| Melanoma,a form of skin cancer arising from malignantlytransformed melanocytes.Due to its resistance to conventional treatments,such as chemotherapy and radiotherapy,and high frequency of metastasis,it has a poor prognosis once metastasis has occurred.With its considerable metastatic potential,melanoma accounts for over 70% of skin cancer-related deaths.There is currently no effective treatment for metastatic melanoma,and five-year survival does not exceed 5% in patients with metastatic disease.How to block the malignant progression of melanoma has been the medical problem in the whole world.The initiation of malignant transformation has been attributed to the process of epithelial-to-mesenchymal transition(EMT)which transforms epithelial carcinoma cells into the more invasive,motile and resistant mesenchymal-like cells.EMT is characterized as a downregulation of epithelial markers,in particular E-cadherin,and an upregulation of mesenchymal markers,particularly N-cadherin.Transforming growth factor-beta(TGF-?)is a major driving force of EMT program invoving in the physiological and pathological process such as development?fibrosis and cancer.In last decade,the anticancer effect of metformin has been reported by many groups.In addition,metformin could inhibit the proliferation?invasion and metastasis of melanoma cell lines in vivo and vitro.the latest studies showed that metformin can inhibit TGF-?1-induced EMT in several tumors,such as prostate,breast and lung cancer,then inhibit tumor progression.Whether it could inhibit TGF-?1-induced EMT in melanoma cells is still unclear.The current resurch discuss the influence of metformin on TGF-?1-induced EMT and invasion in human melanoma cell line 1205 Lu.It will provide a new mechanism for metformin against melanoma and give important clues for the treatment of melanoma.Objective:This experiment investigated the role of TGF-?1 in EMT and invasion of human melanoma cell line 1205 Lu and explored the potential effects of metformin on the process above.Methods:1205Lu melanoma cells were cultured in vitro and treated 48 h with TGF-?1 at 5ng/ml(TGF-?1 group),co-cultured with TGF-?1 at 5ng/ml and metformin at 1mM(TGF-?1+Met group),or untreated(blank contrul group).1.Themorphological ch anges of 1205 Lu cells in each experiment were observed with inverted microscope after 48 h.2.Transwell invasion assay was performed to determine the cell invasioness in each group after 48 h.3.Western-blot was performed to detect the protein expression leves of molecules involved in EMT: Snail(transcription factor),claudin-1(epithelial marker),N-cadherin(mesenchymal marker).4.RT-qPCR was applied to examine the mRNA expression leves of molecules involved in EMT: Snail(transcription factor),claudin-1(epithelial marker),N-cadherin(mesenchymal marker).5.The mean ± standard deviation was calculated for each group,and data were analyzed using one-way analysis of variance(ANOVA)followed by the least significant difference(LSD)test.Statistical analysis was performed using SPSS software,version 19.0.P<0.05 was considered to indicate a statistically significant difference.Results:1.Metformin inhibits TGF-?1-induced 1205 Lu melanoma cells invasion in vitroTranswell invasion assay showed that by the stimulating of 5ng/ml TGF-?1 for 48 h,the number of cells permeating through the basement membrane(412.2±13.427)was significantly more than that in blanck control group(194.1±8.295)(P < 0.05),and the number of invasion cells in Met+TGF-?1 growp(175.3±8.693)was obviously reduced compared with TGF-?1-stimulated cells(P<0.05).2.Metformin inhibits TGF-?1-induced EMT in 1205 Lu melanoma cells2.1 Metformin inhibits TGF-?1-induced EMT-like morphological changes in 1205 Lu melanoma cellsThe morphological changes of 1205 Lu cells in each experiment were observed with microscope.cells in control group were polygonal and uniform distribution.After TGF-?1 stimulated for 48 h,cells changed to fibroblasts-like morphology,similar to EMT-like changes,so TGF-?1 can induce 1205 Lu cells undergo EMT-like changes.Metformin intervention made the cells lose typical mesenchymal-like morphology,closer to the control group in cell morphology,so TGF-?1-induced EMT-like morphological changes were reversed by metformin,as MET-like changes.2.2 Metformin upregulates the expression of claudin-1 in 1205 Lu cells induced by TGF-?1Western-blot and RT-qPCR was used to test the protein and mRNA expression leves of epithelial marker of claudin-1.The results showed that compared with blank control group,the protein and mRNA expressions of claudin-1 in 1205 Lu cells induced by TGF-?1(5ng/ml)alone decreased obviously with statistical significance(P<0.05).And cells treated with the combination of metformin(1mM)and TGF-?1,compared with TGF-?1-stimulated 1205 Lu cells,the claudin-1 protein and mRNA expression leves increased markedly(P<0.05).2.3 Metformin downregulates the expression of Snail,N-cadherin in 1205 Lu cells induced by TGF-?1Analyzing the protein and mRNA expression leves of mesenchymal markers of Snail,N-cadherin in experimental cells by Western-blot and RT-qPCR.Our results demenstrated that compared with blank control group,the protein and mRNA expressions of Snail,N-cadherin in 1205 Lu cells induced by TGF-?1(5ng/ml)alone increased significantly with statistical significance(P<0.05).The leves of Snail,N-cadherin protein and mRNA were suppressed markedly by the combination of metformin(1mM)and TGF-?1,compared with that in TGF-?1 group(P<0.05).Conclusions:1.TGF-?1 can induce melanoma 1205 Lu melanoma cells undergo EMT process and meanwhile enhance the cell invasion in vitro.2.Metformin can inhibit TGF-?1-induced EMT in 1205 Lu melanoma cells and weaken the cell invasion in vitro at the same time. |
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