Study On The Effect And Mechanism Of Glimepiride-Metformin Adduct On Breast Cancer And Prostate Cancer

PurposeGlimepiride-metformin adduct(GMA)is a novel adduct synthesized by glimepiride,metformin and water at a molar molecular mass ratio of 1:1: 1.We examined the effects of GMA on breast cancer and prostate cancer,and preliminarily explored its mechanism of action.MethodsMTT assay,colony formation assay,wound healing assay,immunohistochemistry(IHC),real-time quantitative PCR(RT-qPCR)and Western blot and other techniques were used to study the effects of GMA on breast cancer and prostate cancer,and preliminarily discuss its mechanism of action.The assayal methods are as follows:1.GMA was synthesized by chemical methods.2.Infrared spectroscopy was used to detect the characteristic functional group of glimepiride,metformin hydrochloride,the mixture glimepiride and metformin hydrochloride,and GMA.3.MTT assay was used to detect the effect of GMA on the cellviability of human breast cancer cell lines CAL-148,MDA-MB-453,MDA-MB-231,MCF-7 cells and human breast epithelial cells HBL-100,human prostate cancer cell lines C4-2,PC-3 cells and human normal prostate epithelial cells RWPE-1.4.Colony formation assay was used to detect the effect of GMA on the proliferation of CAL-148,MDA-MB-453,C4-2 and PC-3 cells.5.Wound healing assay was used to detect the effect of GMA on the migration ability of CAL-148,MDA-MB-453,C4-2 and PC-3 cells.6.The orthotopic implantation model was established to observe the inhibitory effect of GMA on tumor growth.7.The expression of Ki67 and AMPK were detected by immunohistochemistry.8.Flow cytometry was used to detect cell cycle and apoptosis of breast cancer and prostate cancer cells.9.RT-qPCR and Western blot were used to detect the levels of AMPK,p53,p21,cyclin D1,CDK4 expression.Results1.Infrared spectroscopy showed that the infrared spectroscopy of GMA was inconsistent with the infrared spectroscopy of equimolar mixture of glimepiride and metformin hydrochloride,indicating that GMA was not simply a physical mixture of glimepiride and metformin hydrochloride but a new adduct.2.GMA inhibited the proliferation and migration of prostate cancer in vitro.3.GMA inhibited the growth of breast cancer in vitro,and inhibited the growth of CAL-148 xenografts in vivo.4.Flow cytometry analysis showed that GMA induced the cycle arrest of breast cancer cells,blocking the cells in G1/S phase,and also induced apoptosis of breast cancer cells;GMA induced the cycle arrest of prostate cancer cells,blocking the cells in the G1/S phase,and also induced the apoptosis of prostate cancer cells.5.Immunohistochemical results showed that GMA up-regulated the expression of AMPK and reduced the expression of Ki67 in CAL-148 xenografts.6.RT-qPCR and Western blot analysis showed that GMA up-regulated the expression of AMPK,p53 and p21,and down-regulated the expression of cyclin D1 and CDK4.ConclusionGMA can inhibit the proliferation of breast cancer cells and prostate cancer cells,and its mechanism of GMA suppressing breast cancer cells may be related to the activation of AMPK,up-regulation of p53,p21,and down-regulation of cyclin D1,CDK4 protein expression levels.