在本研究中，我們期望找到有效抑制黑色素瘤生長以及轉移的天然藥物藉以取代臨床用藥，因此我們將評估白朮內酯I、II及III對A2058人類黑色素瘤細胞的影響。首先我們進行細胞存活率測試，確定其白朮內酯 I、II及III的條件濃度並不會造成細胞直接死亡，接著以細胞侵襲實驗與傷口癒合實驗來測試A2058細胞株的爬行能力，其結果證明白朮內酯I和II有效抑制A2058人類黑色素瘤細胞的侵襲以及轉移，然而白朮內酯III在傷口癒合實驗的結果中，發現其無法有效抑制A2058人類黑色素瘤細胞的爬行能力。進一步我們探討其白朮內酯I和II對於A2058細胞的訊號途徑的調控，在西方墨點法以及酶譜法的結果顯示白朮內酯I和II有效抑制MMP9、MMP2、p-P38以及p-ERK的表現量，使MAPK pathway受到抑制，進而影響A2058細胞株的轉移，另外我們也發現白朮內酯I和II會抑制p-65蛋白進入細胞核中調控其下游基因。由本研究的結果中，我們可以證實白朮內酯I和II會藉由阻斷MAPK pathway進而抑制人類黑色素瘤細胞的轉移以及侵襲，在未來或許可以應用於黑色素瘤的治療方針之一。

Before cancer spread to other places in our body which is known as metastasis, it can be easily cured. In the past published literature, atractylenolide has been proven to have the ability to induce apoptosis, but its effect on the metastasis ability of cancer cells has not yet been proven. Therefore, in this study, we will evaluate the anti-metastatic ability of atractylenolide on A2058 human melanoma cells. First, we used MTT assay to determine that the conditional concentration of atractylenolide I、II and III did not cause direct cell death, and then tested the motility of the A2058 cell line with cell invasion experiments and wound healing assay. The results proved that atractylenolide I and II effectively inhibited A2058 human melanoma invasion and migration. Furthermore, we explored the signal pathway regulated by atractylenolide I and II on A2058 cells. The results of the Western blot and zymography showed that atractylenolide I and II effectively inhibited the expression of MMP9, MMP2, p-P38 and p-ERK, then block the MAPK pathway. From the results of this study, we can confirm the atractylenolide I and II can inhibit the metastasis and invasion of human melanoma cells by blocking the MAPK pathway, which may be one of the treatment guidelines for melanoma in the future.

1.衛生福利部國民健康署編印, 癌症登記報告. 2018.
2.Diepgen, T.L. and V.J.B.J.o.D. Mahler, The epidemiology of skin cancer. 2002. 146: p. 1-6.
3.Balch, C.M., et al., Prognostic factors analysis of 17,600 melanoma patients: validation of the American Joint Committee on Cancer melanoma staging system. 2001. 19(16): p. 3622-3634.
4.Morton, D.L., et al., Technical details of intraoperative lymphatic mapping for early stage melanoma. 1992. 127(4): p. 392-399.
5. Kim, H.Y., et al., Discovery of potential biomarkers in human melanoma cells with different metastatic potential by metabolic and lipidomic profiling. Sci Rep, 2017. 7(1): p. 8864.
6. Su, D.M., et al., Two types of human malignant melanoma cell lines revealed by expression patterns of mitochondrial and survival-apoptosis genes: implications for malignant melanoma therapy. Mol Cancer Ther, 2009. 8(5): p. 1292-304.
7. Quinones, L.G., Garcia-Castro, I., Characterization of human melanoma cell lines according to their migratory properties in vitro. In Vitro Cellular & Developmental Biology-Animal, 2004. 40(1-2): p. 35-42.
8. Vaisanen, A., et al., Prognostic value of MMP-2 immunoreactive protein (72 kD type IV collagenase) in primary skin melanoma. J Pathol, 1998. 186(1): p. 51-8.
9. Kanavy, H.E. and M.R. Gerstenblith, Ultraviolet radiation and melanoma. Semin Cutan Med Surg, 2011. 30(4): p. 222-8.
10. Azoury, S.C. and J.R. Lange, Epidemiology, risk factors, prevention, and early detection of melanoma. Surg Clin North Am, 2014. 94(5): p. 945-62, vii.
11. Jiang, W.G., et al., Tissue invasion and metastasis: Molecular, biological and clinical perspectives. Semin Cancer Biol, 2015. 35 Suppl: p. S244-S275.
12. Gupta, G.P. and J. Massague, Cancer metastasis: building a framework. Cell, 2006. 127(4): p. 679-95.
13. Clark, A.G. and D.M. Vignjevic, Modes of cancer cell invasion and the role of the microenvironment. Curr Opin Cell Biol, 2015. 36: p. 13-22.
14. George Poste , I.J.F., The pathogenesis of cancer metastasis. Nature, 1980: p. 139-146.
15. van Zijl, F., G. Krupitza, and W. Mikulits, Initial steps of metastasis: cell invasion and endothelial transmigration. Mutat Res, 2011. 728(1-2): p. 23-34.
16. Geiger, T.R. and D.S. Peeper, Metastasis mechanisms. Biochim Biophys Acta, 2009. 1796(2): p. 293-308.
17. Su, Z., et al., Apoptosis, autophagy, necroptosis, and cancer metastasis. Mol Cancer, 2015. 14: p. 48.
18. Van Lint, P. and C. Libert, Chemokine and cytokine processing by matrix metalloproteinases and its effect on leukocyte migration and inflammation. J Leukoc Biol, 2007. 82(6): p. 1375-81.
19. Bourboulia, D. and W.G. Stetler-Stevenson, Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): Positive and negative regulators in tumor cell adhesion. Semin Cancer Biol, 2010. 20(3): p. 161-8.
20. Martin, M.D. and L.M. Matrisian, The other side of MMPs: protective roles in tumor progression. Cancer Metastasis Rev, 2007. 26(3-4): p. 717-24.
21. Malemud, C.J., Matrix metalloproteinases (MMPs) in health and disease: an overview. Front Biosci, 2006. 11: p. 1696-701.
22. Redondo, P., et al., Expression and serum levels of MMP-2 and MMP-9 during human melanoma progression. Clin Exp Dermatol, 2005. 30(5): p. 541-5.
23. Li, H., et al., The relationship between MMP-2 and MMP-9 expression levels with breast cancer incidence and prognosis. Oncol Lett, 2017. 14(5): p. 5865-5870.
24. Huang, H., Matrix Metalloproteinase-9 (MMP-9) as a Cancer Biomarker and MMP-9 Biosensors: Recent Advances. Sensors (Basel), 2018. 18(10).
25. Webb, A.H., et al., Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma. BMC Cancer, 2017. 17(1): p. 434.
26. Roomi, M.W., et al., Modulation of MMP-2 and -9 secretion by cytokines, inducers and inhibitors in human melanoma A-2058 cells. Oncol Rep, 2017. 37(6): p. 3681-3687.
27. Salemi, R., et al., MMP-9 as a Candidate Marker of Response to BRAF Inhibitors in Melanoma Patients With BRAF(V600E) Mutation Detected in Circulating-Free DNA. Front Pharmacol, 2018. 9: p. 856.
28. Kim, E.K. and E.J. Choi, Pathological roles of MAPK signaling pathways in human diseases. Biochim Biophys Acta, 2010. 1802(4): p. 396-405.
29. Morrison, D.K., MAP kinase pathways. Cold Spring Harb Perspect Biol, 2012. 4(11).
30. Ye, Y., et al., ERK/GSK3beta signaling is involved in atractylenolide I-induced apoptosis and cell cycle arrest in melanoma cells. Oncol Rep, 2015. 34(3): p. 1543-8.
31. Ye, Y., et al., Atractylenolide II induces G1 cell-cycle arrest and apoptosis in B16 melanoma cells. J Ethnopharmacol, 2011. 136(1): p. 279-82.
32. Fu, X.Q., et al., Inhibition of STAT3 signalling contributes to the antimelanoma action of atractylenolide II. Exp Dermatol, 2014. 23(11): p. 855-7.
33. 段启 , 許., 謝晨, HPLC法測定白术不同炮制品中白术內酯I、II、III. Chinese Traditional and Herbal Drugs, 2008: p. 1343-03.
34. Fu, X.Q., et al., The JAK2/STAT3 pathway is involved in the anti-melanoma effects of atractylenolide I. Exp Dermatol, 2018. 27(2): p. 201-204.