The Role of 15-Lipoxygenase-1- and Cyclooxygenase-2-Derived Lipid Mediators in Endothelial Cell Proliferation

It is a generally accepted paradigm that there is a direct link between inflammation and tumor progression. During inflammation, there is increased formation of lipid hydroperoxides, mediated either non-enzymatically by reactive oxygen species or enzymatically by lipoxygenases (LOs) or cyclooxygenases (COXs). Lipid hydroperoxides undergo further oxidation into oxo-eicosatetraenoic acids (oxo-ETEs), which are produced and released by cells including macrophages and epithelial cells. Therefore, these oxo-ETEs could potentially mediate biological effects in an autocrine and/or a paracrine manner. In addition, oxo-ETEs conjugate intracellular glutathione (GSH) to form adducts which could serve as biomarkers of oxo-ETE formation. In this study, a targeted lipidomics approach combined with stable isotope dilution methodology was employed to identify and quantify lipid hydroperoxides and their metabolites formed in 15-LO-expressing mouse macrophage cell line (R15L cells) and COX-2 expressing cell models (RIES cells and Caco-2 cells) as well as in mouse hematocytes and primary human monocytes. 15-Oxo-5,8,11,13-(Z,Z,Z,E)-ETE (15-oxo-ETE) was identified and characterized as a major eicosanoid produced in both mouse and human macrophage 15-LO pathway. 15-Oxo-ETE was shown to be a metabolite of arachidonic acid (AA)-derived 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). A novel biological activity of 15-oxo-ETE was revealed, which involved inhibition of human umbilical vein endothelial cell (HUVEC) proliferation by suppressing DNA synthesis, implicating a potential anti-angiogenic role of 15-oxo-ETE. In addition to 15-oxo-ETE, another AA-derived eicosanoid 11-oxo-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (11-oxo-ETE), was identified as a major metabolite arising from COX-2-derived from 11(R)-hydroxyl-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (11(R)-HETE) in both rat (RIES) and human (Caco-2) epithelial cell lines. A specific liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM/MS) method revealed that both 11-oxo-ETE and 15-oxo-ETE were secreted in nM concentrations when AA was added to RIES and human Caco-2 cells. Surprisingly, 11(R)-HETE was an excellent substrate for 15-PGDH, with a catalytic efficiency similar to that found for 15(S)-HETE. In addition, it was demonstrated that aspirin significantly stimulated the production of 15(R)-HETE, which was then converted to 15-oxo-ETE by an unknown dehydrogenase (DH). These findings could have significant clinical implications since 15-PGDH is down-regulated during carcinogenesis, which in addition to increasing the pro-proliferative activity of PGE2 would prevent the formation of anti-proliferative 15-oxo-ETE from 15(S)-HETE. However, the formation of 15-oxo-ETE from 15(R)-HETE after aspirin treatment, through a pathway that does not involve 15-PGDH, could help counteract the increased pro-proliferative activity of PGE2.