Inhibition Of The Proliferation Of Bladder Cancer Cell By Metformin And The Mechanism Research

Objective: To evaluate the effects of metformin on human bladder cancer cell and its underlying mechanisms in vitro and in vivo.Methods: 1.Cell Counting Kit-8(CCK-8) was used to investigate the effects of metformin on 253 J and T24 cells' growth;2.Flow cytometry was used to evaluate the cell cycle changes after metformin treatment;3.The possible signaling molecules involved in this process were determined by immunoblot analysis of various proteins;4. Xenograft tumor model:T24 cells were implanted subcutaneously into the right axillary fossa of nude mice to set up bladder tumor xenograft model.The metformin-treat group was intra-peritoneally injected metformin at 200mg/kg/day for 18 days and the control group injected intra-peritoneally normal saline only.The mice were monitored daily for any discomfort and weighed every third day to check for tumor growth;5.The tumors excised from nude mice were fixed in 4% paraformaldehyde for 24 hours and paraffin-embedded. Immunohistochemistry stain for Ki67, VEGF and CD31.Results: 1.In 24 h, 48 h, and 72 h, the proliferation of bladder cancer cells was significantly inhibited compared with control group(P <0.01 or P <0.05); 2.Compared with control group, a significantly increase was observed in the percentage of cells in the G0/G1 phases. In parallel, there was a reduction in the percentage of cells in the S phase(P <0.01 or P <0.05); 3.Western blot analysis found that the expression of phosphorylated AMP-activated protein kinase(AMP-activated protein kinase, AMPK) was increased, while the expression of cyclin D1 was decreased in metformin-treated group.(P <0.01 or P <0.05);4.In bladder tumor xenograft model,metformin inhibits the growth of bladder tumor.Body weight and diet consumption were not influenced by metformin.5. The expression of Ki67 ? VEGF and CD31 are decreased in metformin-treat group.Conclusion:1.Metformin can induce G0/G1 cell cycle arrest and inhibit bladder cancer cells proliferation in vitro.2. Metformin inhibits growth and angiogenesis in bladder Tumors in Vivo.These suggesting that metformin may be a potential therapeutic drug for the treatment of human bladder cancer.