1.Mithramycin A inhibits human epithelial carcinoma cells 1.proliferation and migration involving downregulation of Eps8 expression 2.Mechanism of cell death induced by Caffeic acid on melanoma cells

• Main Authors: Tzi-Peng, 楊子芃
• Other Authors: Chau-Jong Wang
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/99219699772727063271

博士 === 中山醫學大學 === 生化暨生物科技研究所 === 100 === Part1 Mithramycin A is an inhibitor of the binding of the Sp family transcription factor to the GC box. Many studies show that Mithramycin A may reduce the expression of many proto-oncogenes by inhibiting the mRNA and protein synthesis and it has been used as an antibiotic chemotherapy drug for a long time. Recently, Eps8 (EGFR pathway substrate 8) has been revealed to be a novel proto-oncogene related to cellular transformation, Rac activation and actin barbed-end-capping activity. Therefore, the aim of this study was to verify whether Eps8 might be regulated by Mithramycin. Results showed that Mithramycin could reduce the mRNA and protein levels of Eps8 in dose- and time-dependent manners in several cancer cell lines. Furthermore, cell growth and migration ability were also reduced significantly by Mithramycin A treatment. Since Src is a well-known Eps8 activity enhancer, a v-Src transfected IV5 cell line was subjected to Mithramycin A treatment and then analyzed to show that Src expression was unable to restore the Mithramycin-induced decrease in Eps8 expression, cell growth, and migration ability. To further confirm the above mentioned results, the expression of Eps8 was eliminated by a transient transfection with siRNA and subsequent analysis showed that silencing of Eps8 might also lead to a reduced growth and migration ability of cancer cells. These findings suggested that Eps8 was involved in the regulation of growth and motility of cancer cells and Mithramycin A might exert its anticancer ability via a pathway involving the downregulation of Eps8. Part2 Caffeic acid is an organic compound found in plants which also shows antioxidant, immunomodulatory, antiinflammtory and antitumor activities. Melanoma is tumorigenesis of melanocytes which produce melanin in the skin or other organs. It has a bad prognosis and difficult to treat when tumor become malignant. Studies has showed some chemicals or drugs reduced the proliferation of melanoma cells through inducing autophagy. We treat the melanoma cell line B16-F1 with Caffeic acid and find that cells go autophagy and the cell mobility diminished. The autophagy regulators p-Akt is down-regulated, and p-AMPK is up-regulated. FASN (Fatty acid synthase), a tumor related protein which can be activated by Akt and inhibited by AMPK is decreased. Beclin-1 and LC3, the autophagy regulated proteins, are express increasd. For investigate the roles of AMPK, we block the phosphorylation of AMPK by Compound C. Results show the effects of growth and migration abilities inhibited by Caffeic acid was recovery. Autophagy related proteins Beclin-1 and LC3 was recovery partially. It means that AMPK is one of the key regulator intermediate with Caffeic acid induced autophagy. It is worthy for the prophylaxis of melanoma to comprehend the death mechanisms regulated in cells. Our study shows Caffeic acid, the antioxidant ingredients conclude commonly in the plants, inhibit the cell mobility and decrease the cell growth of B16-F1 cells through autophagy. This mechnism is regulated by Akt and AMPK. Although it need more investigates to realize the factors involved, we still get an active principle for tumor inhibition and it is important for chemoprevention of melanoma.