The Role Of G Protein-coupled Estrogen Receptor In The Signaling Pathways Of Type â…¡ Endometrial Carcinoma

Objective: Endometrial cancer is one of the most common malignanttumor in female reproductive system and harm to women’s health. Theincidence and mortality of endometrial cancer have increasingly upward andthe age tends to be younger and younger in our country. According to theClinical observationand the pathological characteristics of endometrialcarcinoma, endometrial cancer is divided into two categories: type Ⅰand typeⅡ. typeⅠis estrogen-dependent endometrial carcinom. We usually think itspathogenesis is duo to the long-term reaction of estrogen without antagonismof progesterone. This kind of tumor have better differentiation, higherexpression of Estrogen Receptor(ER) and Progesterone Receptor(PR), betterprognosis, and higher cure rate of hormonotherapy. Type Ⅱisestrogen-independent endometrial carcinoma, we usually think itspathogenesis has no exactly relationship with estrogen, because of its lowexpression of ER and PR. This kind of tumor have higher malignant degree,poor differentiation, poor prognosis, and hormonotherapy is invalid.Traditionally, Estradiol （E2） start the activation, transcription,translation of the target genes, and then start the activation of correlativesignal pathway to promote the proliferation of tumour cell throughcombination with ER. However, this model does not make effective control inEstradiol-dependent tumor, such as endometrial cancer and breast cancer.Recent research has shown that E2can activate PI3K/Akt (phosphatidylinositol3kinase/protein kinase B) signaling pathway in endometrial cancercells. In ER-positive cells, this effect is ER-dependent. However in HEC-1Acell lines which ER is low expression, this effect is ER-independent. But whatinduces the activation of PI3K/Akt signaling pathway by E2in the endometrialcancer cells which ER-low expression? Revankar found that E2and Tmoxifen (TAM) could activate PI3K/Akt signaling pathway, then trigger the PIP3andAkt gather to nucleus and endoplasmic reticulum in breast cancer SKBr3celllines which GPER is positive expresssion and ER is negative expression, thissuggest that there are some relationship between GPER and the activation ofPI3K/Akt signaling pathway. Further study for the mechanism of GPER in thedevelopment of endometrial carcinoma, especially in the ER (-) endometrialcancer can improve the understanding of E2and ER. Eventually we maydevelop more effective treatments targeted to the signaling pathway.The study selected the Ishikawa cell lines whose estrogen receptor ispositive、HEC-1A cell lines whose estrogen recepter is low expressed andKLE cell lines whose estrogen recepter is negative as the research object, aimto research the function of GPER in the proliferation of Ishikawa、HEC-1Aand KLE cell lines promoted by Estradiol. Estrogen receptor antagonistICI182780, GPER antagonists G15, was used in the study and the downstreampossible signaling pathways was explored as well in vitro experiments. ThenGPER’s mechanism was discussed in the ER different ly expressedendometrial cancer, and may provide the new mentality for the targetedtherapy of endometrial cancer.Methods:1Cell culture: The Ishikawa cells were cultured in nutrient solution of1640, and the HEC-1A and KLE cells were cultured in nutrient solution ofDMEM-F12in vitro, including10%fetal calf serum,100U/ml penicillin and100U/ml streptomycin, at37℃,5%CO2saturated humidity incubator. Thecells were digested by0.25%trypsinase and then subcultured. Cells adhered tothe bottom in monolayer and were chosen to be used in the test at logarithmicgrowth phase.2The MTT method was used to detect the effect of Estradiol、estrogenreceptor antagonist ICI182780and GPER antagonists G15on the growth ofIshikawa cells, HEC-1A cells and KLE cells in different concentrations.3Western Blot was used to detect the influence of Estradiol、ICI182780and G15on the expression of Akt, GPER and Bcl-2protein in Ishikawa cells, HEC-1A cells and KLE cells.Results:1The effect of E2on the proliferation of three types of endometrialcancer cells.Different concentrations of E-102(0mol/L、10mol/L、10-9mol/L、10-8mol/L、10-7mol/L、10-6mol/L) were used to treat three types of cells for24h、48h、72h. OD value was measured and the proliferation were calculated. Theresults suggested that E2effectively promoted the Ishikawa cells proliferationby a dose and time-dependent manner. The effect of E2on the proliferation ofHEC-1A and KLE cells was weaker than Ishikawa cells.2The inhibitory effect of the estrogen receptor antagonist ICI180782onthe proliferation stimulated by Estradiol in three endometrial cancer cellsThree types of cells was pretreated by Estradiol at the concentration of10-6mol/L, then different concentrations of ICI180782(0mol/L、10-9mol/L、10-7mol/L、10-5mol/L) were used to stimulate three types of cells for24h,48h,72h. OD value was measured by MTT, the inhibition rate was calculated.ICI180782obviously inhibited the proliferation of Ishikawa cells stimulatedby Estradiol, by a dose and time-dependent manner. However, ICI180782didn’t inhibite the proliferation of HEC-1A and KLE cells stimulated by E2。3The inhibitory effect of the GPER antagonist G15on the proliferationof endometrial cancer cells stimulated by Estradiol.Three types of cells was pretreated with Estradiol at the concentration of10-6mol/L, then different concentrations of G15(0mol/L、10-9mol/L、10-7mol/L、10-5mol/L) was added to stimulate three types of cells for24h,48h,72h. OD value was measure by MTT, then the inhibition rate was calculated.G15obviously inhibited the proliferation of HEC-1A and KLE cellsstimulated by Estradiol, by a dose-and time-dependent manner. While G15did not inhibite the proliferation of Ishikawa cells stimulated by Estradiol.4The expression of Akt, GPER and Bcl-2protein stimulated byEstradiol measured by Western BlotThree types cells were treated by Estradiol for0h,24h,48h,72h. then theresults showed: The expression of Akt and Bcl-2proteins was increased after the simulation of Estradiol, and increased with the increase of time. Thedifference was statistically significant compared with the control subjects.GPER protein were expressed in the three types of cells. And the expression ofGPER was the highest in KLE cell and the difference was statisticallysignificant in three types cells.5The influence on the epression of Akt, GPER and Bcl-2proteinstimulated by Estradiol and treated by ICI182780and G15in three types ofendometrial carcinoma cellsThree types of endometrial cancer cells were trated by deffrent drugs(10-6mol/L E562+10-mol/L ICI182780、10-mol/L E-52+10mol/LG15) for24h,48h,72h. It was found that ICI182780down regulated the expression ofAkt, Bcl-2and GPER in Ishikawa cells. But there was no effect on theHEC-1A and KLE cells. G15down regulated the expression of Akt, Bcl-2andGPER in HEC-1A and KLE cells. But in Ishikawa cells, G15down regulatedthe expression of GPER, and had no effect on the expression of Akt, Bcl-2.Conclusion:1Estradiol promotes the proliferation of human endometrial carcinomacells both typeⅠand typeⅡ.2Estradiol is mainly dependent on ER activating PI3K/Akt/Bcl-2signaling pathway to promote cell proliferation in typeⅠendometrialcarcinoma; In type Ⅱ endometrial carcinoma estradiol is mainly dependent onGPER activating PI3K/Akt/Bcl-2signaling pathway to inhibite cell apoptosisand promote cell proliferation.3In type Ⅱ endometrial carcinoma, the GPER antagonists G15canblock PI3K/Akt/Bcl-2signaling pathways mediated by GPER to inhibite cellproliferation. So GPER and its antagonist G15may become the new target andnew targeted drugs for treatment of type Ⅱ endometrial cancer.