Curcumin Inhibited The Growth And Proliferation Of Human Eutopic And Ectopic Endometrial Cells And Blood Vessel Formation

ObjectiveTo investigate the effects of Curcumin(Cur) on the proliferation and angiogenesis of ectopic and eutopic endometrial stromal cells from women with endometriosis indometriosis induced by Curcumin(Cur) in vitro.MethodsThe ectopic and eutopic endometrial stromal cells were isolated and cultured in vitro.Then they were identified by vimentin immunocytochemistry stainning.Both the ectopic and eutopic endometrial stromal cells were exposed to Cur in different concentration(0,10μmol/L,20μmol/L,40μmol/L,50μmol/L) for 0～96h.The proliferation and morphological changes were observed by inverted microscope.The growth and inhibition of ectopic and eutopic stromal cells were measured by MTT assay.The apoptosis and cell cycle distribution induced by different concentration of Cur on ectopic and eutopic endometrial stromal cells were assayed by flow cytometric analysis.The expression of VEGF induced by Cur was analyzed by immunocytochemistry.ResultsTwelve cases were successfully cultured in all 14 cases of ectopic endometrial cells.The successful rate was 85.7%.All the 16 cases of eutopic endometrial cells were cultured successfully.The positive rate of vimentin was above 95%in ectopic and eutopic endometrial cells identified by immunocytochemistry staining.The morphous of ectopic and eutopic cells had no obviously difference,the cells became fusiform growing after 24 hours.They became longer followed the time,and arranged to braid appearance.The cells had no clear boundary and contact inhibition.Both ectopic and eutopic endometrial stromal cells had no morphological changes in 10 serial subcultivations.The growth and proliferation depressed in ectopic and eutopic cells after the action of Cur.The cells morphological changes were slightly when exposed to Cur at a concentration of 10μmol/L for 48 hours.There were only individual cells floated in the culture fluid.The number of adherent cell was decreased and the morphological changes was obviously at a concentration of 50μmol/L for 48hours.These changes were significant accompany to the time spread.MTT assay demonstrated that Cur could inhibit the growth and proliferation of ectopic and eutopic endometrial stromal cells in a time-and-dose dependent fashion.The cell inhibition rates of ectopic cells induced by Cur at a concentration of 10μmol/L for 24 h,48 h,72 h,96 h were 33.74%,52.23%,62.04%,68.72%,and those of eutopic cells were 13.13%,30.29%,44.26%,58.14%.The inhibition rates of ectopic cells at a concentration of 1ug/ml were 51.61%,60.64%,70.00%,74.31%,and those of eutopic cells were 24.97%,36.70%,52.68%,67.35%.The inhibition of cell growth induced by Cur in ectopic cells was stronger than in eutopic cells in a time-and-dose dependent fashion(P＜0.05).Annexin V-FITC and PI double stainning showed that Cur could increased the apoptotic and eutopic stromal cells in a dose depend fashion(P＜0.05).These were more obvious in ectopic cells than in eutopic cells(P＜0.05).Cur blocked the cell cycle of the two groups.The number of ectopic cells and eutopic cells were increased in GI stage and decreased in S stage.It was more manifest in the higher concentration(P＜0.05).The percentage of GI stage of ectopic cells was increased from 19.45±3.47 to 34.92±4.14(10μamol/L) and 51.47±5.07(20μmol/L ),and that of eutopic cells increased from 58.50±8.96 to 74.80±6.96(40μmol/L)and 84.35±5.92(50μmol/L).The expression of VEGF in ectopic cells and eutopic cells was significantly decreased after the action of Cur(P＜0.01).The positive rate accumulated points of VEGF in ectopic cells was decreased from 120.80±19.46 to 70.60±5.32(20μmol/L) and 50.80±9.09(50μmol/L),and that in eutopic cells was decreased from 83.00±9.35 to 54.40±5.32(20μmol/L)and 40.60±4.88(50μmol/L).The expression of VEGF was decreased more obvious in ectopic cells than in eutopic cells(P＜0.05)ConclusionCur could signficantly inhibit the proliferation and enhance the apoptosis of ectopic and eutopic endometrial stromal cells from women with endometriosis in a time-and dose dependent fashion.Cur could obviously decrease the expression of VEGF.The actions above were more stronger in ectopic endometrial stromal cells than in eutopic endometrial stromal cells.