Synergistic Efficacy Of Metformin In Combination With Osimertinib In PC9/GR Cells.

Objective:Non small cell lung cancer is one of the leading causes of cancer-related death worldwide.Platinum based chemotherapy regimens provide an effective treatment for patients with non-small cell lung cancer and metastatic cancer,but most patients will inevitably develop disease progression.The discovery of mutant EGFR and the application of the new generation of EGFR-TKI provides an effective new method for the treatment of non-small cell lung cancer,and usher in a new era of personalized treatment of lung cancer.Osimertinib as the representative of the third generation of EGFR-TKI for the generation of drug resistance research and development,in the clinical achieved good results.Metformin is a widely used biguanides,clinical studies have found that it can reduce the incidence of cancer in patients with diabetes and improve the prognosis,and studies have shown that metformin combined with EGFR-TKI can produce synergistic anti-tumor effects.The mechanism of the anti-tumor effect of metformin may be related to the activation of AMPK/mTOR pathway,but the mechanism of metformin is complex,so the mechanism is not clear explanation,still need further research,this experiment aims to study the growth inhibiting effect of osimertinib combined with metformin on gefitinib acquired resistance of non-small cell lung cancer PC9/GR cells and its possible mechanism.Methods:PC9/GR cells were cultured in vitro and divided into osimertinib treatment group and metformin treatment group,osimertinib and metformin in combination treatment group.PC9/GR cells were detected by the(MTT)method in different treatment groups after the treatment of the half inhibitory concentration(IC50)as well as the combination index(CI)value;The changes of apoptosis induced by osimertinib and metformin alone or in combination were detected by the fluorescent microscope and flow cytometry;The change of the p-AMPK and the p-P70S6K protein level were detected by Western blot,and the gray value was analyzed by using ImageJ software.Results:The MTT assay indicated that:IC50 values of osimertinib and metformin on PC9/GR cells were(12.0± 5.53)nmol/L and(4.57±1.28)mmol/L,the combination therapy with osimertinib and metformin shows an synergic effect on proliferative inhibition compared to osimertinib or metformin alone therapy(P<0.05),which shows an synergic effect(CI<1).Fluorescence microscope observed that:compared with the control group,the apoptotic cells increased in both osimertinib and metformin treatment group,and the apoptotic cells increased more obviously in the osimertinib and metformin in combination treatment group.Flow cytometry showed that apoptosis rate of osimertinib treatment group and metformin treatment group was(7.88±1.21)%and(8.38±1.23)%,and in the combination treatment group,the apoptosis rate was(22.72±3.44)%;The results of Western blot showed that:compared with the control group,p-AMPK was significantly up-regulated in both metformin treatment group and two drug combination treatment group;the expression level of p-P70S6K was down-regulated in the osimertinib and metformin mono therapy treatment group,and was more obvious in the combination treatment group compared with the single drug group,the differences were statistically significant(P<0.05).Conclusion:The combination of osimertinib and metformin can significantly inhibit cell proliferation and promote cell apoptosis,which has a synergistic effect.The mechanism may be related to the upregulation of p-AMPK protein and downregulation of p-P70S6K protein in AMPK/mTOR pathway.