Co-silencing Of ERCC1 And BRCA2 Genes Effectively Enhance Cytotoxicity Of Cisplatin To Drug Resistant Lung Cancer Cells

ObjectiveTo investigate the changes of cytotoxicity of cisplatin to drug-resistant lung cancer cell line(A549/DDP)by individually silencing ERCC1 and BRCA2 gene,or co-silencing ERCC1 and BRCA2 genes,and explore the mechanism of cytotoxicity enhance of cisplatin to A549/DDP cells through silencings of the genes.MethodssiRNAs(ERCC1-siRNA and BRCA2-siRNA)targeting ERCC1 and BRCA2 genes were transfected into A549/DDP cells in vitro by using lipofectamine technique.Western blot was used to detect the expression of ERCC1 and BRCA2 protein to confirm transfection efficacy.FANCD2 monoubiquitination and BRCA1 and RAD51 proteins expression following cisplatin treatment were determined in A549/DDP cells and A549 cells before and after siRNA transfections.The CCK-8 and Annexin V/PI flow cytometry assays were carried out to measure the cell proliferation and apoptosis induced by cisplatin,respectively.Immunofluorescence stain was used to detect the imformation of FANCD2 and RAD51 nuclear foci following cisplatin treatment before and after siRNA transfection.Results(1)The expression of ERCC1 and BRCA2 proteins were significantly decreased in A549/DDP cells after transfection of ERCC1-siRNA and BRCA2-siRNA(all P<0.05),indicating that ERCC1 and BRCA2 genes were effectively silenced.(2)In A549/DDP cells,FANCD2 monoubiquitination levels and BRCA1 and RAD51 proteins expressions were increased with the increasing of cisplatin concentration,whereas similar results were not observed in A549 cells.The data suggest that the fanconi(FA)pathway and homologous recombination repair(HRR)pathway were involved in resistant mechanism of A549/DDP to ciplatin.(3)FANCD2 monoubiquitination levels and the formation of FANCD2 nuclear foci induced by cisplatin were significantly decreased in A549/DDP cells post silencing of ERCC1(all P<0.05).BRCA1 and RAD51 proteins expression and RAD51 nuclear foci were significantly reduced after BRCA2 gene silence(all P<0.05),indicating that DNA repair functions by the FA and HRR pathways were supressed.(4)The CCK-8 and the flow cytometry assays showed that individual silencing of ERCC1 or BRCA2 gene inhibited proliferation and promoted apoptosis induced by cisplatin in A549/DDP cells(all P<0.05).Co-silencing of the two genes further enhanced the cytotoxicity of cisplatin to A549/DDP cells compared to silencing of ERCC1 or BRCA2 gene alone(all P<0.05).ConclusionSilencing of ERCC1 and BRCA2 genes by siRNA transfection can increase cytotoxicity of cisplatin to cisplatin-resistant lung cancer cells(A549/DDP).Moreover,co-silencing of the two genes further enhanced killing effect of cisplatin to A549/DDP cells,which were accompanied with downregulations of FANCD2 monoubiquitination levels,BRCA1 and RAD51 expressions and RAD51 foci formation.The results indicate that depletion of ERCC1 synergize with BRCA2 silencing to reverse cisplatin resistance in A549/DDP by inhibiting DNA repair ability of the FA and the HRR pathways.