Study Of Effects Of Estrogen,Progesterone And Estrogen Receptor Antagonist On The Expressions Of ER,PR And P57^kip2 In Human Endometrioid Carcinoma JEC Cells

Objective:To explore the effects of estrogen and progesterone on the expression of estrogen receptor subtype?ER?,ER??,progesterone receptor subtype?PR-A,PR-B?and p57^kip2 protein in human endometrioid carcinoma cells named JEC.To investigate the relationship between female hormone,cell cycle regulation and the development of EC.Methods:The experimental group: The JEC cells?moderately differentiated EC cells?cultured in vitro were interfered with three different concentrations of ?-Estradiol?E₂??10-6 mol/L,10-8 mol/L,10-10 mol/L?and progesterone?P??10-4 mol/L,10-6 mol/L,10-8 mol/L?respectively.The control group: JEC cells were cultured in the culture medium without drug.MTT was used to detect the growth condition of JEC cells after 24,48,72,96 h.After 24,48,72 h,the JEC cells were observed on growth condition and morphological changes by the inverted microscope and transmission electron microscope.Western Blot?WB?was used to detect the expression of ER?,ER?,PR-A,PR-B and p57^kip2 protein in JEC cells after 24,48,72 h.Results: 1.MTT was used to observe the growth condition of JEC cells:With the extension of the time and the increase of the concentration of E₂ and P,the control group and the experimental groups of JEC cells grew gradually?P<0.05?.Three concentrations of E₂ all could promote the growth of JEC cells,and with the increase of the concentration of E₂ to promote more obvious effect?P<0.05?.The low and medium concentrations of P could promote the growth of JEC cells,while the high concentration of P could inhibit the growth of JEC cells?P<0.05?.2.Inverted microscope was used to observe the growth of JEC cells:After 72 h,in the control group,JEC cells were polygonal,long spindle shaped,irregular shape,and the local cells formed a lumen like structure,accompanied by a small amount of apoptosis.Compared with the control group,the cells density of each concentration group of E₂ increased more obviously,and was higher in the high concentration group;In the low and medium concentration of P group,the cells density was increased significantly,and the cells density in the high concentration group was decreased.3.Transmission electron microscope was used to observe the ultrastructural changes of JEC cells:After 72 h,the control group of JEC cells were round or oval,microvilli on cell surface,organelles such as secretory vesicles,mitochondria,endoplasmic reticulum,golgi complex and secondary lysosome were seen in the cytoplasm.The cell nucleus were irregular,with prominent nucleoli.Compared with the control group of cells,in the E₂ group,the secretory vesicles of some cells was not obvious,the endoplasmic reticulum was swollen,and autophagy was observed,in the medium and high concentration groups,microvilli on the surface of cells was swelling,which in the high concentration group was more obvious;Autophagy was found in the cytoplasm of P groups,microvilli and endocrine vesicles in the high concentration group of cells were increased.4.WB was used to detect the expression of ER?,ER?,PR-A,PR-B and p57^kip2 protein in JEC cells:The JEC cells were treated with three different concentrations of E₂ and P.ER?,PR-A,PR-B did not expressed in experimental group or control group,ER? and p57^kip2 expressed in varying degrees.?1?The expression of ER? and p57^kip2 protein after E₂ interference: With the increase of the concentration of E₂ and the extension of the time,the expression of ER? protein was increased and the expression of p57^kip2 protein was decreased?P<0.05?.There was a negative linear correlation between the expression of ER? protein and p57^kip2 protein.?2?The expression of ER? and p57^kip2 protein after P interference.?1?Comparison of the same action time: With the increase of the concentration of P,the expression of ER? protein increased gradually at 24,72 h.At 48 h,the expression of ER? protein in the high concentration group was the highest,and in the medium concentration group was the lowest?P<0.05?.With the increase of the concentration of P,the expression of p57^kip2 protein was increased?P<0.05?.?2?Comparison of the same concentration: With the extension of P interference time,the expression of ER? protein was increased in high concentration group,in the low and medium concentration group,the expression of ER? protein was the highest at 72 h and the lowest at 48h?P<0.05?.The expression of p57^kip2 protein was increased with the extension of P interference time?P<0.05?.There was no correlation between the expression of ER? protein and p57^kip2 protein.Conclusion:The growth and differentiation of endometrioid carcinoma may be regulated by the female hormone and cell cycle.It is possible that E₂ can inhibit the expression of p57^kip2 protein by regulating the expression of ER? protein in JEC cells and relieve the negative effect of p57^kip2 on the cell cycle progression,and promote the growth of JEC cells evidently,which leads to the differentiation of tumor cells develope in the worse direction.P can increase the expression of ER? and p57^kip2 protein in JEC cells,but in the low and medium concentration group,ER? protein promote the growth of JEC cells,while in the high concentration group,p57^kip2 protein can block cell cycle progression,and inhibit the growth of JEC cells,which leads to the differentiation of the tumor cells develope in a better direction.ER?,PR-A and PR-B protein are not expressed in JEC cells,E₂ and P are unable to induce the expression of them.It is possible that combined detection of the expression of ER?,ER?,PR-A,PR-B and p57^kip2 protein in EC is more conducive to assessing the occurrence and development of EC.Objective: To explore the effect of estrogen receptor antagonist on the expression of estrogen receptor subtype?ER?,ER??,progesterone receptor subtype?PR-A,PR-B?and p57^kip2 protein in human endometrioid carcinoma cells named JEC.To investigate the relationship between estrogen regulation,cell cycle regulation and the development of EC.Methods: The experimental group: The JEC cells?moderately differentiated EC cells?cultured in vitro were interfered with ?-Estradiol?E₂??10-6 mol/L?and two types of estrogen receptor antagonists,tamoxifen?TAM?and fulvestrant?ICI182780??10-6 mol/L?.The control group: JEC cells were cultured in the culture medium without drug.MTT was used to detect the growth condition of JEC cells after 24,48,72,96 h.After 24,48,72 h,the JEC cells were observed on growth condition and morphological changes by the light microscopy and electron microscopy.Western Blot?WB?was used to detect the expression of ER?,ER?,PR-A,PR-B and p57^kip2 protein in JEC cells after 24,48,72 h.Results:1.MTT was used to observe the growth condition of JEC cells:With the extension of the drug interference time,the control group and the experimental groups of JEC cells grew gradually?P<0.05?.Compared with the control group,the proliferation ability of JEC cells in E₂ group was enhanced?P<0.05?,the proliferative ability of JEC cells in ICI182780 group was lower?P<0.05?,and the proliferation ability of JEC cells in TAM group was higher?P>0.05?.Compared with the E₂ group,the proliferation ability of JEC cells in E₂+ICI182780 group was lower?P<0.05?,the proliferative ability of JEC cells in E₂+TAM group was lower?P>0.05?.2.Inverted microscope was used to observe the growth of JEC cells:After 72 h,in the control group,JEC cells were polygonal,long spindle shaped,irregular shape,and the local cells formed a lumen like structure,accompanied by a small amount of apoptosis.Compared with the control group,the cells density of E₂ group was increased and the cells density was decreased in ICI182780 group,and the cells density changes of other groups were not obvious;Compared with E₂ group,the cells density of E₂+TAM group and E₂+ICI182780 group were decreased;And the morphological changes of cells in each group were not obvious.3.Transmission electron microscope was used to observe the ultrastructural changes of JEC cells:After 72 h,the control group of JEC cells were round or oval,microvilli on cell surface,organelles such as,mitochondria,endoplasmic reticulum,golgi complex and secondary lysosome were seen in the cytoplasm,secretory vesicles were not obvious,and lipid droplets could be seen occasionally.The cell nucleus were irregular,with prominent nucleoli.Compared with the control group of cells,the pathologic mitosis was easy to see in the E₂ group of cells;Autophagy was increased in the cytoplasm of some cells in ICI182780 group.Compared with the E₂ group of cells,the pathologic mitosis was decreased in the E₂+TAM group and E₂+ICI182780 group.4.WB was used to detect the expression of ER?,ER?,PR-A,PR-B and p57^kip2 protein in JEC cells:ER?,PR-A,PR-B of JEC cells did not expressed in experimental group or control group,ER? and p57^kip2 expressed in varying degrees.?1?The expression of ER? protein in in JEC cells of experimental group or control group:?1?Comparison of the same action time: Compared with the control group,the expression of ER? protein in E₂ group was increased,the expression of protein in ICI182780 group and TAM group were decreased,and the ICI182780 group decreased significantly?P<0.05?.Compared with E₂ group,the expression of ER? protein in E₂+ICI182780 group and E₂+TAM group were decreased,and E₂+ICI182780 group decreased significantly?P<0.05?.?2?Comparison of the same drug groups: With the extension of drug action time,the expression of ER? protein in E₂ group was increased,the expression of protein in ICI182780 group and TAM group were decreased?P<0.05?.?2?The expression of p57^kip2 protein in JEC cells of experimental group or control group:?1?Comparison of the same action time: Compared with the control group,the expression of p57^kip2 protein in E₂ group was decreased,the expression of protein in ICI182780 group and TAM group were increased,and the ICI182780 group increased significantly?P<0.05?.Compared with E₂ group,the expression of p57^kip2 protein in E₂+ICI182780 group and E₂+TAM group were increased,and E₂+ICI182780 group increased significantly?P<0.05?.?2?Comparison of the same drug groups: With the extension of drug action time,the expression of p57^kip2 protein in E₂ group was decreased,the expression of protein in ICI182780 group and TAM group were increased?P<0.05?.There was a negative linear correlation between the expression of ER? protein and p57^kip2 protein after interference of TAM,ICI182780.Conclusion: ICI182780 have a stronger role in antagonizing estrogen,and may induce the expression of p57^kip2 protein by down-regulating the expression of ER? protein in JEC cells,block the cell cycle progression and inhibit the growth of tumor cells.TAM have a weak role in antagonizing estrogen,and it can promote the growth of JEC cells weakly.It is possible that TAM acts as estrogen receptor antagonist and also plays a weak positive role in JEC cells at the same time.ER?,PR-A and PR-B protein are not expressed in JEC cells,TAM and ICI182780 are unable to induce the expression of them.It is suggested that ICI182780 have a stronger role in antagonizing estrogen than TAM in JEC cells,and it is more suitable for the treatment of EC patients.This has important reference value for the selection of EC endocrine therapy drugs.