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Title: [Activation of phosphatidylinositol 3-kinase-protein kinase B (PI3K-PKB) induced by 17beta-estradiol in endometrial carcinoma cell (Ishikawa)].
Author: Guo RX, Wei LH, Wang JL, Sun PM, Sun XL.
Journal: Zhonghua Fu Chan Ke Za Zhi; 2004 Jul; 39(7):469-73. PubMed ID: 15347471.
Abstract:
OBJECTIVE: Cellular response to estradiol is mediated both by estrogen receptor (ER) binding to estrogen response element (ERE) and by non-nuclear actions like activation of signal transduction pathways such as mitogen activated protein kinase (MAPK) pathway. However, the signal transduction of estrogen involving phosphatidylinositol 3-kinase-protein kinase B (PI3K-PKB) is not clear in endometrial carcinoma. Our purpose was to study if PI3K-PKB signaling pathway could be activated rapidly by 17beta-E(2) through non-nuclear action and also, whether PI3K inhibitor, LY294002, could inhibit such non-nuclear action of 17beta-E(2) in endometrial carcinoma cell line Ishikawa. METHODS: Levels of phosphorylated PKB (Ser473 site, p-PKB) and total PKB were examined by western blotting in Ishikawa cells after stimulation with 17beta-E(2) at 1 x 10(-6) mol/L for different time periods and at varied doses for 30 min. Optimal time and appropriate dose for 17beta-E(2) to activate PKB in Ishikawa cells were observed. Inhibitory effect of LY294002 on activation of PKB induced by 17beta-E(2) was also studied. p-PKB/PKB ratio was used to indicate levels of activation of PKB. RESULTS: p-PKB/PKB at 15 min (0.533 +/- 0.029) was significantly higher than the control (0.361 +/- 0.029, P < 0.05). Maximal activation of PKB (maximal levels of p-PKB/PKB) took place at 30 min (0.666 +/- 0.021, P < 0.001) and the activation persisted at least 2 h after stimulation with 1 x 10(-6) mol/L 17beta-E(2) in Ishikawa cells. With increased doses of 17beta-E(2) (vehicle, 1 x 10(-10), 1 x 10(-8), 1 x 10(-6), 1 x 10(-4) mol/L), the activation of PKB (p-PKB/PKB was 0.300 +/- 0.098, 0.312 +/- 0.081, 0.625 +/- 0.100, 0.771 +/- 0.041, 0.902 +/- 0.043 accordingly) increased gradually (in comparison with vehicle, P > 0.05, < 0.05, < 0.05, < 0.001 accordingly). PI3K inhibitor, LY294002 could inhibit the activation of PKB induced by 17beta-E(2) in Ishikawa cells. Levels of PKB (p-PKB/PKB) decreased (0.443 +/- 0.032, 0.415 +/- 0.032, 0.111 +/- 0.035, 0, 0) gradually with increased concentrations (vehicle, 0.1, 10, 50, 100 micro mol/L) of LY294002 (compared with vehicle, P > 0.05, < 0.05, < 0.001, < 0.001) and 50 micro mol/L LY294002 completely blocked the activation of PKB induced by 17beta-E(2). CONCLUSIONS: 17beta-estradiol, by non-nuclear action, can activate promptly PI3K-PKB signaling pathway in endometrial carcinoma cell line, Ishikawa. This action of 17beta-E(2) is PI3K dependent and can be inhibited or blocked by LY294002.

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