Abstract LB-9: Cyclooxygenase-2/15-prostaglandin dehydrogenase derived endogenous canonical NF-κB inhibitors

Abstract

Abstract In colon, breast, and lung cancers, up-regulation of cyclooxygenase-2 (COX-2) and down-regulation of 15-prostaglandin dehydrogenase (15-PGDH) has been hypothesized to lead to a pro-proliferative, angiogenic, and chronically inflamed state. Prostaglandin E2 has been thought to drive the feed forward aspect of this pathology; however, other endogenous lipids may be involved in this process. COXs also mediate the metabolism of arachidonic acid (AA) to hydroxyeicosatetraenoic acids (HETEs), which can then be further oxidized by dehydrogenases to the corresponding keto-products. We have previously established that 11-oxo-eicosatetraenoic acid (oxo-ETE) is a major COX-2/15-PGDH derived metabolites formed from human cells expressing COX-2 treated with AA. 11-oxo-ETE has been identified from human isolates yet currently has no known receptors or known physiologic function. Earlier studies had indicated that molecules possessing a similar α,β-unsaturated ketone moiety may act as canonical Nuclear Factor-κB (NF-κB) inhibitors. Therefore, the present study was aimed to investigate the signaling of this novel COX-2/15-PGDH-derived eicosanoid. The stability and uptake of 11-oxo-ETE into human umbilical vein endothelial cells (HUVECs) was quantified with stable isotope dilution chiral liquid chromatography coupled with electron capture atmospheric pressure chemical-ionization/mass spectrometry. Tumor necrosis factor (TNF)-α induced NF-κB p50/p65 subunit nuclear translocation and consensus DNA binding was reduced by pre-incubation with 11-oxo-ETE. Dosage and reduction in NF-κB were consistent with the levels for the prostaglandin analog 15-deoxy-prostaglandin J2 (15d-PGJ2). Using a HEK293 line stably expressing a firefly luciferase reporter driven by a 5x-NF-κB consensus sequence repeat, 11-oxo-ETE and its methyl ester were shown to significantly inhibit NF-kB signaling induced by TNF-α treatment. Ethacrynic acid (EA) was used in an additional experiment to inhibit cellular glutathione-S-transferases, and this increased the ability of 11-oxo-ETE to inhibit NF-κB signaling in the reporter assay. In vitro biochemical studies also revealed inhibition of the activating kinase Inhibitor of nuclear factor-κB kinase subunit β (≥KKβ) by 11-oxo-ETE. In summary, the present study has established that the COX-2/15-PGDH derived 11-oxo-ETE can enter target endothelial cells and inhibit NF-κB p50/p65 mediated signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-9. doi:1538-7445.AM2012-LB-9