EGFR tyrosine kinase inhibitor HS-10182 increases radiation sensitivity in non-small cell lung cancers with EGFR T790M mutation

摘要

Objective:To investigate the potential of HS-10182,a second-generation epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI),as a radiosensitizer in non-small cell lung cancer(NSCLC).Methods:Two cell lines of NSCLCs,A549 that possesses wild-type(WT)EGFRs and H1975 that possesses EGFR L858R/T790M double mutations,were treated with HS-10182 at various concentrations,and cell viabilities were determined using the MTS assay.The cells were tested by clonogenic survival assays to identify the radiosensitivity of both groups.Western blot was performed to analyze the expression of phosphorylated EGFR,AKT,DNA-dependent protein kinase,and catalytic subunit(DNA-PKcs)proteins.Immunofluorescence analyses were performed to examine the formation and changes in nuclearγ-H2AX foci.Cell apoptosis was examined by flow cytometry and Western blots for cleaved caspase-3,-8,-9,and cleaved poly ADP-ribose polymerase(PARP).Furthermore,we established xenograft models in mice and the effects of different treatments on tumor growth were then assessed.Results:Clonogenic survival assays revealed that HS-10182 significantly enhanced the radiosensitivity of H1975 cells but not A549cells[dose enhancement ratios(DERs)=2.36(P0.05)].Western blot results showed that HS-10182 increased the levels of cleaved caspase-3,-8,-9,and cleaved PARP in H1975 cells but not in A549 cells.In addition,flow cytometry analysis showed that HS-10182 enhanced irradiation-induced apoptosis in H1975.Immunofluorescence results found that HS-10182increased the average number ofγ-H2AX foci after irradiation in H1975 cells,but not in A549 cells.Combined radiation and HS-10182 treatment increased the expression of DNA-PKcs but this increase was more significant in H1975 cells than in A549 cells.Moreover,HS-10182 suppressed the increased expression of Rad50 in H1975 cells in response to irradiation.In vivo experiments found that the combined therapy significantly inhibited tumor growth.Conclusions:HS-10182 enhances the radiosensitivity of H1975 cells which is possibly because that HS-10182 could enhance irradiation-induced apoptosis,increase irradiation-induced DNA damage,and cause a delay in DNA damage repair.Our findings suggest that radiotherapy combined HS-10182 is a novel treatment for lung cancer cells which have acquired the T790M mutation.HS-10182 could be brought to the clinic as a radiosensitizer in NSCLCs with the EGFR T790M mutation.