Effect About Metformin Combined With Pemetrexed On Proliferation,apoptosis And Expression Of Mcl-1 And Noxa In Human Lung Adenocarcinoma A549 Cells

Objective:To invesigate the effects of metformin combined pemetrexed on proliferation,apoptosis and expression of Mcl-1 and Noxa in human lung adenocarcinoma A549 cells in vitro and explored possible mechanism of this effection.Methods:（1）The A549 cells were treated with different doses of metformin（0,2.5,5,10,20,40mmol/L）and pemetrexed（0,0.5,1,2,4,8μmol/L）for48h,CCK8 assay to assess the antiproliferative of the differently treated A549cell lines and detect the half maximal inhibitory concentration（IC50）of metformin（8.759mmol/L）and pemetrexed（1.426μmol/L）,which were used for subsequent experiments;（2）The A549 cells were treated with metformin at IC50,pemetrexed at IC50,and metformin combined pemetrexed by the CCK8assay to assess the antiproliferative of the differently treated A549 cell lines by（24h,36h,48h）;（3）The apoptosis of A549 cells and cell cycle distributions were detected by the flow cytometry;（4）The protein expressions of Mcl-1 and Noxa were detected using Western blot;（5）Linear correlation analysis was used to analyze the correlation between Mcl-1 and Noxa.Results:（1）CCK8 assay to assess the antiproliferative of the differently treated A549 cell lines and detect the proliferation of A549 cells was inhibited by metformin and pemetrexed monotherapy in a dose-dependent manner compared with the control groups（P<0.05）;（2）CCK8 assay to assess the antiproliferative of the differently treated A549 cell lines and detect the combination of metformin and pemetrexed induced a higher percentage of cell proliferation than that of metforminandpemetrexedusedaloneinatime-dependent manner（P<0.05）;（3）The apoptosis rate of A549 cells was detected by the flow cytometry showed that apoptosis rate of metformin combining pemetrexed（30.60±0.36%）wassignificantlyhigherthanthatof metformin（13.32±0.32%）andpemetrexed（12.90±0.13%）group（P<0.05）;（4）The cell cycle distributions was detected by the flow cytometry showed that the G₀/G₁ phase of metformin combining pemetrexed（81.60±0.64%）was significantly higher than that of metformin（66.22±0.54%）and pemetrexed（63.96±1.33%）group（P<0.05）;（5）Western Blot showed that metformin up-regulate the expression of Noxa（0.309±0.041）,and down-regulate the expression of Mcl-1（0.213±0.029）,While pemetrexed could up-regulate the expression of Noxa（0.283±0.068）and down-regulate the expression of Mcl-1（0.349±0.027）;The combination of metformin and pemetrexed up regularly the expression of Noxa（0.569±0.042）,down regularly the expression of Mcl-1（0.087±0.007）compared with metformin and pemetrexed monotherapy.（6）The expression level of Mcl-1 was negatively correlated with the Noxa expression level（P<0.01）.Conclusion:（1）Metformin and pemetrexed could inhibit the proliferation of lung cancer A549 cells in a certain concentration range.The combined inhibiting effect of metformin and pemetrexed on lung cancer A549 cells was better than that of single drug.（2）Metformin and pemetrexed can induce the apoptosis of lung cancer A549 cells,and the combination of the two drugs can induce the apoptosis of lung cancer A549 cells better than single drug.（3）The effect of metformin combined with pemetrexed on proliferation and apoptosis of A549 human lung cancer cells may be related to down-regulation of Mcl-1 protein,up-regulation of Noxa protein expression.