Metformin Promotes Death Receptor Pathway Mediated Apoptosis Of HepG2 Hepatoma Cell Line And Its Mechanism

Background Liver cancer mainly refers to primary liver cancer.The main pathological type is hepatocellular carcinoma,which is a very common malignant tumor in clinic.According to the 2020 edition of global cancer statistics,liver cancer is the sixth most common tumor in the world and the third most common cause of cancer-related deaths.In China,new cases account for about 45%of the world,and related deaths account for about 47%.Metformin is the first-line drug for type II diabetes in clinic.Since the 21st century,people have found that metformin has preventive and therapeutic effects on a variety of tumors,including liver cancer.Objective The occurrence and development of tumor is related to the imbalance of cell apoptosis.Studies have found that death receptors(Fas,TNFR1,DRs)are abnormally expressed on liver cancer cells.This research takes HepG2 hepatoma cell line as the research object to explore whether metformin can pass Fas L/Fas,TNFα/TNFR1 and TRAIL/DRs three exogenous apoptotic pathways promote the apoptosis of hepatoma cells.Methods(1)CCK-8 method was used to detect the changes of cell activity of HepG2 cells treated with different concentrations of metformin at different time;(2)CCK-8 method was used to detect the changes of cell activity of HepG2 cells treated with different concentrations of metformin combined with three pro-apoptotic factors(Fas L,TNFα,TRAIL);(3)The effects of metformin at 10 m M concentration combined with pro-apoptotic factors on apoptosis and caspase-3 activation of HepG2 cells were detected by Annexin V/PI method and caspase-3 kit respectively;(4)Using qPCR to detect the regulatory effect of 10 m M concentration of metformin on death receptor mediated apoptosis pathway related genes in HepG2 cells;(5)Detection of the regulatory effect of different concentrations of metformin on death receptor mediated apoptosis pathway related proteins on HepG2 cells using Western Blot technique.Results(1)HepG2 cells were treated with different concentrations of metformin for 24hours,48 hours,and 72 hours later.There was no significant decrease in cell activity in 1 n M,10 n M,100 n M,1μM,10μM,100μM and 1 m M metformin groups(P>0.05).The cell activity of 10 m M group decreased significantly(P<0.05)and showed a time-dependent(P<0.05).Finally,five concentrations of 1μM,10μM,100μM,1 m M and 10 m M and 48 h of action time were selected as the following experimental conditions.(2)The cell activity in the group of proapoptotic factors only(Fas L:1μg/ml,TNFα:1μg/ml,TRAIL:10ng/ml)and the group of proapoptotic factors combined with different concentrations of metformin were significantly decreased(P<0.05).The cell activity of the combined group is lower than that of the group using proapoptotic factors alone(P<0.05),and this difference will gradually increase with the increase of metformin concentration.(3)Compared with the group using proapoptotic factor alone and the group using 10 m M metformin alone,the apoptosis rate and caspase-3 activity of the combined group of proapoptotic factor and 10 m M metformin were significantly increased(P<0.05).(4)The results of qPCR showed that the mRNA levels of death receptor and ligand genes(m Fas,total Fas,Fas L,DR4,DR5),tumor suppressor p53 gene,mitochondrial pathway Bax gene increased significantly(P<0.05),and the mRNA levels of caspase-8 inhibitor protein FLIP gene,mitochondrial pathway gene(Bcl-2,Bcl-x L)decreased significantly(P<0.05)after treating HepG2 cells with 10 m M metformin for 48hours.(5)Western Blot results show:After treating HepG2 cells with five different concentrations of metformin(1μM,10μM,100μM,1 m M and 10 m M)for 48hours.On the one hand,the expression level of Fas monomer and Fas aggregates protein gradually increased with the increase of metformin concentration,and reached the maximum in the 1 m M group(P<0.05)in the first four concentration groups.However,the expression level of Fas monomer in 10 m M group was not significantly higher than that in 1 m M group,while the expression level of Fas aggregates was reduced to no statistical difference from that in control group.On the other hand,the expression level of death receptor protein(DR4,DR5),transcription factor p53,mitochondrial pathway protein Bax increased gradually;the expression level of caspase-8 inhibitor protein FLIP_L,mitochondrial pathway protein(Bcl-2,Bcl-x L),p Ser536-NF-κB p65 in the NF-κB pathway decreased gradually.Conclusion On the one hand,metformin can promote the transcription and protein expression of Fas,DR4,DR5 and Bax genes,down-regulate the transcription and protein expression of FLIP,Bcl-2 and Bcl-x L genes,and then activate caspase-3 to induce apoptosis,which may be related to the up-regulation of p53 mRNA and protein expression level by metformin.On the other hand,metformin may inhibit cell activity by reducing the phosphorylation level of NF-κB p65 at Ser536,and ultimately promote the death receptor pathway to mediate apoptosis of HepG2 hepatoma cell lines.