| IntroductionEndometrial carcinoma is one of the most common malignant tumors of female genital tract. It accounts for 20 - 30% of all female malignant tumors and the incidence has continued to increase since 1980s. Long - term stimulation of estrogen without inhibition by progesterone may play an important role for endometrial carcinoma. The treatment of endometrial carcinoma with progesterone has been introduced for about 50 years . With the advance of molecule tumor study, some studies about how progesterone is involved in the inhibition of endometrial carcinoma have been taken in vitro and in animal models, but the mechanism is still not clear. Traditionally , progesterone regulates transcription factor and synthesizes specific protein after combining with genomic progesterone receptor to suppress cell proliferation of tumor cells. Recent study shows that progesterone can educe volant nongenomic action through combining with membrane progesterone receptor of many kinds of cellular surface and activate ERK signal trans-duction pathway.Materials and methods1. Main agenda and equipmentIshikawa cell lines;progesterone;fetal bovine serum;RPMI1640 culture fliud;progesterone nuclear receptor pharmacon RU486;mouse anti man phosphor - ERK polyclone antibody;mouse anti man phosphor - ERK polyclone antibody;mouse anti man total ERK polyclone antibody;mouse anti man Cy-clinDl polyclone antibody;biotin labeling dis - antibody superclean bench;carbon dioxide constanr tenperature incubaton;inverted phase contrast microscope;overdrive refrigerated centrifuge;homogenate machine;spectralumenosity merer;multifunctional electrophotesis apparatus2. Empirical method2.1 Cell cultureIshikawa cells were grown in RPMI1640 containing lOOu/ml PC lOOU/ml SM 10% FBS understandard cell cultured conditions (humidified atmosphere, 5%co2,37cC) passaged as usual. Cells were serial subcultivated with 0. 25% pamcreation when cells grow fully.2. 2 Drug action and protein extraction1) Cells were seeded to the 25 cm2 culture flask with the cell density of 1 x lOVml. Desert culture fluid when cells spread to 85% , and in the deprived blood serum DMEM culture fluid for 24 hours. Cells were randomly divided into four groups:CD Time dependence of progesterone to ERK^CyclinDl protein;cells were treated with progesterone for 0^ 5^ 15 N 30 ^ 60^ 120 minutes respectively.(2) Concentration dependence of progesterone to ERK^CyelinDl protein : cells were treated with different dose of progesterone(1 X 10~\l x 10~\l x 10 "7 J x 10 ~\l x 10 "5 4 x 10 ~4mol/L) respectively for 30 minutes.(3) The vir - repression of RU486 to the action of progesterone in Ishikawa cells:Intervent cells with 0^P 1 x 10"8mol/L^ P 1 x 10"8mol/L + RU486 1 x 10~6mol/LNRU486 1 xlO"6mol/L respectively for 30 minutes .2) Extraction of total cellular protein.Desert drugs and bathe cells for 3 times and add cell disruption (50mmol/ L Tris2HCU10 mmol/L EDTA. 150mmol/L NaCl.1% Tritonx 2100^0. 02% NaN3^PMSF 0. lmg/ml). Spited cells on the ice and scrape cells quickly. Put cells in the refrigerator of -70T! after determine the concentration of protein.3 SDS - PAGE and Western blottingResults1. The time dependence of progesterone to total ERK, p - ERK and Cy-clinDl protein;The level of p - ERK was significantly lower than the control af-ter stimulated by progesterone at 5min. The lowest inactivation of p - ERK took place at 30min,and the level of them increased gradually at 60min and 120min after stimulation of 1 x 10~8mol/L progesterone in Ishikawa cells. The level of CyclinDl protein was lower than at 0 - 15min, and increased at 60min and 120min.2. The concentration dependence of progesterone on total ERK, p -ERK andCyclinDl protein;With increased doses of progesterone, the inactivation of p-ERK and CyclinDl protein increased gradually.3. The vir - repression of RU486 to the action of progesterone in Ishikawa cells: RU486 could not inhibit the inactivation of p - ERK and CyclinDl protein induced by progesterone in Ishikawa cells.DiscussionProgesterone is belong to steroid hormone superfamily. Traditional views include that progesterone combines with progesterone receptor located in cytoplasm and cell nucleus, enhances or suppresses target genetic transcription, produces specific proteins to induce classic genemic action. The studies in these years show that progesterone can combine with membrane progesterone receptor located cellular membrane to induce nongenomic action. ERK is an important member of mitogen - activated protein kinase superfamily. It is firstly phosphorylated by some kinds of growth factors , cell factors , tumor promotional factors , and then causes the expression of specific 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. Now there are a lot of studies a-bout the mechanism of activation of ERK, but there is few reports about the mechanism of it's inactivation.Our finding shows that there was expression of p - ERK protein and CyclinDl protein when there was no intervention on Ishikawa cell lines. The expression of p - ERK and CyclinDl protein was inhibited obviously after the action of progesterone for 5 minutes. This action was strongest at 30 min. It indicates that the effect of progesterone is not genomic action because it can't finishthe process of transcription and translation. Studies indicate that steroid hormone combines with receptor located in cellular nucleus and forms hormone - receptor complex . They begin to transcript after 10 minutes, and then translate and synthesize protein . So the quick action of progesterone is not through genomic progesterone receptor, but through membrane progesterone receptor. Our study shows that the quantity of CyclinDl protein was not changed. It begins to descend after 30 minutes. It was because that the process of transcription and translation need some time . So the expression of CyclinDl protein falls after the expression of p - ERK. Previous research shows that CyclinDl protein can produce a marked effect as the downstream protein of p - ERK. So we can presume that progesterone could affect ERK/CyclinDl signal transduction pathway and conduce to its depressant effect on cellular nucleus to induce volant and non-genomic action. It is possible that progesterone affects CyclinDl protein through another pathway. It needs further research in our future study.RU486 is a kind of artificial genomic progesterone receptor antagonist. In our study, when progesterone and RU486 were interfered in Ishikawa cell lines simultaneously, the effect of progesterone was not prevented . It further demonstrates that the action of progesterone was not through cellular nucleus. There must have certain membrane progesterone receptors in cellular membrane.It is not clear why the expression of p - ERK and cyclinDl protein incre-assed gradually after adding progesterone on Ishikawa cell lines. Foreign scholars confirm that genomic progesterone receptor could activate MAPK signal transduction pathway through traditional genomic action which leads to the increased quantity of p - ERK protein. Probably progesterone could combine with genomic progesterone receptor to generate this effect in Ishikawa cell lines. Progesterone activates p - ERK and CyclinDl protein and maintains the quantity of this two proteins to a higher level after the volant nongenomic action.Conclusion1 Progesterone can inhibit ERK/CyclinDl protein through nongenomic action in Ishikawa cell lines.2 The nongenomic action of progesterone generates through membrane progesterone receptor.3 The action of nongenomic action of progesterone in Ishikawa cell lines possess the time and concentration dependence. |
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