Effect Of 17β-estradiol On Proliferation And Expressions Of P21 Ras And P-erk In Endometrial Carcinoma Cells

Background and Objective:Endometrial carcinoma is one of the three common malignant tumors which affects women's health and has a tendency of obvious increase in recent years. The incidence of endometrial carcinoma has been the first one of the female genital duct malignant tumors in some developed countries of Europe and America. Recently,the generally acknowledged that "long-term stimulation of estrogen without inhibition of progesterone"may play an important role for Endometrial carcinoma, but the molecular mechanism of estrogen how to affect cells' proliferation has not exactly definited now.Traditionally, estrogen regulates transcription factor and synthesizes specific protein after combining with genomic estrogen receptor which called "genomic transcription action".This process usually needs several hours to synthesize the protein,so we also call it "long-time effect".Recent study shows that estrogen can induce volant nongenomic transcription action through combining with membrane estrogen receptor of many kinds of cellular surface like the growth factor and activate the signal transduction pathways. This course usually takes several minutes that we can't explain it by the classical genomic transcription action, therefore we call it "rapid activation" or"nongenomic transcription action". Related researches have already confirmed that the signal transduction pathway of phosphatidylinositol 3-kinase-protein kinase B( PI3K/ Akt ) can be induced by 17β-estradiol in endometrial carcinoma cells to make them grow quickly.Whether can the estrogen activate the mitogen-activated protein kinases(MAPK)signal transduction pathway in endometrial carcinoma cells and make them proliferated is rarely reported in China until now.P21ras is a membrane protein that coded by the oncogene ras and the molecular weight is 21 KD which plays an important role in the MAPK signal transduction pathway.P-ERK is the activated form of extracellular signal-regulated kinase ( ERK). ERK is a kind of protein kinase belong to the MAPK family which includs Serine and Threonine and guided by Proline protein.ERK locates in cytoplasm usually.When it is activated, it permeates into karyolemma and conducts the signals from the plasm of the cell into cellular nucleus. It can phosphorylate some transcription factors,activate the expression of the immediate-early gene c-jun and c-fos, induce the expression of the oncogene cyclinD₁ and the c-myc. The expression of the cyclin D₁ can urge the cell to convert from the G₁ phase to the S phase and cause the cell propagate out of control finally. In addition, the P-ERK can ascend the expression of cyclinE, and denature the inhibition protein P27^KIP to regulate the cell apotosis from several levels and promote the occurrence of the rumor.According to the above consideration,our studies observe the influence of 17P-estradiol on proliferation, cell cycle progression, expressions of P21ras and P-ERK in endometrial carcinoma cells (Ishikawa and HEC-1A) and to explore the possibility of endometrial carcinoma induced by estradiol.Materials and Methods:1,Materials: The Ishikawa cell (high expression of ER) comes from well divided endometrial adenocarcinoma ; The HEC-1A cell (low expression of ER) comes from the American ATCC cell database. Both are from University of Peking people hospital gynecology laboratory. Ishikawa and HEC-1A cells were cultured in DMEM containing 100U/ml PC, 100U/ml SM, 10% FBS under standard cell cultured conditions (humidified atmosphere,37℃,5 % CO₂ ) as usual.Cells were serial subcultivated with 0 .25 % pancreatin when cells grow fully.2,Methods:(1) The effect of estradiol on proliferation were examined by monoterazolium (MTT) assay in Ishikawa and HEC-1 A cells after stimulation with varied doses of E₂ (0,10^-10mol/L,10^-8mol/L,10^-6mol/L,10^-4mol/L)for different times(24, 48, 72h ).(2) The effect of estradiol on cell cycle distrubion of endometrial carcinoma cell lines, Ishikawa and HEC-1 A were examined by fluorescence-activated cell sorting technique after stimulation with varied doses of E₂ (0,10^-8mol/L,10^-6mol/L)for the same time 24h .(3)The activity of P21ras and P-ERK were examined by immunoprecipitation in Ishikawa and HEC-1 A cell after stimulation with 10^-6mol/L E₂ for different times(omin, 5min,15min,30min,1h,2h) to observe the optimal time and with varied doses of E₂ (10^-10mol/L,10^-8mol/L,10^-6mol/L,10^-4mol/L)for the optimal time .Then to analyses the time effect,the dose effect,the dependability of P21ras and P-ERK and the relation with ER.3,All analysises were treated by the SPSS statistical package program 13.0. .The result of proliferation,and cell cycle by one way analysis of variance; the expression of P21ras and P-ERK by t or t'Test, their relationship by Spearman. Statistically significant level was considered as "alpha equals 0.05" (α=0.05).Results:1,With increased concentrations of E₂, the proliferation of Ishikawa cell increasedgradually and in a time-dependent manner(F≥32.93,P≤0.001). Cell cycle distributionanalysis revealed that percentage of Ishikawa cell at G₀-G₁ phase (F=54.552, P<0. 001)decreased and percentage of S phase cells increased significantly (F=40.88, P=0. 000),whereas that of HEC-1 A cell didn't show significant alteration(P>0.05).2,The maximal activation of P21ras and P-ERK took place at 30min in Ishikawacell(74.00±2.5495, 68.00±4.8166)and 15min in HEC-1A cell (78.00±3.1623,74.00±2.3022) after stimulation with 10^-6mol / L E₂ in HEC-1A cell. With increaseddoses of E₂, the activation of P21ras and P-ERK increased gradually,present inconcentration-dependent manner.3,Under different time ,different concentrations of E2,there were significant positive correlations between the expression of P21ras and P-ERK (r≥0.049, p≤0.027, r>0.047,p<0.036) ,the expression of P21ras between Ishikawa and HEC-1A cells was negative correlation (r<-0.049,p<0.030) , the expression of P-ERK between Ishikawa and HEC-1A cells was negative correlation too. (r≤-0.048,p≤0.032) .Conclusion:1,17P-estradiol could promote the cell proliferation and the cell cycle distrubion of endometrial carcinoma cell Ishikawa line.The difference between the two cell lines might be due to the different level of the expression of ER in them.The estradiol could promote the cell proliferation through "genomic transcription action" and "nongenomic transcription action" in Ishikawa ,but in HEC-1A it could only do this by the "nongenomic transcription action".2,17β-estradiol could activate the expression of P21ras and P-ERK in endometrial carcinoma cells( Ishikawa and HEC-1A) and in concentration-dependent manner,besides the expression of P21ras and P-ERK were significant positive correlations.This indicated that estradiol could activate the mitogen-activated protein kinases(MAPK)signal transduction pathway quickly by the way of nongenomic transcription action to promote the occurrence of endometrial carcinoma . 3,The expression of P21ras and P-ERK were negative correlations with ER.Maybe the nuclear estrogen receptor and membrane receptor competitivly combined with the E₂ ,that weakened the effect of E₂ through the membrane receptor to activate the MAPK pathway.This provided a new treatment target to the endometrial carcinoma by blocking the signal transduction pathway.