The Mechanism By Which Metformin Inhibits Proliferation And Migration Of Ovarian Cancer Cells

Objective: This study is to investigate the mechanism by which the novel anti-tumor drug metformin inhibits proliferation and migration of ovarian cancer cells.Through the study,we expect to identifiy the targeting pathway and molecules of metformin in ovarian cancer cells,and lay the foundation for application of metformin for ovarian cencer targeted therapy.Methods:(1)Ovarian cancer HO8910/SKOV3 cells were treated with metformin at various concentrations.CCK8 and Ed U kits were used to detect proliferation and DNA replication of ovarian cancer cells.Transwell method was used to detect migration of ovarian cancer cells.In addition,Western blot was used to detect the changes of cell proliferation and migration related proteins with different concentrations of metformin.(2)The changes of LSD1 m RNA level in ovarian cancer cell HO8910/SKOV3 treated with metformin were detected by q RT-PCR to determine whether metformin affects transcription of LSD1.In addition,ovarian cancer HO8910/SKOV3 cells were treated with metformin and the effect of metformin on LSD1 protein level was detected by Western blot.The amount of the methylated H3K4 protein was determined for evaluation of the LSD1 demethylation activity.(3)To determine whether metformin regulates the PI3K/AKT pathway in ovarian cancer cells,the effect of metformin on phosphorylation of AKT,combined with stimulation with EGF or inhibition with a PI3 K inhibitor,was detected by Western blot.(4)LSD1 knockdown cell lines HO8910-sh LSD1 and SKOV3-sh LSD1 were established using the lentiviral sh RNA expression system.The effect of the LSD1 knockdown with or without treatment of metformain on proliferation and DNA replication was detected using the CCK8 and Ed U kits,respectively.The effect on cell migration was detected using the Transwell assay.In addition,Western blot was used to detect expression of the proteins related to cell proliferation and migration.Results:(1)The data from the detection by the CCK8 or Ed U kit demonstrated that metformin inhibited the proliferation and DNA replication of ovarian cancer HO8910/SKOV3 cells.The Transwell assay showed that metformin significantly inhibited the migration of ovarian cancer cells.(2)The results of q RT-PCR showed that metformin had no significant effect on the LSD1 m RNA level in the HO8910/SKOV3 ovarian cancer cells.However,western blot showed that treatment with metformin caused a significant decrease in the LSD1 protein level in the ovarian cancer cells.The methylated H3K4me2,an LSD1 substrate,was increased upon treatment with metformin,indicating that metformin inhibits the demethylation activity of LSD1.(3)Metformin inhibited the PI3K/AKT signaling pathway in the ovarian cancer cells.Furthermore,metformin inhibited the EGF-indued activation of the PI3K/AKT pathway and enhanced the inhibitory effect of the PI3 K inhibitor on the phosphorylation of AKT,suggesting that metformin specifically attack the PI3K/AKT signaling pathway.(4)Knockdown of LSD1 in ovarian cancer cell lines HO8910 and SKOV3 by the LSD1 sh RNAs inhibited cell proliferation and migration.Treatment with metformin in the knockdown cell lines further increased the inhibitory effect of LSD1 knockdown on proliferation and migration.Conclusions:(1)Metformin inhibits proliferation and migration of ovarian cancer HO8910/SKOV3 cells.(2)Metformin had no significant effect on the LSD1 m RNA level in ovarian cancer HO8910/SKOV3 cells.Metformin down-regulates the LSD1 protein level,and the amount of H3K4me2,a methylated protein that is a specific substrate of LSD1.(3)Metformin inhibits the PI3K/AKT pathway,eliminates the stimulatory effect of EGF on the PI3K/AKt,and enhances the inhibitory effect of a PI3 K inhibitor on PI3K/AKT pathway.Metformin may down-regulate the LSD1 protein level through inhibition of the PI3K/AKT pathway.(4)Metformin enhances the inhibitory effect of LSD1 knockdown on ovarian cell proliferation and migration.