318206a221
[4] 沈默殺手：肺癌 - 肺腺癌介紹。問8健康新聞網。取自https://reurl.cc/n8A6v
[5] Detterbeck, F. C., Boffa, D. J., Kim, A. W., & Tanoue, L. T. (2017). The eighth edition lung cancer stage classification. Chest, 151(1), 193-203.
doi: 10.1016/j.chest.2016.10.010.
[6] Rami-Porta, R., Crowley, J. J., & Goldstraw, P. (2009). Review the revised TNM staging system for lung cancer. Ann Thorac Cardiovasc Surg, 15(1), 5.
[7] Hanahan, D., & Weinberg, R. A. (2000). The hallmarks of cancer. cell, 100(1), 57-70. doi.org/10.1016/S0092-8674(00)81683-9
[8] 癌症生物學 (Cancer Biology) 專題 (上)。INVESTIGATOR。取自https://reurl.cc/40vGv/
[9] Korthuis RJ, Anderson DC, Granger DN (1994). Role of neutrophil-endothelial cell adhesion in inflammatory disorders. J Crit Care. 9 (1): 47–71. doi:10.1016/0883-9441(94)90032-9
[10] Tokito, A., & Jougasaki, M. (2016). Matrix Metalloproteinases in Non-Neoplastic Disorders. International journal of molecular sciences, 17(7), 1178. doi:10.3390/ijms17071178
[11] Kalluri, R., & Weinberg, R. A. (2009). The basics of epithelial-mesenchymal transition. The Journal of clinical investigation, 119(6), 1420–1428. doi:10.1172/JCI39104
[12] Shen, J., Zhou, Q., Li, P., Wang, Z., Liu, S., He, C., … Xiao, P. (2017). Update on Phytochemistry and Pharmacology of Naturally Occurring Resveratrol Oligomers. Molecules (Basel, Switzerland), 22(12), 2050. doi:10.3390/molecules22122050
[13] Wang, H., Zhang, H., Tang, L., Chen, H., Wu, C., Zhao, M., ... & Liu, G. (2013). Resveratrol inhibits TGF-β1-induced epithelial-to-mesenchymal transition and suppresses lung cancer invasion and metastasis. Toxicology, 303, 139-146.
doi: 10.1016 / j.tox.2012.09.017
[14] Cucciolla, V., Borriello, A., Oliva, A., Galletti, P., Zappia, V., Della, R, F. (2007). Resveratrol: from basic science to the clinic. Cell cacle, 6(20), 2495-2510
doi: 10.4161/cc.6.20.4815
[15] Nivelle, L., Aires, V., Rioult, D., Martiny, L., Tarpin, M., & Delmas, D. (2018). Molecular analysis of differential antiproliferative activity of resveratrol, epsilon viniferin and labruscol on melanoma cells and normal dermal cells. Food and chemical toxicology, 116, 323-334. doi: 10.1016/j.fct.2018.04.043
[16] Cheng, K., Liu, X., Chen, L., Lv, J. M., Qu, F. J., Pan, X. W., ... & Xu, D. F. (2018). α-Viniferin activates autophagic apoptosis and cell death by reducing glucocorticoid receptor expression in castration-resistant prostate cancer cells. Medical Oncology, 35(7), 105. doi: 10.1007/s12032-018-1163-y
[17] Kulanthaivel, P., Janzen, W. P., Ballas, L. M., Jiang, J. B., Hu, C. Q., Darges, J. W., ... & Adams, L. M. (1995). Naturally Occurring Protein Kinase C Inhibitors; II1. Isolation of Oligomeric Stilbenes from Caragana sinica2. Planta medica, 61(01), 41-44. doi: 10.1055/s-2006-957996
[18] Zhi S, Guang X. (1998) Growth inhibitory effect of stilbenoids on lung cancer lines. Acta Academiae Medicinae Sinicae.;25:327–330
[19] Sung SH, Kang SY, Lee KY, et al. (2002). (+)-Alpha-viniferin, a stilbene trimer from Caragana chamlague, inhibits acetylcholinesterase. Biological & pharmaceutical bulletin.;25(1):125–127. doi:org/10.1248/bpb.25.125
[20] Tian, C. Y., Hu, C. Q., Xu, G., & Song, H. Y. (2002). Assessment of estrogenic activity of natural compounds using improved E-screen assay. Acta Pharmacologica Sinica, 23(6), 572-576.
[21] IL1B interleukin 1 beta(2017 Sep 24). Retrieved from https://reurl.cc/32pgO
[22] Lopez-Castejon, G., & Brough, D. (2011). Understanding the mechanism of IL-1β secretion. Cytokine & growth factor reviews, 22(4), 189–195. doi:10.1016/j.cytogfr.2011.10.001
[23] Dennler, S., Goumans, MJ., ten, Dijke, P. (2002). Transforming growth factor beta signal transduction. Journal of leukocyte blology, 71(5), 731-740.
doi: 10.1189/jlb.71.5.731
[24] Caja, L., Dituri, F., Mancarella, S., Caballero-Diaz, D., Moustakas, A., Giannelli, G., & Fabregat, I. (2018). TGF-β and the Tissue Microenvironment: Relevance in Fibrosis and Cancer. International journal of molecular sciences, 19(5), 1294. doi:10.3390/ijms19051294
[25] Lee, M. K., Pardoux, C., Hall, M. C., Lee, P. S., Warburton, D., Qing, J., … Derynck, R. (2007). TGF-beta activates Erk MAP kinase signalling through direct phosphorylation of ShcA. The EMBO journal, 26(17), 3957–3967. doi:10.1038/sj.emboj.7601818
[26] Heldin, C. H., & Moustakas, A. (2016). Signaling Receptors for TGF-β Family Members. Cold Spring Harbor perspectives in biology, 8(8), a022053. doi:10.1101/cshperspect.a022053
[27] Morikawa, M., Derynck, R., & Miyazono, K. (2016). TGF-β and the TGF-β Family: Context-Dependent Roles in Cell and Tissue Physiology. Cold Spring Harbor perspectives in biology, 8(5), a021873. doi:10.1101/cshperspect.a021873
[28] Pardali, E., Sanchez-Duffhues, G., Gomez-Puerto, M. C., & Ten Dijke, P. (2017). TGF-β-Induced Endothelial-Mesenchymal Transition in Fibrotic Diseases. International journal of molecular sciences, 18(10), 2157. doi:10.3390/ijms18102157
[29] Hata, A., & Chen, Y. G. (2016). TGF-β Signaling from Receptors to Smads. Cold Spring Harbor perspectives in biology, 8(9), a022061. doi:10.1101/cshperspect.a022061
[30] Atfi, A., Buisine, M., Mazars A., Gespach C. (1997). Induction of apoptosis by DPC4, a transcriptional factor regulated by transforming growth factor-beta through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling pathway. The Journal of Biological Chemistry, 272(40), 24731-24734.
doi: 10.1074/jbc.272.40.24731
[31] Siegel, PM., Massagué, J. (2003). Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nature Reviews Cancer, 3(11), 807-821.
doi: 10.1038/nrc1208
[32] Hannon, GJ., Beach, D. (1994). p15INK4B is a potential effector of TGF-beta-induced cell cycle arrest. Nature, 371, 257-261. doi: 10.1038/371257a0
[33] Zhang, J., Lu, C., Zhang, J., Kang, J., Cao, C., Li, M. (2013) Involvement of ZEB1 and E-cadherin in the invasion of lung squamous cell carcinoma. Mol Biol Rep, 40(2), 949-956. doi: 10.1007/s11033-012-2136-4
[34] Larsen, J. E., Nathan, V., Osborne, J. K., Farrow, R. K., Deb, D., Sullivan, J. P., Dospoy, P. D., Augustyn, A., Hight, S. K., Sato, M., Girard, L., Behrens, C., Wistuba, I. I., Gazdar, A. F., Hayward, N. K., … Minna, J. D. (2016). ZEB1 drives epithelial-to-mesenchymal transition in lung cancer. The Journal of clinical investigation, 126(9), 3219-35. doi:10.1172/JCI76725
[35] Dohadwala, M., Yang, SC., Luo, J., Sharma, S., Batra, RK., Huang, M.,…Dubinett, SM. (2006). Cyclooxygenase-2-dependent regulation of E-cadherin: prostaglandin E(2) induces transcriptional repressors ZEB1 and snail in non-small cell lung cancer. Cancer Research. 66(10), 5338-5345
doi: 10.1158/0008-5472.CAN-05-3635
[36] Pickup, M. W., Owens, P., & Moses, H. L. (2017). TGF-β, Bone Morphogenetic Protein, and Activin Signaling and the Tumor Microenvironment. Cold Spring Harbor perspectives in biology, 9(5), a022285. doi:10.1101/cshperspect.a022285
[37] Inman, G. J., Nicolás, F. J., Callahan, J. F., Harling, J. D., Gaster, L. M., Reith, A. D., ... & Hill, C. S. (2002). SB-431542 is a potent and specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Molecular pharmacology, 62(1), 65-74. doi.org/10.1124/mol.62.1.65
[38] Spaderna, S., Schmalhofer, O., Wahlbuhl, M., Dimmler, A., Bauer, K., Sultan, A., ... & Kirchner, T. (2008). The transcriptional repressor ZEB1 promotes metastasis and loss of cell polarity in cancer. Cancer research, 68(2), 537-544. doi: 10.1158/0008-5472.CAN-07-5682.
[39] Takeyama, Y., Sato, M., Horio, M., Hase, T., Yoshida, K., Yokoyama, T., ... & Minna, J. D. (2010). Knockdown of ZEB1, a master epithelial-to-mesenchymal transition (EMT) gene, suppresses anchorage-independent cell growth of lung cancer cells. Cancer letters, 296(2), 216-224. doi: 10.1016/j.canlet.2010.04.008