Expression Of Glycogen Synthase Kinase-3β In Endometrial Adenocarcinoma And The Regulating Action Of Progestin

Objectives:To investigate the expression of Glycogen synthase kinase-3 (GSK-3β) in endometrial adenocarcinoma,the effects of GSK-3βon the cell proliferation and neoplasm invasiveness of endometrial adenocarcinoma cell lines,and the regulating action of progestin on GSK-3β.Materials and Methods:GSK-3βexpression in paraffin embedded specimen of 25 patients of endometrial adenocarcinoma and 9 patients of normal endomerium were detected by IHC.GSK-3βmRNA expression in endometrial adenocarcinoma differentiated cell lines Ishikawa,HEC-1-A and undifferentiated cell line KLE were measured by Real Time PCR,and GSK-3βprotein expression were measured by Western Blot.Human endometrial adenocarcinoma cell lines Ishikawa,HEC-1-A and KLE were transfected with GSK-3βsiRNA.The expression of GSK-3βand caspase-3 protein were examined by Western blot.The proliferation of cells transfected with GSK-3βsiRNA or negative control siRNA were observed through BrdU incorporation assay while cell cycle distribution and apoptotic rate were detected by Flow cytometry(FCM).The neoplasm invasiveness of cells was observed through Matrigel invasion assay and Gelatin zymography.Ishikawa cell line were grown in medium depleted of steroid for 3 days. Treated by 10^-6mol/L,10^-7 mol/L and 10^-8mol/L MPA,cell viability was determined by MTT method at 24 h,48 h or 72 h treatment.The expression of AKT and pGSK-3β^Tyr216 protein were measured by Western blot at 48h treatment.MMP-2 and MMp-9 activity were detected by Gelatin zymography.Endometrial adenocarcinoma differentiated cell Ishikawa were transfected with AKT siRNA.The expression of AKT and GSK-3βmRNA were measured by Real Time PCR.The expression of AKT and GSK-3βprotein were examined by Western blot.The cell cycle distribution and apoptotic rate were detected by Flow cytometry(FCM).The expression of pCyclinD1^Thr286 and pCaspase-8^Thr62 protein were examined by Western blot.Results:IHC results of paraffin embedded specimen showed that compared with normal endomerium,endometrial adenocarcinoma was characteristic of high GSK-3βexpression.In endometrial adenocarcinoma,expression of GSK-3βwas correlated with clinical stage.Compared with StageⅠ,high GSK-3βexpression was observed in patients of StageⅡ-Ⅳ.While compared with Grade 1(Well differentiated) and Grade 2(Moderately differentiated), tumour Grade 3(poorly differentiated) showed high GSK-3βexpression.For high risks patients(Grade 3,deepmyometrial invasion,cervical extension), GSK-3βwas detected high expression compared with patients without high risks.Compared with differentiated Ishikawa cell line,HEC-1-A and KLE cell lines were detected of high GSK-3βexpression.The GSK-3βsiRNA had high inhibitory effect on the expression of GSK-3βgene.Compared with cells transfected with negative control siRNA or non-transfected cells,the cell proliferation of Ishikawa and HEC-1-A cells transfected with GSK-3βsiRNA was decreased significantly(P＜0.05).The proportion of cells in S phase was reduced and the apoptotic rate was increased as while as the expression of caspase-3 protein increased.But there was no statistics difference in different group of KLE cells.When GSK-3βexpression was blocked by RNA interference,cell invasiveness was downregulated(P＜0.05),and the expression of MMP-2 was decreased.10^-8mol/L MPA reduced the expression of pGSK-3β^Tyr216 protein in Ishikawa cell line(P＜0.05).10^-8mol/L MPA inhibited Ishikawa cell proliferation,but 10^-6mol/L MPA had no effect on the Ishikawa cell proliferation,10^-8mol/L MPA reduced MMP-2 and MMP-9 expression in Ishikawa cell,on the contrary,10^-8mol/L MPA elevated MMP-2 and MMP-9 expression in Ishikawa cell.MPA 10^-6mol/L,10^-7mol/L and 10^-8mol/L all inhibited AKT expression in Ishikawa cell.When AKT expression was blocked by RNA interference, pGSK-3β^Tyr216 protein expression was improved(P＜0.05).Compared with cells transfected with negative control siRNA or non-transfected cells,the cell proliferation of Ishikawa cells transfected with AKT siRNA was decreased significantly(P＜0.05).The proportion of cells in S phase was reduced as while as the expression of pCyclinD1^Thr286 protein elevated and the apoptotic rate was increased as while as the expression of pCaspase-8^Thr62 protein increased.Conclusions:Compared with normal endometrium,endometrial adenocarcinoma was characteristic of high GSK-3βexpression.Its high invasiveness could be attributed to its high GSK-3βexpression.GSK-3βcould promote the cell proliferation and neoplasm invasiveness of the differentiated cell lines Ishikawa and HEC-1-A,but had no effect on the undifferentiated cell line KLE.10^-8mol/L MPA inhibited the Ishikawa cell line AKT and GSK-3 expression,inhibited the cell viability and MMP-2,-9 expression.While 10^-6mol/L MPA inhibited AKT expression,but had no effect on neither GSK-3βexpression nor the Ishikawa cell viability,while promoted mmp-2,-9 expression.AKT could downregulated Ishikawa cell line GSK-3βexpression,while promoted the cell viability and the expression of MMP-2,-9.Thus downregulated GSK-3βwas suggested to play an essential role in progestin therapy,and might serve as a novel target in the treatment of endometriod adenocarcinoma.