Metformin Inhibits Cisplatin Resistance In Endometrial Cancer And Its Possible Related Mechanisms

Background: Endometrial Carcinoma(EC),which accounts for about 7% of female malignancies,is one of the three major gynecological malignancies.The current treatment for endometrial cancer is mainly surgery,combined with radiotherapy,chemotherapy and endocrine therapy.Most ECs require chemotherapy based on cisplatin combined with radiotherapy as supplementary treatment.Once EC develops resistance,the healing is extremely poor,and the average survival time is less than 12 months.Therefore,looking for drugs and methods to reverse the drug resistance of endometrial cancer is an important means to improve the efficacy of chemotherapy.Metformin has anti-tumor effects.A large number of documents have confirmed that metformin can inhibit the proliferation of endometrial cancer and improve the survival rate.Therefore,we explored whether metformin can reverse the chemotherapy resistance of endometrial cancer.OBJECTIVE:This article uses endometrial cancer cell Ishikawa as a model to establish an endometrial cancer cell Ishikawa cisplatin-resistant strain(Ishikawa/DDP),to explore whether metformin can reverse the cisplatin resistance of endometrial cancer,and whether its mechanism is related to AKT Expression related.Methods:1).Using endometrial cancer cell Ishikawa as a model,Ishikawa cell line was induced and treated with cisplatin concentration of 10μM,20μM,40μM,60μM,80μM,100μM in a gradual and incremental manner.It took 2 months to successfully establish Ishikawa/DDP,CCK-8 method to detect the inhibition rate of endometrial cancer Ishikawa cell line treated with different concentrations of cisplatin,and calculate the IC50 value according to the formula;2).Q-PCR to detect the expression level of AKT1 mRNA in Ishikawa cell line and Ishikawa/DDP;3).After treating the Ishikawa/DDP cell line with the control group(0m M),0.25 m M,0.5m M,1 m M,2 m M,4 m M and 8 m M concentration of metformin for 24 h,48h and 72 h respectively,the cell viability was detected by the CCK-8 method After treating the Ishikawa/DDP cell line with 0.25 m M,0.5 m M metformin for 24 hours,PI flow cytometry was used to detect cell apoptosis and Western-blot to detect the expression of Bcl-2,Bax protein and AKT1.Results:1.Overexpression of AKT in Ishikawa/DDP After Ishikawa/DDP is established,the AKT1 mRNA expression in Ishikawa cells and Ishikawa/DDP is detected by Q-PCR.Compared with Ishikawa cells,the expression of AKT1 mRNA in Ishikawa/DDP cells is significantly increased(P<0.001).It shows that AKT1 is overexpressed in Ishikawa/DDP cells.2.Metformin inhibits the viability of Ishikawa/DDP cells and promotes apoptosis.The CCK-8 method was used to detect the viability of metformin on Ishikawa/DDP cell lines,and it was found that metformin reduced the viability of Ishikawa/DDP cells(P<0.01);PI flow cytometry detection Apoptosis,it was found that metformin increased G0-G1,decreased S phase,and decreased G2-M phase of drug-resistant cells(P<0.01).3.Metformin inhibits the proliferation of Ishikawa/DDP cell line and promotes its apoptosis,which may be related to the up-regulation of BAX and down-regulation of BCL-2;24 hours after metformin treatment of Ishikawa/DDP cell line,Western Blot(WB)detects BCL-in Ishikawa/DDP 2 and the expression of BAX,it was found that the anti-apoptotic protein BCL-2 decreased,and the pro-apoptotic protein BAX increased.4.Metformin inhibits the viability of the Ishikawa/DDP cell line and promotes its apoptosis,which may be related to reducing the level of AKT1 mRNA and inhibiting its expression.After treating the Ishikawa/DDP cell line with metformin for 24 hours,the mRNA expression of AKT1 was detected by Q-PCR,and the protein expression of AKT1 was detected by WB;the data showed that metformin significantly reduced the mRNA and protein levels of AKT1 in Ishikawa cisplatin-resistant strains.Conclusion:Metformin can inhibit the viability of the Ishikawa/DDP cell line and promote its apoptosis;it may be related to the up-regulation of BAX,down-regulation of BCL-2,and reduction of the mRNA level of AKT1 in the Ishikawa/DDP cell line and inhibiting its protein expression.