Metformin Induced Apoptosis Of Nasopharyngeal Carcinoma Cell Line CNE-1 Via Inhibiting IGF-1R

Objective:We investigated the effect of metformin on the proliferation and apoptosis of NPC cell line CNE-1,the effect of metformin on the expression of Insulin-like growth factor 1 receptor(IGF-1R),also observed the effect of Insulin-like growth factor 1(IGF-1,the specific agonist of IGF-1R) on the proliferation of NPC cell line CNE-1 and the expression of IGF-1R,in order to explore the influence of metformin on CNE-1 and the role of IGF-1R in that process.Methods:1.The proliferative activity of the cells in each group(0-80 concentration gradient) was measured by CCK8 assay,the apoptosis of the cells in each group(0-40 concentration gradient) was analyzed by flow cytometry with Annexin-V/PI double staining method,and the expression of bax,bcl-2 mRNA in each group(0-20 concentration gradient) was detected by real-time fluorescence quantitative PCR(qPCR) method,in order to detect the 24-hours influence of different concentration of metformin(0 mmol/L,5mmol/L,10mmol/L,20mmol/L,40mmol/L,80mmol/L) on the proliferation and apoptosis of nasopharyngeal carcinoma cell CNE-1.2.The expression level of IGF-1R mRNA was detected by qPCR,and the expression level of IGF-1R protein was detected by immuno-protein blot(WB),in order to detect the effect of different concentrations of metformin(0mmol/L,5mmol/L,10mmol/L,20mmol/L) on the expression of IGF-1R in nasopharyngeal carcinoma cell line CNE-1 after 24 h treatment.3.In order to elucidate the relationship between the IGF-1R and the proliferation of nasopharyngeal carcinoma cell line CNE-1,and further explain the role of IGF-1R in inhibiting the proliferation of nasopharyngeal carcinoma cells by metformin,the cells were divided into three groups: control group,IGF-1(100ng/ml) group and metformin(20mmol/L) combined with IGF-1(100ng/ml) group.After 24 h treatment,the proliferation activity of the cells was measured by CCK8 method,and theexpression of IGF-1R protein was detected by WB method.Results:1.After treated with metformin(in different concentrations) for 24 h,CCK8 assay showed that the cell proliferation activity was lower than that of the control group(P<0.05 except for the 5mM group);the results of flow cytometry showed that the apoptosis rate of metformin group was higher than that of control group(P<0.05 except for the 5mM group);the results of qPCR suggested that after the treatment of metformin,the expression level of pro-apoptotic gene—bax mRNA was up-regulated while the expression level of anti-apoptotic gene—bcl-2 mRNA was down-regulated as compared with that of control group(P<0.05).2.After treated with metformin(in different concentrations) for 24 h,both the results of qPCR and WB showed that the expression level of IGF-1R was lower than that of the control group(P<0.05).3.The results of CCK8 assay showed that the proliferation activity of CNE-1cells treated with IGF-1 was higher than that of control group(P<0.05),while the proliferation activity of CNE-1 cells treated with metformin and IGF-1 was significantly lower than that of IGF-1 group(P<0.05);the WB results showed that the level of IGF-1R protein in CNE-1 cells treated with IGF-1 was higher than that in control group(P < 0.05),while the expression level of IGF-1R protein in the combined group was lower than that in the IGF-1 group(P<0.05).These results suggest that metformin can reverse the cell-stimulating effect of IGF-1.Conclusion:Metformin can inhibit the proliferation of nasopharyngeal carcinoma cell line CNE-1 and induce its apoptosis,which is related to the down-regulation of IGF-1R.