Effect And Regulation Of Metformin By Regulating Progesterone Receptor Isoform B In Endometrial Cancer Subline Of Progestin Resistance

BackgroundEndometrial cancer is one of the most frequently occurring gynecologic malignant tumors around the world. Surgery is still the initial treatment for patients with early stage endometrial cancer. But for young patients, how to preserve their fertility has always been a problem to solve, which is a great challenge to the traditional treatment of endometrial cancer. Currently, progestins have been widely used for the young patients of early-stage endometrial cancer who are persistant to preserve their fertility functions. However, the response rate of the long-term progestogen therapy is not very satisfying. Progestin-resistance in endometrial cancer cells may be the fundamental reason for the failure of progesterone therapy. Therefore, how to improve the resistance of progesterone and to improve the effect of progesterone therapy of endometrial cancer is currently a difficult problem to solve in clinical practice. ObjectiveThis paper applied the cell proliferation experiment(CCK-8) to analysis the effect of metformin on the proliferation of endometrial cancer subline of progestin-resistance. Besides, western blot technique was used to analysis the effect of metformin in endometrial cancer subline of progestin-resistance by PRB signaling pathways, in order to provide a new thought on improving the progesterone resistance, delaying the progress of endometrial carcer and to improve the prognosis of endometrial cancer. Methods1. Ishikawa endometrial cancer cells were cultured for a long period in the presence of the synthetic medroxyprogesterone 17-acetate(MPA), thereby generating a subline refractory to the growth-suppressive effects of MPA.2. The effect of metformin(l, 5, 10, 20, 40, 60 μmol/L) or MPA(1, 10, 20, 40, 60, 70, 80 mmol/L) on proliferation of the MPA-resistant subline and parental Ishikawa cells was detected by CCK-8 method.3. Western blotting technique was used to detect the effect of metformin or/and MPA on the expression of PR-B in the MPA-resistant subline and parental Ishikawa cells.4. Statistical analysis: the SPSS Statistical package program 17.0 for all analysis.All measurement data was presented as means ± standard errors. T-test was used when comparing the two groups. One-way ANOVA was used to compare the mean among groups when the data met the condition of homogeneity of variance and normality. LSD-t test was used to analysis the means between two groups. And rank sum test was used when neither of the two conditions was satisfied. Differences with P-values of less than 0.05 were considered significant. Results1.The MPA-resistant subline showed growth stimulation rather than inhibition in the parental Ishikawa cells after low MPA concentration treatment. The doubling Time of MPA-R-Ishikawa cell line is(43±4) hours and that of Ishikawa cell line is(47±3) hours. No changes in doubling time were observed.2. Low concentration(1, 5 μmol/L) of MPA promoted the growth of MPA-R-Ishikawa cells(by 3%, 13%) and inhibited the growth of Ishikawa cells(by 16%, 30%).High concentration(10, 20, 40, 60 μmol/L) of MPA inhibited the growth of MPA-R-Ishikawa cells(by4%, 3%, 9%, 40%) and the growth of Ishikawa cells(by 41%, 55%, 65%, 66%). At the same concentration, the difference between the two cell inhibition rates were statistically significant(χ2= 17.29, P = 0.000). Metformin(1, 10, 20, 40, 60, 70, 80mmol/L) inhibited the growth of MPA-resistant subline(by-10%, 20%, 56%, 89%, 97%, 98%, 99%) greater than parental Ishikawa cells(by-6%, 19%, 37%, 54%, 70%, 72%, 83%), and the inhibitory effect was dose-dependent.3.western blot assay showed that for Ishikawa cells, the protein expression levels of PR-B in metformin group and MPA + metformin group were both higher than the control group, and the differences were statistically significant(F=112.97, P <0.05). For MPA-R-Ishikawa cells, the protein expression levels of PR-B in metformin group and MPA + metformin group were also higher than the control group, and the differences were also statistically significant(F=598.64, P <0.05). The protein expression levels of PR-B of Ishikawa cells in the control group is lower than that in MPA-R-Ishikawa cells, and the differences were also statistically significant(t=10.21, P <0.05).ConclusionMetformin may regulate the progestin-resistance in endometrial cancer by increasing the expression of PR-B.