The Anti-NSCLC Activity And Mechanism Of Shikonin Combined With EGFR-TKIs

Objective:Epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs)have become the first-line therapy drugs for non-small cell lung cancer(NSCLC)patients with EGFR-activating mutations.However,NSCLC patients who are initially response to EGFR-TKIs will develop acquired resistance about 1 year.Therefore,potential chemosensitizers are required to sensitize NSCLC cells to EGFR-TKIs treatment.In this study,we plan to explore the anti-NSCLC effect by combining shikonin with EGFR-TKIs and uncover the potential mechanism.Methods:(1)SRB assay was used to detect cell proliferation activity.(2)Long term anti-proliferation activity of NSCLC cells were determined by colony formation assay.(3)PI staining combined with flow cytometric analysis was used to explore apoptosis.(4)Mitochondrial membrane depolarization was investigated by JC-1 staining.(5)The expression of proteins was detected by western blot.(6)Human lung cancer A549 xenografts were established for detecting antitumor activity in vivo.Results:(1)Anti-NSCLC effect of shikonin combined with gefitinib in vitro and in vivoShikonin sensitized the antiproliferative effect of gefitinib in NSCLC cells.The increased apoptosis induced by shikonin plus gefitinib was accompanied by mitochondrial depolarization,Mel-1 degradation,Caspase-3 activation and PARP cleavage in NSCLC cells.Moreover,the enhanced anti-NSCLC efficacy of shikonin combined with gefitinib were validated in human lung cancer A549 xenografts nude mice model.NAC was demonstrated to protect cells from apoptosis induced by shikonin plus gefitinib.Shikonin increased the expression levels of p-eif2a(Ser 51)and ATF-4 in the absence of gefitinib,and this effect was markedly enhanced in the presence of gefitinib.Thus,the data of the present study suggested that ROS-mediated ER stress was involved in shikonin plus gefitinib-induced apoptosis in NSCLC cells.(2)Anti-NSCLC effect of shikonin combined with erlotinib in vitro and in vivoShikonin sensitized the antiproliferative effect of erlotinib in NSCLC cells.The increased apoptosis induced by shikonin plus erlotinib was accompanied by mitochondrial depolarization,Mel-1 degradation,Caspase-3 activation and PARP cleavage in NSCLC cells.Moreover,the enhanced anti-NSCLC efficacy of shikonin combined with erlotinib were validated in human lung cancer A549 xenografts nude mice model.NAC was demonstrated to protect cells from apoptosis induced by shikonin plus erlotinib.Shikonin increased the expression levels of p-eif2a(Ser 51)and ATF-4 in the absence of erlotinib,and this efifect was markedly enhanced in the presence of erlotinib.Thus,the data of the present study suggested that ROS-mediated ER stress was involved in shikonin plus erlotinib-induced apoptosis in NSCLC cells.(3)Anti-NSCLC effect of shikonin combined with AZD9291 in vitroShikonin sensitized the antiproliferative effect of AZD9291 in NSCLC cells.The increased apoptosis induced by shikonin plus AZD9291 was accompanied by Mcl-1 degradation,Caspase-3 activation and PARP cleavage in NSCLC cells.Moreover,the enhanced anti-NSCLC efficacy of shikonin combined with AZD9291 were validated in clinical patient samples.NAC and GSH were demonstrated to protect cells from anti-proliferation and apoptosis induced by shikonin plus AZD9291.Shikonin increased the expression levels of p-eif2?(Ser 51)and ATF-4 in the absence of AZD9291,and this effect was markedly enhanced in the presence of AZD9291.Thus,the data of the present study suggested that ROS-mediated ER stress was involved in shikonin plus AZD9291-induced apoptosis in NSCLC cells.Conclusion:Shikonin could sensitize the anti-NSCLC efifect of EGFR-TKIs in NSCLC cells.ROS-mediated ER stress was involved in shikonin plus EGFR-TKIs-induced apoptosis in NSCLC cells.Shikonin might be a potential therapeutic candidate for NSCLC patients,and shikonin combining with EGFR-TKIs might be an attractive chemotherapy strategy for NSCLC treatment.