vity directly, simultaneously. Using phosphor Akt polyclonal antibody on western blot and Akt kinase activity assay, Akt activation stimulated by M estrogen after exposure at different lengths of time and that by varied concentrations of estrogen were investigated in two endometrial carcinoma cell lines. A rapid activation of Akt was observed by this steroid. Within min, estrogen induced a significant increase of Akt phosphorylation and the peak level of P Akt could be observed at min in Ishikawa cells and min in HEC A cells and persisted for at least h in both cell lines , whichwas in parallel with Akt kinase activity assay. Akt activation increased gradually with increased concentrations of the estrogen showing a dose dependent manner in both cells . Comparable expression levels of Akt mRNA at different time points in each cell line after stimulation with M E suggesting non transcriptional effect involvement To test if Akt activation by estrogen in the two cell lines was by transcriptional or non transcriptional effect, we detected changes of Akt mRNA expression at min, min or min and h after stimulation with M E in the two cell lines.
As shown in Fig Akt and mRNAs were strongly expressed Quizartinib selleckchem but Akt mRNA was only weakly expressed in the both cell lines. None of the Akt, or mRNA expression changed at any of the different time points in either Ishikawa or HEC A cells. Effect of LY, a specific PIK inhibitor, on estrogen induced activation of Akt Akt activation and Akt kinase activity decreased with increasing doses of LY, and they were completely blocked when the concentration ofLYwas augmented to M . Similar results were obtained when Akt enzyme activity was assessed. Correlation between ER and E induced activation of Akt ER antagonist, ICI , was employed to observe its effect on E induced Akt activation.
After cotreating the cells with both ICI and E for min or min , with increasing concentrations of ICI , levels of activated Akt decreased gradually to about basal levels in Ishikawa but no change was observed in HEC A Discussion Icariin Here, we reported for the first time that estradiol, through non transcriptional action, can activate promptly PIK Akt signaling pathway and this action of E appears to be ER dependent in ER positive endometrial carcinoma cell line, such as Ishikawa and ER independent as shown in poorly expressed ER endometrial carcinoma cell line, HEC A. We tested ER and status in the two cell lines and approved that ER were positively expressed in Ishikawa cells but poorly expressed in HEC A cells, which were in accordance with the other reports . Estrogen can activate Akt by non transcriptional mechanism in endometrial cancer cells According to the traditional model, steroid hormones including estrogen bind to intracellular receptors and sub