| Background Endometrial Cancer(EC) is one of the three most common female reproductive tract malignant tumors,and its incidence has been increasing in recent years.The Amerian Cancer Society estimates that nearly 52,630 new cases of endometrial carcinoma may be diagnosed in 2014,and at the same time there are 8,590 cases of death[1]. Obesity, diabetes, and insulin resistance are well-known risk factors that drive the development of endometrial cancer[2,3]. Unfortunately, obesity is not only a risk factor for endometrial cancer, but also may be associated with an increased risk of death from this disease[4]. Surgery is the initial treatment for patients with early stage disease.For the young patients who have polycystic ovarian syndrome(PCOS) or irregular menstruation or a long time not ovulation. How to preserve their fertility has always been a difficulty of treatment[5].Progestins, especially synthetic progestins such as medrox-yprogesterone acetate(MPA), have long been widely used for the advanced patients or recurrent patients or the young patients who want to preserve their fertility. However, the response rate to this treatment has not been very satisfied, varying from 16% to 34% of the effective long-term progestogen therapy[6]. Some research suggests that progestin-resistant in endometrial cancer cells is thefundamental reason of treatment failure[7]. How to improve the resistance of progesterone, enhance the effect of progesterone therapy of endometrial carcinoma is currently facing a difficult problem in clinic. So looking for a drug to relieve the progress of endometrial carcinoma or improve the prognosis of endometrial carcinoma is a problem that should be solved in the clinical treatment nowadays. Tumor cells can secrete numerous growth factors, among them epidermal growth factor(EGF) and insulin-like growth factor(IGF) is important to promote epithelial hyperplasia, is closely related to tumor cell apoptosis and proliferation. EGFs is the meaning of epidermal growth factor system, which is made up of EGF and its receptor(skin factor receptors EGFR)[8]. EGF receptor(EGFR) family is one of transmembrane receptor tyrosine kinases, and it consists of four members.They are Erb B-1, also called EGFR,because EGFR is the expression product of protooncogene c-erb B1;Erb B-2(also called HER2 or neu), Erb B3(also called HER3) and Erb B4(also called HER4).They can regulate cell proliferation,motility, differentiation, and survival by modulating the expression of many cell growth-related genes that control cell cycle progression[9]. Numerous reports have indicated that EGFR is over-expressed in various solid tumors including endometrial cancer and has been associated with a worse prognosis[10]. Research of progesterone resistance’s mechanism in endometrial cancer cells, some reports claim that progesterone resistance associated with excessive activation of EGFR, but its specific regulating mechanism is still unclear[11].In recent years, studies have shown that over-expressed of epidermal growth factor receptor(EGFR) can reduce the sensitivity of endometrial cancer cells of progesterone, makes it produce drug resistance, induce epithelium-interstitial transformation in endometrial cancer cells. High EGFR expression can lead to sustained activation of its downstream signaling molecules,while the pathway inhibitor AG1478 can effectively restrain the activation of the important signal molecules in the pathway[12]. Epidemiological studies have found that approximately 11.1% of endometrial cancer with diabetes mellitus. The relationship between endometrial carcinoma and diabetes has been confirmed by cohort and case-control researchs, which combined with type 2 diabetes can increase the risk of endometrial cancer one to three times [13].Metformin is an antidiabetic medication from the biguanide class that is widely used as the first line treatment of type 2 diabetes. In the field of gynecology, metformin is widely used in the treatment of polycystic ovary syndrome to improve insulin resistance. Study found that metformin can improve the progesterone treatment of early endometrial atypical hyperplasia and endometrial carcinoma [14-17]. Our previous research demonstrated that metformin through activation of AMP activated protein kinase(AMPK), thereby inhibiting excessive activation of AKT/m TOR signaling pathway to suppress the proliferation of endometrial cancer cells in vitro culture, and promote the expression of progesterone receptor. In addition, insulin-like growth factor(IGF)-I and IGF-II can promote cell proliferation in endometrial cancer cells, while metformin reverses this effect and inhibits cell proliferation in endometrial cancer cells. The vivo experiments of nude mice have verified the metformin can significantly inhibit the proliferation of endometrial cancer cells[18,19]. The above experiments prompt that metformin can inhibit the growth of endometrial cancer cells in vivo and in vitro, and could improve the resistance of progesterone, enhance the clinical treatment effect of progesterone. The human endometrial carcinoma cell lines Ishikawa 、HEC-1A and AN3 CA were used. The aim of this study was to investigate the regulatory mechanisms of EGFR and ERK signaling by metformin in endometrial cancer cells and to determine the effects of metformin combined with or without EGFR-TK inhibitor on cell proliferation.Objective This paper applied the cell proliferation experiment(CCK-8) to analysis the effect of metformin on cell proliferation in endometrial cancer.We applied fluorescence quantitative PCR and western blot technique to analysis the effect of metformin in endometrial cancer cells by EGF signaling pathways, in order to provide a new train of thought on improving the progesterone resistance, relieving the progress of endometrial carcinoma and to improve the prognosis of endometrial carcinoma.Methods 1. Cell proliferation was assessed after exposure to metformin and/or the EGFR inhibitor AG1478 by cell proliferation experiment(CCK-8)in endometrial cancer cells. 2. EGFR m RNA was determinted by RT-PCR on the effect of different concentrations of metformin in endometrial cancer cells. 3. Application of protein immunoblot(Western blot) method to detecthe protein level of EGFR、p-EGFR、ERK and p-EGFR under the action of metformin in endometrial cancer cells. 4. Statistical analysis: the SPSS Statistical package program 17.0 for all analysis.All data are presented as means ± standard errors(SEs). One-way ANOVA was used to compare EGFR m RNA and protein,downstream pathway protein. LSD-t test was used in the means between two groups. Differences with P-values of less than 0.05 were considered significant.All blots represent three independent experiments.Results 1. Both of metformin and EGFR inhibitor AG1478 can inhibit cell proliferation in endometrial cancer cells, and at the same time metformin enhanced the inhibitory effects of EGFR inhibitor AG1478 on cell proliferation(P<0.05). 2. Metformin downregulated EGFR m RNA in a concentration-dependent manner in the three cell lines(P<0.05). 3. Metformin effects on endometrial carcinoma after different time, the protein expression levels of total EGFR and ERK have no significant change(P>0.05),while the protein expression levels of phosphorylation EGFR and phosphorylation ERK have been reduced,and the effect is in a time-dependent manner(P<0.05).Conclusion 1. Metformin can significantly inhibit cell proliferation in endometrial cancer cells, and at the same time metformin can enhanced the inhibitory effects of EGFRinhibitor AG1478 on cell proliferation. 2. Metformin may regulate the EGFR signaling pathway that inhibition of EGFR expression of phosphorylated proteins that regulate ERK signaling pathway, inhibition of ERK protein phosphorylation and then inhibit the proliferation of endometrial cancer cells. |
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