PPARδ signaling mediates the cytotoxicity of DHA in H9c2 cells.

Docosahexaenoic acid (22:6n3, DHA) is an n-3 polyunsaturated fatty acid (PUFA) known to affect numerous biological functions. While DHA possesses many properties that impact cell survival such as suppressing cell growth and inducing apoptosis, the exact molecular and cellular mechanism(s) remain unknown. Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that regulate many cell pathways including cell death. As DHA acts as a ligand to PPARs the aim of this study was to examine the involvement of PPARδ in DHA-mediated cytotoxicity toward H9c2 cells. Treatment with DHA (100μM) resulted in a significant decline in cell viability, cellular metabolic activity and total antioxidant capacity coinciding with increased total proteasome activities and activity of released lactate dehydrogenase (LDH). No changes in reactive oxygen species (ROS) production or accumulation of lipid peroxidation products were observed but DHA promoted apoptotic cell death as detected by flow cytometry, increased caspase-3 activity and decreased phosphorylation of Akt. Importantly, DHA enhanced PPARδ DNA binding activity in H9c2 cells strongly signifying that the cytotoxic effect of DHA might be mediated via PPARδ signaling. Co-treatment with the selective PPARδ antagonist GSK 3787 (1μM) abolished the cytotoxic effects of DHA in H9c2 cells. Cytotoxic effects of DHA were attenuated by co-treatment with myriocin, a selective inhibitor of serine palmitoyl transferase (SPT), preventing de novo ceramide biosynthesis. LC/MS analysis revealed that treatment with DHA resulted in the accumulation of ceramide, which was blocked by GSK 3787. Interestingly, inhibition of cytochrome P450 (CYP) oxidase with MS-PPOH (50μM) abolished DHA-mediated cytotoxicity suggesting downstream metabolites as the active mediators. We further demonstrate that CYP oxidase metabolites of DHA, methyl epoxy docosapentaenoate (EDP methyl esters, 1μM) (mix 1:1:1:1:1:1; 4,5-, 7,8-, 10,11-, 13,14-, 16,17- and 19,20-EDP methyl esters) and 19,20-EDP cause cytotoxicity via activation of PPARδ signaling leading to increased levels of intracellular ceramide. These results illustrate novel pathways for DHA-induced cytotoxicity that suggest an important role for CYP-derived metabolites, EDPs.