| Background Cadmium(Cd)is a well-known human carcinogen.Cadmium can cause abnormal DNA methylation,inhibit cell apoptosis and DNA repair,and these factors can increase the risk of cancer.Ample epidemiological investigations indicated that chronic Cd exposure increased risk of malignant phenotype in prostate cancer.Several studies,both in vitro and in vivo studies have validated that chronic Cd exposure initiated and promoted the transformation of normal human prostate epithelial cells to malignant phenotypes,which consequently developed to aggressive tumors with highly metastatic potential.However,the signaling mechanisms involved are not fully clarified.Endoplasmic reticulum stress is considered to be a new target for the control of epithelial-mesenchymal transition.Previous in vitro cell experiments have shown that Cd can cause endoplasmic reticulum stress in cells,and in vivo experiments have also shown that Cd exposure can cause endoplasmic reticulum stress in the placenta.Therefore,we speculate whether Cd can induce endoplasmic reticulum stress in prostate cancer cells,thereby inducing cells to undergo epithelial-mesenchymal transition to improve migration and invasion capabilities.Subject In this study,we aimed to elucidate the role of chronic Cd exposure epithelial-mesenchymal transition(EMT)and thus malignant phenotypic changes of prostate cancer cells.Methods Two prostate cancer cell lines PC-3 and DU145 were used,which were continuously exposed to a non-toxic level(0.5 or 2 μM)of Cd for up to 3 months.Transwell experiment detected cell migration and invasion ability;Western-blotting or RT-PCR were used to detected the expression levels of EMT-related proteins(such as E-cadherin and vimentin)in the two cell line proteins,and the expression levels of proteins and genes related to signaling mechanisms.PC-3 and DU145 cells were cultured with 2 μM Cd in the absence or presence of NAC for 2 weeks,Transwell experiment was used to detect the effects of NAC on cell migration and invasion,and Western-blotting was used to detect the expression of EMT-related proteins to explore the influence of NAC on the malignant transformation of cell phenotype.Results PC-3 and DU145 cells which were continuously exposed to Cd for up to 3months,resulting in significantly promoted migration and invasion of the cells accompanied with the increased expression of metalloproteinases(e.g.,MMP-2).These phenotypic changes were considered to be the consequence of enhanced EMT as evidenced by diminished expression of E-cadherin and increased vimentin expression.Regarding the mechanisms of Cd-induced EMT,we found Smad3 was activated but without upregulation of TGF-β.Alternatively we found endoplasmic reticulum(ER)stress of prostate cancer cells was significantly evoked by Cd exposure,with the decreased expression of heat shock protein Bip and increased phosphorylation of ER stress sensor Inositol-requiring enzyme-1α(IRE1α).The elevated ER stress by Cd exposure was parallel with the increase of intracellular ROS generation,which could be nullified by ROS scavenger N-acetylcysteine(NAC).Removal of ROS by NAC significantly reduced ER stress in prostate cancer cells exposed to Cd,followed by the decrease of Smad3 phosphorylation and expression of nuclear Snail,a critical transcription factors for EMT,resulting the restoration of E-cadherin and suppression of vimentin expression.Consequently,the malignant phenotypic changes of prostate cancer cells caused by chronic Cd exposure were markedly inhibited by NAC treatment.Conclusion These findings confirmed the vital role of Cd on inducing malignant transformation of prostate cancer cells,and clearly indicated a new TGF-β independent,ROS mediated ER stress/Smad signaling pathway in chronic Cd exposure-induced EMT of prostate cancer cells,which suggests a preventive/therapeutic strategy for improving the prognosis of prostate cancer by targeting ER stress and utilizing antioxidants such as NAC. |
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