Cell Injury Of Cx43 Protein Expression And It’s Mechanism Regulated By Oxidative Stress Of As₂O₃ In Bladder Cancer Cells

Objectives:To investigate the effect of As₂O₃ on human bladder cancer cells injury through oxidative stress and discuss its possible molecular mechanism.To discuss its possible molecular mechanisms,we selected the 5637 cell line to study the effects of Cx43 on As₂O₃ and oxidative stress on cells in vitro.Methods:5637 cells were cultured in 5%CO₂ medium with 5%fetal bovine serum and 1%penicillin/streptomycin in RPMI1640 medium at 37°C.The cells are epithelial-like adherent growth and are passaged every 2-3 days.Cytotoxicity was observed by detecting the amount of dehydrogenase released to the outside of the cell.5637 cells were seeded into 96-well plates.After the stimulation,the supernatant was collected,incubated with the same volume of CCK-8 buffer for 1 hour at room temperature,and the OD value of each well（detection wavelength:450 nm）was measured by a microplate reader.Record the results and plot the time curve and the measurement curve.After treatment,the cells are differentiated between living cells and dead cells using different fluorescent colors under a fluorescence microscope.Living cells are capable of hydrolyzing calcein AM by intracellular esterase to produce green fluorescence.Instead,PI enters the cell through the damaged cell membrane of dead cells and interacts with the nucleic acid,emitting red fluorescence.The images were acquired by Olympus’CCD camera.The steps of protein extraction and Western blot analysis are described in the previous literature.The extracted Cx43protein tissue was loaded onto a 10%SDS-polyacrylamide gel and electrophoretically transferred to a PVDF membrane.Then,by blocking,primary antibody and secondary antibody incubation,the chemiluminescence reagent was developed after washing the secondary antibody,and the gel analysis system scanned each band of protein,and the image analysis software was used to determine the gray value of the band.--actin was used as an internal reference analysis.Results:（1）As₂O₃ significantly inhibited the growth of human bladder cancer 5637cells,as indicated by the increased PI-positive red dead cells and reduced calcein-positive living cells.（2）As₂O₃ promoted the expression of Cx43 on human bladder cancer 5637 cells;Cx43 inhibitor could reduce the injury of As₂O₃ on human bladder cancer 5637 cells.（3）As₂O₃ can highly express Cx43 in bladder cancer cells through oxidative stress,and the high expression of Cx43 can be reversed by using antioxidants.Conclusions:As₂O₃ can induce cell damage through high expression of Cx43 in bladder cancer cells through oxidative stress.The application of As₂O₃ may become a new development direction of bladder perfusion therapy.