| Objective:Metformin is commonly used in the treatment of type 2 diabetes mellitus.In recent years,metformin has been shown to inhibit the growth of malignant tumors and enhance the efficacy of chemotherapy.In the malignant tumor of Gynecology,the highest mortality rate is ovarian cancer.The key point of this disease is to find out the effective measures to prevent and reverse the drug resistance.Studies have indicated that the formation and development of malignant tumors are closely related to IGF-1R,and insulin-like growth factor system(IGF-I,IGF-1R and AKT)is associated with drug resistance in ovarian cancer.So we aim by in vitro and in vivo studies of metformin on IGF system resistant ovarian cancer cells to explore the mechanism of the role of epithelial ovarian cancer chemotherapy drug metformin,adjuvant therapy for a new ovarian cancer.Methods:(1)By in situ hybridization and immunohistochemistry and immunofluorescence detection of epithelial ovarian cancer,benign tumor tissue,the expression of MRP2 protein and mRNA in normal ovarian tissues,and analyze the relationship between the biological characteristics of ovarian cancer;(2)CCK-8 assay was used to detect the proliferation ability of cells and draw the growth curve;(3)CP70 cell cycle and apoptosis were detected by flow cytometry and AnnexinV/PI double staining;(4)Changes of IGF-1,IGF-1R,AKT protein expression in MRP2 and related signaling pathway of IGF cells treated with different concentrations of metformin were detected by Western blot after Western blot analysis of CP70 cells;(5)Changes of MRP2 and IGF-1,IGF-1R,AKT,p-IGF1,p-IGF1R,p-AKT gene expression in CP70 cells were detected by fluorescence quantitative PCR;(6)Objective to establish cisplatin resistant human ovarian cancer cell line CP70 and non drug resistant A2780 cell model and to analyze the effect of metformin on the expression of MRP2 and the inhibition of tumor in nude mice.Results:(1)The positive expression of MRP2 protein expression in normal ovarian tissue,benign ovarian tissue and epithelial ovarian cancer rates were 0(0/5),20%(2/10)and 58%(14/25),the difference was statistically significant,P<0.05;the expression of MRP2 gene expression in normal ovarian tissue,benign ovarian tissues and ovarian epithelial cancer the rate was 0(0/5),10%(1/10)and 48%(12/25),the difference was statistically significant,P<0.05.Expression of MRP2 and MRP2 mRNA in epithelial ovarian carcinoma is not affected by the patient’s age,pathological stage,the difference was not statistically significant(P>0.05),but the expression and pathological type,lymph node metastasis(P<0.05).(2)The effect of metformin on ovarian cancer cell line A2780 and drug resistant cell line CP70 was increased.After 24h treatment of metformin treated CP70 cells by CCK-8 method,the resistance to cisplatin decreased significantly,and the concentration dependence.(3)The cell cycle distribution of CP70 along with the increased concentration of metformin changed significantly and showed the percentage of G0/G1 phase increased significantly 10mM/ml compared with 0.01mM/ml group(F=69.514,P<0.05),there was statistical significance of natural 100mmol/L apoptosis rate and the concentration of metformin 10mmo1/L concentration difference(F=79.814,P<0.05).(4)Detection of CP70 cells treated with metformin 48h Western blotting,found that IGF-1,IGF-1R,AKT expression decreased gradually with the increase of the concentration of metformin,p-IGF1,P-IGF1R,P-AKT decreased compared to IGF-1,IGF-1R,AKT is more obvious,the two have obvious concentration dependence.The protein group and beta-actin relative gray ratio compared with the 0 mM/ml concentration of metformin,the difference was statistically significant(P<0.05),of which 0 mM/ml + metformin significantly inhibited the combined application of the blocking agent LY294002 group 10 mM/ml on ovarian cancer cells CP70 cells.(5)Metformin can affect MRP2 and IGF-1,IGF-1R,AKT,p-IGF1,p-IGF1R,p-AKT gene expression,and in a concentration dependent manner,thereby inhibiting the proliferation of CP70 cells was statistically significant in metformin group and normal control group expression of different concentration(P<0.05).(6)Observation of A2780 group and CP70 group the tumor volume and growth,DDP + metformin group tumor volume was significantly smaller than the control group and the treatment group A2780+ with cisplatin,cisplatin + metformin inhibition rate was 59.16%,CP70+ + cisplatin inhibition rate of metformin was 79.03%,obviously higher than the simple use of cisplatin.The difference was statistically significant(P<0.05).(7)In vivo experiments showed that metformin inhibited the expression of MRP2 protein,and the combination of cisplatin plus metformin two drugs had higher inhibition rate on A2780 and CP70 cells compared with the single use,and the two drugs showed synergistic effect.Conclusions:(1)The expression of MRP2 protein in epithelial ovarian cancer was positively correlated with the type of ovarian cancer and lymph node metastasis.(2)A certain concentration of metformin can enhance the sensitivity of cisplatin to ovarian cancer cell line CP70.(3)Metformin inhibits the expression of IGF,IGF-1R and AKT receptors in the IGF signaling pathway to inhibit the resistance of MRP2 cells to cisplatin in ovarian cancer cells.(4)In vivo experiments showed that metformin inhibited the inhibitory effect of cisplatin on ovarian cancer A2780 and CP70 cells by inhibiting the MRP2 protein,and the inhibition effect of cisplatin combined with metformin was better. |
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