[Activation of phosphatidylinositol 3-kinase-protein kinase B (PI3K-PKB) induced by 17beta-estradiol in endometrial carcinoma cell (Ishikawa)].
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.
Guo
Rui-Xia
RX
Department of Gynecology, People's Hospital, Peking University, Beijing 100044, China.
Wei
Li-Hui
LH
Wang
Jian-Liu
JL
Sun
Peng-Ming
PM
Sun
Xiu-Li
XL
D002867:Chromones / Q000494:pharmacology; D016889:Endometrial Neoplasms / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011960:Receptors, Estrogen / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured
D002867:Chromones; D004791:Enzyme Inhibitors; D004958:Estradiol; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011960:Receptors, Estrogen; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one