Effect Of Angiotensin â…¡ Receptor Inhibitor On Proliferation And Apoptosis Of Estrogen Induced Human Endometrial Carcinoma Cells

High level estrogen stimulation without antagonism of progestogen has close relationship with endometrial carcinoma. The mechanism is mainly about estrogen receptor-dependent effect, while less about estrogen receptor-independent effect. Recently,study shows that AngiotensinⅡreceptor 1 (AT1-R) can activate MAPK of SKBR3 breast cancer cell line with negative expressed estrogen receptor and promote the proliferation of breast cancer cell. According to this,we presume that whether estrogen can promote the proliferation of ER low-expressed endometrial carcinoma cell via AT1-R. We proceed the study from estrogen receptor-independent mechanism, and explore molecule mechanism of endometrial carcinoma from a new point of view,hoping to control estrogen receptor-independent effect and suppressing the cell cycle. So as to provide new theory of the treatment of ER negative-expressed endometrial carcinoma.ObjectiveImmunocytochemical assay,MTT assay,fluorescence activated cell sorting technique and Western blot were applied to detect the expression of AT1-R,PI3K,p-Akt and ERK protein and the relationship among them in HEC-1A cell. To explore the effects of estrogen and AngiotensinⅡreceptor 1 inhibitor(saralasin) on cell proliferation,cell cycle and apoptosis in HEC-1A cell.Materials and Methods1. materialsendometrial carcinoma cell line HEC-1A;fetal bovine serum; McCoy's 5a nutrient medium;water-solubility 17β-estrogen; AT1-R antagonist (saralasin) estrogen receptor antagonist (ICI182780); AT1-R antibody,PI3K antibody,p-Akt antibody,ERK antibody;PI; Annexin V-FITC apoptosis detection kit;carbon dioxide homeothermia incubator; inverted phase contrast microscope; laser confocal microscopy; flow cytometer; speeding refrigerated centrifuge; electrophoresis apparatus; automatic gel-image formation analysator.2. MethodsHEC-1A cell is cultured in McCoy's 5a nutrient medium which contains 10% fetal bovine serum,100U/ml penicillin and 100U/ml streptomycin in 5%CO2 homeothermia incubator. When HEC-1A cell grow to over 95%,passaging it by 0.25% pancreatic enzyme.Immunocytochemical assay was applied to detect the expression of AT1-R,PI3K,p-Akt and ERK protein in HEC-1A cell. The effects of estrogen and saralasin on cell proliferation, cell cycle distribution and apoptosis of HEC-1A cell were detected by MTT assay and fluorescence activated cell sorting technique.The expression of ERK and p-Akt protein in HEC-1A cell were analyzed by Western blot after treated by estrogen and saralasin.3. Statistical AnalysisSPSS 13.0 software was employed to analyze all data. Statistical evaluation was performed using One Way ANOVA, LSD-t test. Data was presented by (?)±s, P<0.05 was considered as statistical significance.Results1,Expression of AT1-R, PI3K. p-Akt and ERK protein in HEC-1A cell.AT1-R,PI3K,p-Akt,and ERK protein are observed by laser confocal microscopy. Outcome shows that there is green fluorescence in the cytoplasm and nucleus.Nucleus are blue-stained.ATl-R,PI3K and ERK protein are all positive expressed in the cytoplasm, while p-Akt protein is positive expressed in the nucleus.2,The effect of estrogen and saralasin on proliferation of HEC-1A.MTT showed that estrogen can promote the proliferation of HEC-1A after 5 min.This effect can last for 15min. Saralasin obviously inhibited the proliferation of estrogen induced HEC-1A cell after 10min,while ICI182780 could not inhibit the proliferation of estrogen induced HEC-1A cell.3,The effect of estrogen and saralasin on cell cycle and apoptosis of HEC-1A.Estrogen decreased G0-G1 phase proportion and increased S phase proportion significantly,with small number of apoptotic cells after 10min. Saralasin obviously induced apoptosis of estrogen induced HEC-1A cell, increased GO-G1 phase proportion and decreased S phase proportion.ICI182780 had no obviously effect on cell cycle and apoptosis of estrogen induced HEC-1A cell.4,The effect of estrogen and saralasin on the expression of ERK and p-Akt protein in HEC-1A cell.Estrogen could up-regulated the expression of ERK1/2 protein after 10min.While saralasin down-regulated the expression of ERK1/2 protein after 10min. ICI182780 had no obvious effect on the expression of ERK1/2 protein.All the drugs had no obvious effect on the expression of P-Akt protein. Conclusions1,Estrogen can promote the proliferation of HEC-1A cell. Estrogen receptor Inhibitor ICI182780 has no obvious effect on proliferation,cell cycle and apoptosis of estrogen induced HEC-1A cell. So we suppose that the effect of estrogen on HEC-1A cell is ER-independent.2,AT1-R is strong expressed in HEC-1A cell.After inhibiting AT1-R,the proliferation of HEC-1A cell decreased significantly, G0-G1 phase proportion increased and S phase proportion decreased.The AT1-R inhibitor can also induce apoptosis of estrogen induced HEC-1A cell and up-regulated the expression of ERK1/2 protein. So we think that estrogen can promote proliferation of HEC-1A cell via AT1-R.The mechanism maybe activation of MAPK signaling pathway.3,Estrogen receptor Inhibitor has no obvious effect on HEC-1A cell. AT1-R inhibitor can inhibit proliferation of HEC-1A cell.For ER low-expressed endometrial carcinoma, AT1-R inhibitor can provide new theory of the treatment.