Disulfiram Inhibited TGF-?-induced Epithelial-mesenchymal Transition In Lung Cancer Cells And Its Mechanism

Purpose: During these years,lung cancer remains the most commonly diagnosed cancer and the leading cause of cancer mortality in the world.Metastasis has been demonstrated to be one of the most life-threatening pathological events in lung cancer.It is suggested that epithelial carcinoma cells achieved migratory and invasive properties through the epithelial-to-mesenchymal transition(EMT)programme which plays a key role in promoting successful completion of the metastatic cascade and disseminate to distant tissues.Disulfiram(DSF),an anti-alcoholism drug,has attracted increasing interests in recent years due to its promising and broad-spectrum anticancer activity.In this study,we investigated the potential effect of disulfiram on TGF-?-induced EMT in lung cancer and its molecular mechanism.Methods: Human non-small cell line A549 was used to perform the following experiments.EMT in A549 cells was induced by TGF-? and DSF was added to cells before TGF-? induction to inhibit EMT.MTT assay was performed to test the cytotoxicity of DSF.The morphology changes were observed through a microscope,and the effect of DSF on migration ability was detected by the wound healing test and transwell migration assay.The effect of DSF on cell migration was further tested by transwell invasion assay.Western blotting was used to detect the expression of EMT-associated proteins in A549 cells and the expression of Snail,ERK1/2 and p-ERK1/2 to analyze the possible mechanism of DSF inhibiting the EMT programme.The ERK pathway inhibitor U0126 was used to further confirm the effect of DSF on inhibition of EMT.Results: 1.MTT assay showed that DSF had no apparent cytotoxicity at concentrations of 1 ?M,2 ?M,5 ?M,10 ?M,and 20 ?M.2.TGF-? induced EMT in A549 cells and the cell morphology converted from the typical epithelial cells to stromal-like cells,whereas the cell morphology was reversed after introducing different concentrations of DSF treatment.3.The results of the wound healing test and transwell assay demonstrated that TGF-? promoted cell migration and invasion ability,however,this effect was inhibited by DSF.4.TGF-? suppressed the expression of E-cadherin which is an epithelial marker and increased the expression of the mesenchymal marker including Vimentin,N-cadherin.These effects can be inhibited by DSF in a dosedependent manner.5.The expression of Snail,ERK1/2 and p-ERK1/2 was blocked by DSF treatment.6.U0126 had a similar effect on EMT of A549 cells.Conclusion: These results indicated that Disulfiram inhibited EMT of A549 induced by TGF-? through suppressing the ERK-Snail pathway,suggesting the potential of DSF used for clinical treatment against lung cancer.