Figure 3.

PGE₂-induced activation of the PI3K-AKT and CREB pathway is required for COX-2 induction. (A, B) HCC827 cells and A549 cells were stimulated with 10 µM PGE₂ at the indicated time points. The activation of downstream signals of PGE₂ was analyzed by immunoblotting. (C, D) HCC827 cells were pretreated with PKA inhibitor H89 for 30 min and then treated with PGE₂ for an additional 30 min or 24 h. Phosphorylation of CREB (30 min) and COX-2 expression (24 h) were evaluated by Western blotting. H89 inhibits phosphorylation of CREB and PGE₂-induced COX-2 expression. (E) HCC827 cells were transfected with siRNA-control or siRNA-CREB for 48 h, and the cells were treated with or without PGE₂ for 24 h. COX-2 expression was evaluated by immunoblotting. Knockdown of CREB abrogates PGE₂-mediated upregulation of COX-2 expression. (F, G) HCC827 cells were pretreated with either PI3K inhibitor LY294002 or AKT inhibitor GDC0068 for 30 min and then treated with PGE₂ for an additional 24 h. COX-2 expression was evaluated by immunoblotting. Inhibition of PI3K-AKT reduces PGE₂-mediated COX-2 expression. (H) ex vivo human lung adenocarcinoma specimens were cultured with PGE₂ and PI3K-AKT inhibitors COX-2 mRNA expression was assessed by qPCR (***P < 0.001). The data represent tumor specimens from four individual lung cancer patients. (I, J) HCC827 cells were pretreated with the PI3K inhibitor LY294002 or AKT inhibitor GDC0068 for 30 min and then treated with PGE₂ for an additional 30min. CREB phosphorylation was evaluated by immunoblotting. PGE₂-induced phosphorylation of CREB is diminished by inhibition of PI3K-AKT. (K, L) HCC827 cells and A549 cells were cultured with PGE₂ for the indicated time intervals. Phosphorylation of ATK 1, AKT2, and AKT3 was detected by Western blotting using specific antibodies against each of the AKT isoforms. AKT1 is activated in both cell lines in response to PGE₂ stimulation.