Studys On The Anti-Tumor Effect And The Mechanism Of Metformin In Human Endometrial Carcinoma Ishikawa Cells

Objective:To observe the effect of Metformin on expression of REDD1(regulated in development and DNA damage responses-1),p-AKT ser 473,AKT with the phosphorylation level of AKT and REDD1,AKT,mTOR mRNA expressions in human endometrial carcinoma cell lines Ishikawa cell in vitro, and the role of possible REDD1 / AKT /mTOR signaling pathway,to investigate the potential effect of Merformin on migration of human endometrial carcinoma cells, and the role t REDD1 and p-AKT and mTOR have played in this.Methods:Human endometrial carcinoma Ishikawa cells were recoveried, and cultured in a humidifier 5 % CO2 incubator at 370 C. The RPMI 1640 medium contained 10% fetal bovine serum(FBS). Inoculating the cells into 25 cm2 culture bottle with 5 x 105 cells each bottle when the cells confluenced. The medium was changed every 2 days.When 90% confluenced,the Ishikawa cells was inoculated into serum-free RPMI1640 medium for 12 hours, and then carried out the following drug intervention respectively: 1 mmol/l?5 mmol/l?10 mmol/l meiformin intervened for 48 hours, the same volum of serum-free RPMI 1640 culture acts as the control group.After intervention, Ishikawa cells migration was measured by wound healing assay and Transwell assay, then total RNA and protein was extracted. The expression of gene AKT,mTOR and REDD1 was detected by Quantitative Real-time PCR techniques.Expression levels of protein AKT and p-AKT and REDD1 were examined by Western-blot.Results:(1) Metformin inhibited endometrial carcinoma Ishikawa cells migration. With the increase of concentration of metformin, inhibition of cell migration rate has a tendency to increase. At the concentration of 10 mmol/l, metformin has the strongest inhibitory effects of cell migration.The difference was statistically significant(P <0.05).( 2) Metformin can upregulate the expression of REDD1 and inhibite theexpression of mTOR,but has minimal effect on the expression of AKT. Metformin can also down-regulation of AKT phosphorylation. The expression of 1mmol/l ?5mmol/l?10 mmol/l REDD1 mRNA is 2.97 times ?3.25 times?4.87 times than that of control group, respectively, the difference was statistically significant(P <0.05).The expression of 1mmol/l?5mmol/l?10 mmol/l mTOR mRNA is 0.70 times ?0.54 times ? 0.37 times than that of control group, respectively, the difference was statistically significant(P <0.05). The expression of 1mmol/l?5mmol/l?10 mmol/l AKT mRNA is 1.10 times,1.02 times,1.00 times than that of control group, the difference was not statistically significant.( 3) Metformin can increase the expression of REDD1 protein which is in the REDD1 / AKT /mTOR signaling pathway, and inhibit p-AKT protein expression,but has minimal effect on the expression of AKT. The expression of 1mmol/l,5mmol/l,10 mmol/l REDD1 protein is3.05 times ?5.51 times?11.92 times than that of control group, respectively, the difference was statistically significant(P <0.05); The expression of 1mmol/l ?5mmol/l?10 mmol/l p-AKT protein is 0.70 times ?0.51 times?0.20 times than that of control group, respectively.The most significant effect was in the concentration of10 mmol/l of both protein. The difference was statistically significant(P <0.05). AKT phosphorylation can also inhibit by Metformin,the levels of AKT phosphorylation is0.62 times,0.36 times,0.15 times than that of control group. The expression of1mmol/l,5mmol/l,10mmol/l AKT protein is 1.06 times,1.03 times,1.07 times than that of control group, the difference was not statistically significant.Conclusion:1.Metformin inhibited endometrial carcinoma Ishikawa cells migration. With the increase of concentration of metformin, inhibition of cell migration rate has a tendency to increase. At the concentration of 10 mmol/l, metformin has the strongest inhibitory effects of cell migration.2.Metformin upregulated the expression of REDD1, downregulated the expression of mTOR and the levels of AKT phosphorylation. Suggesting that expression of REDD1,mTOR and the levels of AKT phosphorylation may play an important role in the process of human endometrial carcinoma Ishikawa cells migration, and REDD1 / AKT/mTOR signaling pathway is involved in this process.3.Metformin may promote the expression of REDD1/AKT/ mTOR signaling pathway associated protein REDD1 and down-regulation of AKT phosphorylation and also inhibit the associated mTOR mRNA to exert its inhibition effect of the Ishikawa cells migration.