Aspirin-triggered lipoxins (15-epi-LX) are generated by the human lung adenocarcinoma cell line (A549)-neutrophil interactions and are potent inhibitors of cell proliferation

Abstract

Background: The mechanism by which aspirin (ASA) acts to protect against human cancer is not yet known. We recently showed that ASA triggers the formation of a new series of potent bioactive eicosanoids, 15-epi-lipoxins (15-epi-LXs or ASA-triggered LX [ATL]), during interactions between prostaglandin endoperoxide synthase-2 (PGHS-2) in endothelial cells and 5- lipoxygenase (LO) in leukocytes. Here, we investigated the trans-cellular biosynthesis of these eicosanoids during costimulation of the human tumor A549 cell line (alveolar type II epithelial cells) and neutrophils, and evaluated their impact on cell proliferation. Materials and Methods: A549 cells in isolated neutrophils were coincubated and mRNA expression levels of key enzymes in eicosanoid biosynthesis were measured. In addition, product formation was analyzed by physical methods. The effect of LX on cell proliferation was determined by using a soluble microculture tetrazolium (MTT) assay and by measuring [3H]-thymidine incorporation. Results: Interleukin-1β (IL-1β)-primed A549 cells showed selective elevation in the levels of PGHS-2 mRNA and generated 15-hydroxyeicosatetraenoic acid (15- HETE). ASA markedly increased 15-HETE formation by A549 cells, while treatment with an inhibitor of cytochrome P450 reduced by ~50%, implicating both PGHS-2-and cytochrome P450-initiated routes in 15-HETE biosynthesis in these cells. Maximal production of 15-HETE from endogenous sources occurred within 24 hr or cytokine (IL-1β) exposure and declined thereafter. Chiral analysis revealed that ~85% of ASA-triggered epithelial-derived 15-HETE carries its carbon 15 alcohol group in the R configuration. Costimulation of ASA-treated A549 cells and polymorphonuclear neutrophilic leukocytes (PMN) led to production of both LXA4, and LXB4 as well as 15-epi-LXA4 and 15- epi-LXB4 (9.5 ± 0.5 ng LX/107 A549 cells). 15-epi-LXA4 accounted for ~88% of the total amount of LXA4 produced. In addition to LXs, stimulation of A549 cells and PMN also liberated substantial amounts (77.2 ± 8.2 ng/107 A549 cells) of peptidoleukotrienes (pLTs), which were not generated by either cell type alone. Addition of ASA to these co-incubations led to an increase in the amounts of LXs generated that was paralleled by a decreased in pLTs. LXA4, LXB4, 15-epi-LXA4 and 15-epi-LXB4, as well as dexamethasone, inhibited cell proliferation at 100 nM range with a rank order of activity of 15-epi-LXB4 >>> LXB4 > dexamethasone ≤ 15-epi-LXA4 > LXA4. Conclusions: These results indicate that ASA promotes the formation of antoproliferative 15-epi-LXs by epithelial cell-leukocyte interactions. Moreover, they suggest that these novel eicosanoids, when generated within the microenvironment of tissues, may contribute to ASA's therapeutic role in decreasing the risk of human cancer.