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Cyclooxygenase-2 (COX-2) is an enzyme expressed in the majority of human primary carcinomas and a large body of evidence suggests that COX-2 and its synthesized product PGE2, play a role in tumor invasion, angiogenesis, resistance to apoptosis, and suppression of host immunity. We investigated the effects of PGE2on COX-2 expression in human pancreatic cancer cells. COX-2 was expressed in 4/9 pancreatic cancer cell lines as determined by immunoblotting. COX-2 was highly expressed in L3.6pl cells, and its expression was further enhanced by PGE2 treatment. To determine which receptor subtype was responsible for PGE2-induced COX-2 expression, AH-6809 and SC-19220, an EP1/EP2 and an EP1-receptor antagonists respectively, were administered 30 min before the addition of PGE2. Pretreatment with AH-6809 but not with SC-19220 reduced COX-2 expression indicating EP2 but not EP4 is responsible for PGE2 action. Since the activation of EP2 leads to an increase in intracellular cAMP, the ability of PGE2 to stimulate adenyl cyclase was investigated. PGE2 increased cAMP levels in L3.6pl cells in a time and concentration-dependent manner. To determine whether increased PKA activity is responsible for COX-2 expression induced by PGE2, immuncytochemistry and confocal microscopy were performed. A redistribution of the catalytic subunit from a cytoplasmic distribution in untreated cells to a nuclear localization in PGE2treated cells was observed. Confocal microscopy also confirmed a PGE2-induced increase in CREB phosphorylation compared to the low basal levels found in unstimulated cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays also confirmed that PGE2 increased CREB DNA binding, where as H-89 and AH-6809 both inhibited CREB binding to a CRE located in the COX-2 promoter. Consistent with COX-2 playing a role in tumor proliferation, H-89, AH-6809, celecoxib, NS-398 and indomethacin, all reduced pancreatic cancer cell growth as measured by 3H-thymidine incorporation. In summary, we demonstrate that CREB is involved in PGE2-induced expression of COX-2 in L3.6pl pancreatic cancer cells mainly through activation of EP2 receptors and PKA-dependent CREB phosphorylation. Elucidating the pathways that control COX-2 expression may lead to a better understanding of its dysregulation in pancreatic carcinomas and facilitate novel therapeutic development.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]