The Non-nuclear Effects Of Estrogen In The Proliferation Of Endometrial Carcinoma Cells

Object:To detect the expression of estrogen receptor alpha in endometrial carcinoma tissues, to correlate this data with morphology and staging of the endometrial carcinoma, and to explore the existence of the ER alpha on the plasma membrane of the endometrial cell lines.Methods : Immunohistochemical reactions for estrgen receptor alpha via the Streptavidin-Biotin-Peroxidase Complex method in 33 endometrial carcinoma tissue samples were campared with the mopholgical data, disease stage and the depth of myometrial invasion. The immunofluorescence of the estrgen receptor alpha on the plasma membrane of endometrial carcinoma cell lines Ishikawa and HEC-1B by flow cytometry.Results:The expression of ER alpha were decreased with the low degree of tumor differentiation, and the clinical advanced stage and deep myometrial invasion. And the location of the ER alpha was altered with progression of the disease. The expression rate of ERαin the plasma increased significantly in the endometrial cancer with severe myometrial invasion.(p=0.024). There was a little portion of the Ishikawa cells which has ERαexpressing on the plasma membrane (3%). And the fluorescence intensity of membrance ERαon Ishikawa cells increased with the application of estradiol, but decreased 24 hours after the treatment with estradiol. And ERα's fluorescence intensity increased gradually by the treatment of E2-BSA for 1hour and 24hours. There was no fluorescence intensity of membrane ERαon the HEC-1B cells.Conclusion: The difference of the expression level and the location of the ERαbetween the different differentiation, clinical stage and myoinvasion of the endometrial carcinoma indicate the ERα's role in the progression of the disease. And there is a little part of ERαexpressing on the plasma membrane of the Ishikawa cells. The expression of ERαon plasma membrane is regulated by estradiol. Object:To detect the role of nonnuclear effects of estrogen in the proliferation of the endometrial carcinoma cells. To explore its possible pathway by studying the effects of inhibitors on the estrogen stimulated proliferation of endometrial cancer cells.Methods:The cell proliferatin of endometrial carcinoma cell lines Ishikawa and HEC-1B were measured by using MTT methods after treated with estradiol, E2-BSA of different concentration, or after preincubation with ICI 182780, PD 98059 and HB-EGF neutralizing antibody.Results:The tumor cells'proliferation were significant when Ishikawa cells were treated with 1×10-8M estradiol and E2-BSA, and when HEC-1B cells were treated with 1×10-7M estradiol. The proliferation of Ishikawa cells induced by estradiol and E2-BSA could be inhibited by ICI 182780 and PD 98059, but HB-EGF neutralizing antibody only suppress the E2-BSA induced proliferation, and have no effect on the estradiol induced proliferation. And all the inhibitors have no effects on HEC-1B cells. Conclusion: Our data show that the impermeable membrane estradiol—E2-BSA has the same effect with estradiol on endometrial carcinoma cells, imply that the non-nuclear effects of estrogen might promot growth of endometrial tumor cells. And this action could be interfered by ER antagnon—ICI 182780, suggestion that in ER positive endometrial carcinoma cell line Ishikawa, the non-nuclear effects of estrogen on proliferation is ER dependent. Also, the inhibition to the E2-BSA stimulated cell growth by PD 98059 and HB-EGF neutralizing antibody, imply that the MAPK signal pathway and HB-EGF might involve in the proliferation actions of the estrogen's non-nuclear effects, Object:The activation of extracellular signal-regulated kinase(ERK1/2), intracellular free Ca2+ concentration and the secretion of heparin-binding epidermal growth factor-like growth factor(HB-EGF) were measured to investigate the signaling pathway activated rapidly by non-nuclear action of estradiol.Methods:Two endometrial carcninoma cell lines, Ishikawa and HEC-1B cells were incubated with estradiol and E2-BSA, then the activation of ERK1/2 was measured by means of western blotting expreiments; the intracellular free Ca2+ concentration ([Ca2+]i) of Fura-3 loaded tumor cells was examined by immunofluorescent labeling and confocal microscopy; the amount of HB-EGF in the supernatants were assayed by ELISA. The inhibitory effects of ICI 182780 and HB-EGF neutralizing antibody on the activation of ERK1/2 were observed, which were incubated previously in the estradiol and E2-BSA treated endometrial cell lines. And the ICI 182780 actions on [Ca2+] i increased by estradiol and E2-BSA were also detected.Results:Estradiol and E2-BSA induced a significant increase of ERK1/2 phosphorylation, with a maximun activity at 1×10-8M in Ishikawa or 1×10-7M in HEC-1B cells. At the concentration of 1×10-8M, estradiol and E2-BSA activate the ERK1/2 2 minutes after treatment in Ishikawa and HEC-1B cells, and reach the maximum phosphorylation after 15 min and 30 min respectively. ICI 182780 could only inhibit the activation of ERK1/2 by estradiol and E2-BSA sitmulation in Ishikawa cells. And in this cell line, the ERK1/2 phosphorylation induced by E2-BSA was significantly inhibited by the HB-EGF neutralizing antibody.E2 and E2-BSA induced a rapid rise in the intracellular free Ca2+ concentration of Ishikawa cells, and these changes in [Ca2+]i could be inhibited by ICI 182780. The increase of [Ca2+]i in HEC-1B cells induced by E2 and E2-BSA was slower and lower compared with in Ishikawa, and ICI 182780 have no effect on it. Only E2-BSA enhanced HB-EGF secretion in Ishikawa cells, and ICI 182780 could block this action.Conclusion:Like E2, plasma membrane-impermeable E2-BSA could activation the ERK1/2 and increase the intracellular free Ca2+ concentration of endometrial caicinoma cells. These non-nuclear effects of estrogen could be inhibited by ICI 182780 in Ishikawa cells, indicated that in the ER positive endometrial cells, the non-nuclear effects of estrogen were mediated by ER. The increase secretion of HB-EGF in Ishikwa, as well as the inhibitory effects of HB-EGF neutralizing antibody on the activation of ERK1/2 induced by E2-BSA, suggestion HB-EGF might be one part of the signal transduction pathway activated by the non-nuclear effect of estrogen in ER positve endometrial cancer cells.