Antitumor Effect And Mechanism Of Metformin On Human Lung Cancer HCC827 Cells

Objective: Metformin is one of the antidiabetic drugs in the world,It is also a novel apoptosis inducer.Recent studies showed that metformin may be associated with reduced incidence and improved prognosis of certain cancers,and a growing body of evidence had confirmed that the drug may have the potential to be an anti-cancer drug.Therefore,our study is to explore the anticancer effect and the possible mechanism of metformin on human lung cancer cell line HCC827.Methods: The effect of different concentrations of metformin(5 mM,10 mM,20 mM,40 mM,80 mM)on proliferation of HCC827 cells was detected by CCK-8 assay at 24 h,48 h and 72 h.DAPI staining was used to detect apoptosis.Mitochondrial membrane potential was detected by JC-1 kit.The apoptosis rate of the cells was detected by flow cytometry quantitative analysis.The level of reactive oxygen(ROS)was detected using the luorescent dye with DCFH-DA and observed under confocal laser scanning microscopy.Dihydroethidium fluorescence probe was used to detect superoxide anion.Lipid oxidation detection kit is used to detect the level of MDA.Real-time PCR was used to detect the level of BAX and BCL-2 mRNA transcription.Western blot was used to detect the expression of protein such us AMPK/mTOR,BAX,BCL2 and p-H2 A.X.Results:1.Effect of metformin on the proliferation of HCC827 cellsWe examined the inhibitory effect of metformine on HCC827 cells growth.Metformin significantly inhibited the growth of the cells in the concentration-dependent and time-dependent manner.2.DAPI staining for apoptosisThe shape of cell in control grough was circle and evenly dyed.However,the cells treated with metformin at different concentrations showed the typical apoptotic features such as smaller cells,condensed chromatin,highly aggregated,marginalized and even apoptotic bodies.3.Mitochondrial membrane potential was detected by JC-1 kitGreen/Red fluorescence ratio in metformin treatment group was higher than that in control group(P<0.01),suggesting that metformine decreased mitochondrial membrane potential.4.The effect of metformin on the apoptosis of HCC827 cells was detected by flow cytometryThe HCC827 cells were collected to detect apoptosis after treated with metformin for 48 h.The results showed that there was a significant increase in the apoptotic rate in the metformin group(P<0.01),and the percentage of early apoptotic cells increased with the increase of the concentration.5.Effect of metformin on ROS and MDA in HCC827 cellsCompared with the control group,The level of ROS and MDA were increased after metformine administration(P<0.05).6.Metformin increased the expression of p-H2 A.X in HCC827 cellsMetformin induced the increase of ROS and cause DNA damage in HCC827 cells.Metformin increased the expression of DNA damage marker p-H2 A.X(P<0.01).7.The effect of metformin on AMPK/mTOR protein pathwayCompared with the control group,metformin up-regulated p-AMPK in HCC827 cells after metformin administration for 48 h(P<0.05),but there was no significant difference in p-mTOR level.8.The effect of metformin on apoptosis related genesCompared with the control group,the expression of BAX was increased and its mRNA expression was also increased.The expression of BCL-2 was decreased and its mRNA expression was decreased after metformin administration for 48 h.Conclusions:1.Metformin inhibits the proliferation of human lung cancer cell lineHCC827 in a time-and dose-dependent manner.2.Metformin can induce apoptosis in HCC827 cells.Metformin-induced apoptosis may be related to the AMPK pathway,up-regulation of BAX/BCL-2expression and induction of ROS production...