Role Comparison Of GST Isoenzymes In Cisplatin-resistence And Migration Of Tumor Cells

Most chemotherapy failures involving the invasion and metastasis of cancers are related to drug resistance,and up-regulation of GST isozymes is associated with drug-resistance in many cancer cells.EDEA?Ethanediame bisethacrynic amide?synthesized was an inhibitor of GST with the selectivity as GSTM>GSTA>GSTP.Previous work found that it sensitized drug-resistant cancer cells SKOV3/DDP and the tumor-loading mice,and also A549/DDP cells but was null in the tumor-loading mice,while no sensitization for SGC7901/DDP cells.These results indicated different drug-resistant cancer cells had different expression set of GST isozymes.The designment of inhibitors specific for different GST isozymes responsible for cisplatin-resistance in different types of cancer cells is challenged by the technology and the cost.The identification of GST isozyme?s?essential for drug-resistance in common solid cancer cells as the traget is important in practice.Three cisplatin-resistant solid cancer cell lines A549/DDP,SKOV3/DDP,and SGC7901/DDP were selected.The expression of GST isoenzymes was detected by western-blot,and their activity was differentiated by the inhibition of EDEA;Potent siRNA specific for GSTP1,GSTM2 and GSTA1 were screened and transfected in A549/DDP,SKOV3/DDP and SGC7901/DDP to detect IC₅₀0 of cisplatin by CCK8,followed by the flow cytometry and Hoechst 33343 staining to detect the apoptosis.Cytotoxicity of HNE after silencing of GSTP1,GSTM2 and GSTA1,or ihibition by EDEA was detected by CCK8 to infer the related mechanism.In addition,wound healing assay and transwell assay were used to detected the migration of A549/DDP transfected with siRNA of GST isoenzymes or inhibition by EDEA,and proteins related was detected by western blot.GSTP1,GSTA1 were down-regulated while GSTM2 up-regulated in A549/DDP,GSTP1,GSTM2 and GSTA1 were decreased in SGC7901/DDP while increased in SKOV3/DDP,and GST activity was enhanced in cisplatin-resisitant cells.The activity of GSTM mostly susceptible to inhibition of EDEA was highest in SKOV3/DDP while lower in SGC7901/DDP and the lowest in A549/DDP.The activity of GSTA less susceptible to inhibition of EDEA was highestly expressed in A549/DDP.In A549/DDP,the silence of GSTA1 leaded to 5-fold decrease whereas that of GSTM2 resulted 2-fold decrease in IC50 of cisplatin.In SKOV3/DDP,the silences of GSTM2 and GSTA1 leaded to 2-fold decrease in IC50 of cisplatin.In SGC7901/DDP,the silence of GSTA1made 6-fold decrease and that of GSTP1 resulted 4-fold decrease and while of GSTM2 gave 1.3-fold reduction in cisplatin IC50.Flow cytometry and Hoechst 33343 staining found that the apoptosis were significant increased after the silences of GST isozymes combining with DDP.The silences of GST isozymes or inhibition of EDEA decreased IC₅₀ of HNE.Wound healing assay and transwell assay showed either EDEA inhibiton or the silence of GSTM2 increased the migration of A549/DDP in comparison with the control groups,while no effects with the silence GSTP1 or GSTA1.Western blot showed that the mechanism was probably related to activiation of EGFR and thus AKT/PI3K signal pathway.In conclusion,the expression set of GST isoenzymes related to drug-resistance varied with different cisplatin-resistant solid cancer cell lines.The experiment results with EDEA inhibiton and silences of GST isoenzymes suggested that GSTA1 predominated over GSTM and GSTP in cisplatin-resistance of solid cancer cell lines.GSTA1 has the promise as a target for counteract drug-resistance of common cancer cells.The blocking of GSTM2 resulted the enhanced migration of A549/DPP cells,devaluating GSTM as a target for sensitization of cancer cells to drugs.