Study On The Role Of ERK5/AP-1 Signal Pathway In Benzidine-induced Epithelial-Mesenchymal Transition In SV-40 Immortalized Human Urothelial Cells And The Mechanism Of Curcumin's Intervention

Objectives Bladder cancer is the most common cancer of urinary tract.Overexposure to benzidine has been manifested as an important cause of bladder cancer.However,the molecular mechanism of benzidine-induced malignancy is still insufficiently interpreted.Epithelial-mesenchymal transition(EMT)is a crucial pathophysiological process in embryonic development as well as initiation and development of epithelium-originated malignant tumors.The role of extracellular regulated protein kinase 5(ERK5)in benzidine-meditated bladder cancer development has not been explored.Curcumin has been proved to have anti-cancer activities,however,few studies have been conducted to detect the interventional activities of curcumin on benzidine-related bladder EMT.The objective of present study was to investigate the mechanism of benzidine induced malignancy in human normal urothelial cells as well as chemical preventive effect of curcumin on this processMethods SV-HUC-1 cells were exposed to different concentrations of benzidine for 4days,then MTT assay was applied to detect cell viability after 4 days culture.Invert microscope was used to screen morphology changes of cells after benzidine treatment.Wound healing assay was used to assess changes in cells' migratory capacity.Changes in invasive ability were measured by transwell assay.Western blot and RT-PCR were applied to detect protein and RNA expression levels of EMT related markers.Immuno?uorescence was performed to detected expression levels of EMT markers.Activation of ERK5 signal pathway and AP-1 pathway were detected by western blot.Specific ERK5 inhibitor and curcumin were performed to detect the influence on benzidine-induced EMT.Results We screened benzidine at concentrations of 0 0.001 0.005 0.01 0.05 and 0.1 ?M and curcumin at 1?M as experimental doses by MTT assay.Benzidine induced morphological change from epithelial to spinal-like mesenchymal shape.SV-HUC-1cells were stretched to longer and thinner shape,part of which generated pseudopodium.Wound healing assay revealed enhanced cell migratory capacity.Invasive ability was reinforced as showed by the results of matrigel invasive assay and the numbers of cells were boosted significantly.Benzidine exposure downregulated protein levels of epithelial markers E-cadherin and ZO-1,upregulated protein levels of mesenchymal markers vimentin,N-cadherin,MMP-2 and MMP-9.m RNA levels of E-cadherin and ZO-1 were decreased while m RNA levels of vimentin and N-cadherin were increased.Benzidine exposure induced activation of ERK5 signal pathway as protein level of p-ERK5 was elevated and total–ERK5 was deceased.Levels of p-c-fos and p-c-jun,memb-ers of AP-1 family,were upregulated after benzidine treatment.ERK5 inhibitor,XMD8-92 inhibits ERK5/AP-1 activity,leads to inversion of benzidine-induced SV-HUC-1 cells' morphological changes.Inhibition of ERK5 restrained benzidine-enhanced migratory ability in Sv-Hu C-1 cells as determined by wound healing assay.Inhibition of ERK5 restrained benzidine-promoted invasive capacity as detected by Transwell invasion assay.Cultivation with ERK5 inhibitor resulted in upregulated protein levels of E-cadherin and ZO-1 and downregulated protein levels of vimentin and N-cadherin.Cultivation with ERK5 inhibitor induced the m RNA levels of E-cadherin and ZO-1 and deduced m RNA levels of vimentin and N-cadherin.Curcumin attenuated benzidine-induced cell morphology changes.Curcumin reversed benzidine-elevated cell migratory capacity.Treatment with benzidine weakened benzidine-enhanced cell invasive ability as detected by Transwell invasive assay.Cultivation with curcumin leads to elevation of expression levels of E-cadherin and ZO-1 as well as decreased expression levels of vimentin and N-cadherin as determined by western blotting.)Cultivation with curcumin upregulated m RNA levels of E-cadherin and ZO-1,meanwhile downregulated vimentin and N-cadherin m RNA levels.Curcumin suppressed ERK5/AP-1 activity.Benzidine-induced increase of p-ERK5,p-c-Fos and p-c-Jun was reversed by curcuminConclusion Benzidine induces EMT in SV-HUC-1 cells.ERK5 positively regulates benzidine-induced EMT.Curcumin inhibits ERK5/AP1 activity,leading to reverse of benzidine-meditated SC-HUC-1,which may be an promising way in cancer treateatmen...