Study On The Effect Of Metformin On EGFR-TKI Resistance In Non-small Cell Lung Cancer At Different Sugar Concentrations

BackgroundThe Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor(EGFR-TKI)is a molecularly targeted drug for the treatment of non-small-cell lung cancer(NSCLC)with EGFR gene variations.NSCLC,but its resistance gradually appears as the duration of drug usage increases.In addition to its traditional hypoglycemic effect,metformin has been found to inhibit the development of different malignancies.In recent years,more and more studies have shown that metformin can also combine with different antitumor drugs to exert anti-tumor effects and delay the development of drug resistance in the latter,but the strength of its effects is still controversial.The group’s previous study found that the difference of glucose concentration in the environment may be the reason for the strength of the antitumor effect of metformin.It is hypothesized that the glucose level of NSCLC patients may influence the strength of the synergistic effect of metformin with EGFR-TKI drugs as well as the development of drug resistance in the latter.ObjectiveTo research the effects of different glucose levels of metformin on the growth and glucose metabolism of EGFR-TKI-resistant cells in NSCLC,and to further discuss the possible mechanism of delaying EGFR-TKI resistance,and to investigate new therapeutic ideas for the future development of obtained EGFR-TKI resistance in NSCLC patients.Methods1.Establishment and activity assay of each cell line under different glucose concentrations(1)In this experiment,PC-9 cells(EGFR exon 19 deletion mutation,EGFR-TKI sensitive cell line)were used to establish gefitinib resistant cell lines named PC9Ge/L,PC9Ge/M and PC9Ge/H by increasing gefitinib concentrations(0.01μM-0.02μM)at low,medium and high glucose concentrations,respectively.These three cell lines were also given2.5mM and 5mM(the concentrations derived by the group)metforminib and named PC9(G+2.5M)/L,PC9(G+5M)/L;PC9(G+2.5M)/M,PC9(G+5M)/M;PC9(G+2.5M)/H,PC9(G+5M)/H.(2)The growth status of each group of cells was observed using an inverted phase contrast microscope.(3)The CCK-8 method was used to detect the percentage of cellular activity of each group of cells under the effect of different gefitinib concentrations,and the inhibition curves were plotted to calculate the resistance index of each group of cells to gefitinib.(4)Flow cytometry to detect the apoptosis and cycle distribution of each group of cells.2.Assay of gluconeogenesis in each group of cells(1)The concentration of ATP in each group of cells was measured using the ATP assay kit.(2)The relative mRNA expression of key enzymes in the process of gluconeogenesis and the expression level of PKM2 protein in each group of cells were measured using qPCR assay and Western-Blot method.(3)The lactate content of each group of cells was measured using the lactate assay kit.3.Detection of IGF-1R signalling pathway in each group of cells The relative mRNA expression levels of IGF-1R and AKT and the protein expression levels of p-IGF-1R,IGF-1R,p-AKT and AKT in each group of cells were detected using qPCR assay and Western-Blot method.Results1.Establishment and activity assay of each cell line at different sugar concentrations(1)As observed by inverted phase contrast microscopy,the lower the sugar concentration of the medium,the more significant the induction of drug resistance in PC-9 cells(sensitive strain)while adding metformin changed their cell morphology and inhibited their growth rate,and the higher the metformin concentration,the slower their growth rate.(2)The inhibition curves and resistance index showed that metformin delayed cell resistance in low and medium sugar cultures,and the lower the sugar concentration and the higher the metformin concentration,the more significant this effect was.The effect of metformin on EGFR-TKI resistance was not obvious in high glucose culture.(3)In low glucose culture,the apoptosis rate in PC9(G+2.5M)/L and PC9(G+5M)/L groups was significantly higher than that in PC9Ge/L group,and the higher the metformin concentration,the more significant the increase in apoptosis rate.The apoptosis rate of PC9(G+5M)/H group was slightly higher than that of PC9Ge/H group in high glucose culture.The percentage of G0/G1 phase cells in the PC9(G+5M)group was higher than that in the PC9 Ge group in low and medium glucose culture,but there was no obvious difference in the cell cycle distribution between the groups in high glucose culture.2.Detection of the role of sugar metabolism in each group of cells(1)In low glucose culture,the ATP concentration of PC9(G+2.5M)/L and PC9(G+5M)/L groups was lower than that of PC9Ge/L group,and the higher the metformin concentration,the more significant the decrease in ATP concentration.(2)The mRNA expression levels of key enzymes of glycolysis and tricarboxylic acid cycle and PKM2 protein expression in EGFR-TKI-resistant cells affected by metformin were reduced in low glucose culture compared with PC9Ge/L;the mRNA expression levels of hexokinase,phosphofructokinase-1 and citrate synthase were reduced in PC9(G+5M)/M group compared with PC9Ge/M group in medium glucose culture;the mRNA expression levels of hexokinase,phosphofructokinase-1 and citrate synthase were lower in the medium and high sugar groups compared to the PC9Ge/M group;no obvious differences were observed in the high sugar culture.(3)Lactate concentration increased in the PC9(G+2.5M)and PC9(G+5M)groups compared to PC9 Ge when cultured in medium and high glucose,and the increase in lactate concentration was more significant with higher metformin concentration;no obvious difference was observed in low glucose culture.3.Detection of IGF-1R signalling pathway in each group of cells(1)The mRNA expression levels of IGF-1R and AKT in EGFR-TKI-resistant cells affected by metformin were reduced in low and medium glucose culture compared with the PC9Ge/L group;there was no significant difference in high glucose culture.(2)In low glucose culture,the expression of p-IGF-1R,IGF-1R,p-AKT and AKT protein was reduced in both PC9(G+2.5M)/L group and PC9(G+5M)/L group compared to PC9Ge/L,and it became concentration-dependent;in medium glucose culture,compared to PC9Ge/M cell group,the expression of AKT protein in PC9(G+2.5M)/M group The expression of AKT protein was reduced in the PC9(G+2.5M)/M group and IGF-1R protein was reduced in the PC9(G+5M)/M group compared to the PC9Ge/M group;no significant difference was observed in the high glucose culture.Conclusions1.At low ambient glucose concentrations,metformin inhibits the growth and proliferation of PC-9 EGFR-TKI-resistant cells,ATP production,gluconeogenesis and the IGF-1R signalling pathway,thereby enhancing the sensitivity of PC-9 cells to EGFR-TKI and delaying their drug resistance.These effects of metformin diminish with increasing environmental glucose concentrations.2.The mechanism by which metformin delays the development of resistance to EGFRTKI in PC-9 cells at low ambient glucose concentrations may be related to the inhibition of abnormal activation of the IGF-1R pathway.