Study Of Effects Of Estrogen And Its Receptor Antagonist On The Morphological Changes And The Expression Of P57^kip2,Cyclin D1 And CDK4 In Human Endometrioid Carcinoma Ishikawa Cells

Objective: To investigate the effects of estrogen and its receptor antagonists on the expressions of p57^kip2,Cyclin D1,CDK4 and morphological changes in human endometrioid carcinoma?EC?cells named Ishikawa?highly differentiated?in vitro.To explore the relationship between female hormonal regulation,cell cycle regulation and the development of EC,to provide some experimental evidence for the endocrine treatment of endometrial cancer.Methods: Ishikawa cells were cultured in vitro.?1?Study of the expressions of p57^kip2,Cyclin D1 and CDK4 and morphological changes in Ishikawa cells after estrogen intervention: The cells treated with three different concentrations of E2?10-6,10-8,10-10 mol / L?were used as the experimental group,and the cells were cultured in the same amount of complete culture medium without any drug as the control group.?2?Study of the expressions of p57^kip2,Cyclin D1 and CDK4 and morphological changes in Ishikawa cells after estrogen receptor antagonist intervention:The cells treated with estrogen receptor?ER?antagonist[ Tamoxifen?TAM??10-6 mol / L?,Faslodex?ICI182780??10-6 mol / L?],E2?10-6 mol / L?,E2 + TAM and E2 + ICI182780 were used as the experimental group.Cells were cultured with the same amount of complete medium without any drug as a control group.Ishikawa cells were cultured for 24,48,72,96 hour after,the proliferation of cells were detected by MTT.After 24,72 hour,the cells were observed on morphological changes by the inverted microscope and transmission electron microscope. RT-PCR was used to detect the expressions of p57^kip2,Cyclin D1 and CDK4 m RNA in cells after 24,72 hour.Western Blot?WB?was used to detect the expressions of p57^kip2,Cyclin D1 and CDK4 protein in cells after 24,48,72 hour.Result:1.MTT was used to observe the proliferation of Ishikawa cell?1?Effect of estrogen on the proliferation in Ishikawa cells: Compared with the control group,the proliferation of Ishikawa cells in low and middle concentration of E2 significantly increased?P<0.05?,and the proliferation of cells in high concentration group not obvious?P> 0.05?.With the prolongation of E2 effect,in the low concentration group,no significant difference found between 72 hour and 96 hour,while the proliferation of other groups showed an increasing trend?P<0.05?.?2?Effect of estrogen receptor antagonist on the proliferation in Ishikawa cells: Compared with the control group,the proliferation activity of Ishikawa cells in E2 group,ICI182780 group and E2 + ICI182780 group were decreased,the E2 + ICI182780 group decreased significantly?P<0.05?,while the proliferation of TAM group,E2 + TAM group were increased.Compared with E2 group,the proliferation of E2 + ICI182780 group was decreased?P<0.05?,and the proliferation of E2 + TAM group was enhanced.The proliferative activity of the experimental groups and the control group were increased with the prolongation of the time of action of the drug.?P<0.05?.2.Inverted microscope was used to observe the growth of Ishikawa cells?1?Effect of estrogen on the growth in Ishikawa cells: Compared with the control group,the density of Ishikawa cells were increased in E2 low and middle concentration groups.The density of Ishikawa cells in high concentration groups was decreased.The morphological changes of each group were not obvious.?2?Effect of estrogen receptor antagonist on the growth in Ishikawa cells:?1?Compared with the control group,the density of Ishikawa cells in E2 group,ICI182780 group and E2 + ICI182780 group were decreased significantly,while E2 + ICI182780 group decreased more obviously.TAM group was increased slightly.?2?Compared with E2 group,the cell density of E2 + ICI182780 group was decreased and E2 + TAM group was increased.The morphological changes of each group were not obvious.3.Transmission electron microscope was used to observe the ultrastructural changes of Ishikawa cells?1?Effect of estrogen on the ultrastructure in Ishikawa cells: Compared with the control group,in E2 low and middle concentration groups,mitochondria and endoplasmic reticulum of some Ishikawa cells were swelled slightly,swelling degree increased with the prolongation of E2.In E2 high concentration groups,the mitochondria of more Ishikawa cells were swollen obviously,which showed the phenomenon of apoptosis.With the prolongation of E2 effect,the mitochondria and endoplasmic reticulum of cells were swollen and vacuolized diffusely,and the apoptosis was increased.?2?Effect of estrogen receptor antagonist on the ultrastructure in Ishikawa cells: Compared with the control group,in E2 group,endoplasmic reticulum and mitochondria of some cells were swollen obviously,and the endoplasmic reticulum in different groups were swollen to some degree and showed apoptosis.Compared with the E2 group,in TAM group and ICI182780 group,the endoplasmic reticulum was swollen mildly,and the E2 + TAM and E2 + ICI182780 group was swollen obviously.4.Western Blot was used to detect the expressions of p57^kip2,Cyclin D1,CDK4 protein in Ishikawa cells?1?Effects of estrogen on the expressions of p57^kip2,Cyclin D1 and CDK4 protein in Ishikawa cells: The expressions of p57^kip2,Cyclin D1 and CDK4 protein was increased,with the increase of the concentration of E2 and the extension of the time?P<0.05?.The expressions of p57^kip2,Cyclin D1 and CDK4 protein were all higher in high concentration group.While the expressions of p57^kip2 protein was the lowest in low concentration group.The expressions of Cyclin D1 and CDK4 protein were the lowest expression in control group.There was a positive correlation between the expressions of the Cyclin D1 protein and CDK4 protein?P<0.05?.?2?Effects of estrogen receptor antagonist on the expressions of p57^kip2,Cyclin D1 and CDK4 protein in Ishikawa cells:?1?With the prolongation of drug effect time,the expression of p57^kip2 protein in E2 group,ICI182780 group and E2 + ICI182780 group were increased gradually?P<0.05?.The expression of p57^kip2 protein in TAM group and E2 + TAM group were decreased gradually,especially the expression of p57^kip2 protein in E2 + ICI182780 group was highest?P<0.05?,and lowest in TAM group.With the prolongation of drug effect time,the expressions of Cyclin D1 and CDK4 protein in E2 group,ICI182780 group and E2 + ICI182780 group were decreased gradually?P<0.05?.The expressions of Cyclin D1 and CDK4 protein in TAM group,E2 + TAM were increased gradually.Especially the expressions of Cyclin D1 and CDK4 protein were lowest in E2 + ICI182780 group?P <0.05?,and highest in TAM group.?2?Compared with E2 group,the expression of p57^kip2 protein in E2 + ICI182780 group was increased and decreased in E2 + TAM group?P <0.05?.The expressions of Cyclin D1 and CDK4 protein wre decreased in E2 + ICI182780 group and increased in E2 + TAM group?P<0.05?.5.RT-PCR was used to detect of the expressions of p57^kip2,Cyclin D1,CDK4 m RNA in Ishikawa cells?1?Effects of estrogen on the expressions of p57^kip2,Cyclin D1 and CDK4 m RNA in Ishikawa cells: With the prolongation of E2 effect time,the expression of p57^kip2 m RNA in low and middle concentration groups was decreased gradually,and in high concentration groups was increased gradually?P<0.05?.While the expression of Cyclin D1 m RNA in low and middle concentration group was increased gradually,and in high concentration groups was decreased gradually?P<0.05?.The expression of p57^kip2 m RNA in the high concentration group was highest,and in the middle concentration group was lowest at 24 and 72h?P<0.05?.The expression of Cyclin D1 m RNA in the middle concentration group was highest and in the high concentration group was lowest at 24 and 72 hour?P<0.05?.?2?Effects of estrogen receptor antagonist on the expressions of p57^kip2,Cyclin D1 m RNA in Ishikawa cells: With the prolongation of drugs effect time,the expression of p57^kip2 m RNA in ICI182780 group and E2 + ICI182780 group was increased gradually?P<0.05?, and in E2 group,TAM group and E2 + TAM group was decreased gradually?P<0.05?.The expression of Cyclin D1 m RNA in E2 group,ICI182780 group and E2 + ICI182780 group was decreased gradually?P<0.05?,and in TAM group and E2 + TAM group was increased gradually.Compared with the control group,the expression of p57^kip2 m RNA in E2 group,ICI182780 group,E2 + TAM group and E2 + ICI182780 group was increased?P<0.05?,especially in E2 + ICI182780 group was highest,while in TAM group was lowest.The expression of Cyclin D1 m RNA in E2,ICI182780 group and E2 + ICI182780 group was decreased,especially in E2 + ICI182780 group was lowest?P<0.05?.The expression of Cyclin D1 m RNA in TAM group and E2 + TAM group was increased,especially in E2 + ICI182780 group was highest?P>0.05?.Compared with E2 group,the expression of p57^kip2 m RNA in E2 + ICI182780 group was increased,and in E2 + TAM group was decreased?P<0.05?.The expression of Cyclin D1 m RNA in E2 + TAM group was increased,and in E2 + ICI182780 group was decreased?P<0.05?.conclusion:?1?With the increase of the concentration of E2 and the extension of the time,the expressions of p57^kip2,Cyclin D1 and CDK4 protein were increased gradually.?2?When the concentration of estrogen is low,the expressions of Cyclin D1 and CDK4 may be increased mainly,which play a positive role in the regulation of cell cycle progression and promote the proliferation of Ishikawa cells.A higher estrogen concentration may negatively regulate the cell cycle progression and arrest the cells in the G1 phase,thereby inhibiting the proliferation of Ishikawa cells and leading to obvious cell damage.?3?The application of ER antagonist ICI182780 alone or in combination with estrogen could inhibit the proliferation of Ishikawa cells by inducing the expression of p57^kip2,down-regulating the expressions of Cyclin D1 and CDK4,hinder the cell cycle progression.?4?The application of TAM alone or in combination with estrogen may play a weak estrogen-like role by up-regulating the expressions of Cyclin D1 and CDK4,downregulating the expressions of p57^kip2,and weakening promote Ishikawa cells.?5?ICI182780 may be more suitable than TAM for endocrine therapy in patients with EC,which provides some experimental evidence for the selection of EC endocrine therapy.