Regulation of Apoptosis in Eicosapentaenoic Acid-Treated HL-60 Cells

Background Neutrophil apoptosis is an important physiological process in the resolution of pulmonary inflammation. Previous studies have shown that eicosapentaenoic acid (EPA; 20:5n-3) increases the rate of apoptosis in a concentration- and time-dependent manner in HL-60 cells. However, it is not known if the EPA-induced apoptosis involves the lipoxygenase (LO) and cyclooxygenase (COX) enzymes or the downstream metabolic products of these enzymes. Thus, the objective of this study was to determine the effects of inhibitors LO and COX enzymes on apoptosis, viability, and necrosis in EPA-treated HL-60 cells. Materials and methods Cells were incubated with 50 μ m EPA in the presence of an enzyme inhibitor (1–10 μ m ) for 12 h. Compounds were used to inhibit COX 1 and 2 (ibuprofen), 5-, 12-, 15-LO (NDGA), 12-LO (baicalein), 5-LO (AA-861), and 5-LO activating protein (MK-886). Eicosanoid (0.001–1.0 μ m ) add-back experiments were also conducted; LTB4 and 5-HETE with 5-LO inhibition and 12-HETE with 12-LO inhibition. Flow cytometry was used to assess apoptosis. Results Inhibition of COX 1 and 2 had no effect on apoptosis. Inhibition of 5-LO and 12-LO significantly increased apoptosis in EPA-treated HL-60 cells. Addition of LTB4 reduced apoptosis to levels significantly lower than in HL-60 cells treated with EPA alone; 5-HETE and 12-HETE also lowered apoptosis to control levels. Conclusions These data indicate that inhibition of LO, particularly 5-LO, increased apoptosis in EPA-treated HL-60 cells. Furthermore, this study demonstrated that the products of the LO enzymes, particularly LTB4 , are critical in the regulation of apoptosis in EPA-treated HL-60 cells.