Synergistic Effect And Mechanisms Of AZD9291 Combined With STAT3 Inhibitors On H1975 Lung Cancer Cell

Objective:Now epidermal growth factor receptor tyrosine kinase inhibitor?EGFR-TKI?is one of rhe first-line treatment of advanced non-small cell lung cancer?NSCLC?and it has been developed to the third generation for clinical use.Currently AZD9291?osimertinib;Tagrisso?is the only FDA-approved third-generation inhibitor,which was mainly used in patients with T790M mutation.Although the effects was significant,resistence often occurred in most patients who had been treated after 9-13 months.Various causes of drug resistance have been elucidated,but there might be other unknown mechanisms.Some studies indicated that STAT3 protein was overexpressed in various of tumor tissues.In this study,we explored the changes of pathways after T790M mutant H1975 lung cancer cells treated with AZD9291,and the effects or mechanisms of STAT3 inhibitor combined with AZD9291.Methods:?1?H1975 cells were co-cultured with various concentrations of AZD9291 for 72hours,then the expression levels of STAT3,p-STAT3,ERK,p-ERT,AKT,p-AKT proteins were detected by Western-Blot assay.?2?The effects of AZD9291 and/or STAT3 inhibitor on H1975 cells were detected by MTT assay.The combination index?CI?was used to assess the anti-proliferative effects.?3?H1975 cells were co-cultured with either agent alone or combination for 72 hours,then the apoptosis rate and cell cycle were measured by FCM assay.?4?The expression levels of STAT3 and p-STAT3 proteins were detected by Western-Blot assay after H1975 cells were treated with AZD9291 combined with STAT3 inhibitor for 72h.Results:?1?Result showed that AZD9291 decreased the expression levels of p-ERK and p-AKT proteins.However,the expression of p-STAT3 was upregulated.?2?MTT assay showed that the inhibitory effect on the proliferation of H1975 cells was significantly higher than that of AZD9291 monotherapy group and STAT3 monotherapy group?P<0.05?,which showed synergistic effect?CI value<1?.?3?STAT3 inhibitor combined with AZD9291 decreased the expression levels of p-STAT3 proteins compared with AZD9291 alone.?4?Compared with the single drug group,Flow cytometry analysis showed that combination of the two drugs showed stronger induction of apoptosis and the cell cycle was significantly blocked in the G₀/G₁ phase.Conclusions:?1?AZD9291 combined with STAT3 inhibitor have synergistic anti-tumor effects on H1975 cells.?2?The expressions of p-STAT3 decreased by STAT3 inhibitors may be contributed to synergistic anti-tumor effects.