The Research Of Ebselen Block And Reverse The Multidrug Resistance Mediated By HIF-1α In A549 Cell Line

Objective:To investigate the mechanism of cisplatin resistance in A549 cell line,the multi-drug resistance blockade by ebselen in A549 cell lines,and the multidrug resistance by ebselen in A549/DDP cells.Investigate the role of hypoxia inducible factor-1 in ebselen inhibition drug resistance and reversal of multidrug resistance caused by cisplatin.Methods:Different concentrations of cisplatin and ebselen were applied to A549cells and A549/DDP cells to determine the cell morphology and biochemical indicators,and to detect the expression level of related proteins and the similarities and differences of multidrug resistance genes.MTT assay was used to determine cell viability and half mortality.Changes in cell morphology were observed using an inverted microscope.Scratches were used as a measure of tumor migration.ROS content was measured by DCFH-DA probe method,glutathione peroxidase activity was detected by GSH-PX kit,apoptosis was detected by hoechst 33342,and fluorescence localization of P-gp and HIF-αwas performed by immunofluorescence labeling method.The expression levels of P-gp,HIF-1α,Akt,PI3K,Bax,Bcl-2,E-cadherin,and Caspase-3 proteins were detected by immunoblotting.In A549 cells,the hypoxic model was created with cobalt dichloride,and estradiol interfered with HIF-1α,each expression of HIF-1αand P-gp proteins in the cells were observed.In A549/DDP cells,the gene level was detected by PCR;HIF-1αwas interfered by small RNA,and the protein expression levels of HIF-1αand P-gp were detected by immunofluorescence.Results:As concentration of cisplatin increased,the damage of A549 cells is aggravated and dose dependency.At the same time,the content of reactive oxygen species in injured A549 cells was significantly increased,cell viability was decreased,and apoptosis appeared in shrinkage and rounding.In addition,cisplatin can increase the expression of P-gp,HIF-1α,Akt,and PI3K in A549 cells,and HIF-1αtranslocates from the cytoplasm to the nucleus;E-cadherin downregulates,the migration rate increases,and the ratio of Bax/Bcl-2 increases,apoptotic cells increased.Cobalt chloride regulated the expression of HIF-1αincrease,but without changing the P-gp level.After estradiol interfered with HIF-1α,the protein levels of HIF-1αand P-gp in A549 cells decreased significantly.When cisplatin was used together with 4mmol·L^-1ebselen,the cell migration rate decreased,the number of apoptotic cells increased,ROS decreased,HIF-1α,P-gp decreased,Caspase-3 increased,and Bax/Bcl-2 ratio increased.E-cadherin increased.Ebselen reduced the reactive oxygen species in A549/DDP cells and decreased the expression of P-gp and HIF-1αproteins.The combination of ebselen and cisplatin upregulates Caspase-3 protein,and the ratio of Bax/Bcl-2 increases greatly.In the A549/DDP cells,the expression of HIF-1αand P-gp decreased significantly after small RNA of HIF-1αinterference;4mmol·L^-1ebselen significantly reduced the ROS content and down-regulated HIF-1,P-gp.The level of protein expression;combined with cisplatin,4mmol·L^-11 ebselen can significantly inhibit cell viability and reduce the concentration of half maximalinhibitory.Conclusion:Cisplatin can lead to the increase of ROS in A549 cells,which leads to the increase of HIF-1αand P-gp,results the drug resistance.The simultaneous use of cisplatin and Ebselen reduces the formation of A549 drug resistance by inducing ROS and HIF-1αin A549 cells,synergizes with the apoptosis induced by cisplatin,and inhibits tumor migration.It can break the active oxygen homeostasis in A549/DDP cells,reduce the stability of HIF-1α,reduce the expression of P-gp,and then reverse its multidrug resistance.