PPARδ signaling mediates the cytotoxicity of DHA in H9c2 cells

Abstract

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.

Keywords: Apoptosis; Ceramide; Docosahexaenoic acid; Epoxydocosapentaenoic acids; H9c2 cells; PPARδ.

Fig. 1

Effect of DHA exposure on cellular viability and metabolic activity. (A) Cell viability was estimated based on the Trypan Blue exclusion method. H9c2 cells were treated with 0 or 100 µM of DHA, and assessed at 24 h. (B) Cellular metabolic activity was estimated by the oxidation of MTT to formazan. (C) The percentage of LDH release was quantified colorimetrically. (D) Proteasomal activity was assessed by the detection of 7-amino-4-methylcourmarin (AMC) in cell lysates post cleavage from the peptide LLVY-AMC. (E) PPARδ DNA binding was quantified using an ELISA kit. Values are expressed as mean ± SEM; N = 3; *p < 0.05 DHA 100 µM vs. control, #p < 0.05 DHA 100 µM vs. DHA 100 µM and GSK 3787 1 µM.

Fig. 2

Effect of DHA on oxidative stress. (A) Histogram representing the generation of reactive oxygen species in H9c2 cells treated with 0, 1, 10 or 100 µM of DHA at 6, 12 and 24 h. (B) Lipid peroxidation was determined by the accumulation of TBA-active products after 24 h of treatment. H9c2 cells treated with 0 or 100 µM of DHA with and without GSK 3787 (1 µM). (C) Total antioxidant capacity was measured in H9c2 cells following 24 h treatment with 0 or 100 µM of DHA with and without GSK 3787 (1 µM). Values are expressed as mean ± SEM; N = 3; *p < 0.05 DHA 100 µM vs. control, #p < 0.05 DHA 100 µM vs. DHA 100 µM and GSK 3787 1 µM.

Fig. 3

DHA-induced apoptosis mediated via PPARδ-signaling. (A–D) Representative bivariate dot plots of Annexin-V FITC and propidium iodide binding. Apoptotic cells were assessed by combining values falling in the upper and lower right quadrants. (E) Histogram summarizing flow cytometry data demonstrating induction of DHA-induced apoptosis. (F) Cells were harvested and subjected to western blotting to assay the level of p-AKT (upper insert), and quantified (lower insert). (G) Caspase-3 activity was 19,20-EDP with or without GSK 3787 1 µM and assessed at 24 h. (C) Cell viability and (D) cellular metabolic activity were measured. (E) PPARδ DNA binding was quantified using an ELISA kit. Values are expressed as mean ± SEM; N = 3; *p < 0.05 EDPs or 19,20-EDP 1 µM vs. control, #p < 0.05 19,20-EDP 1 µM vs. 19,20-EDP 1 µM and GSK 3787 1 µM. (F) LC/MS analysis was employed to measure the accumulation of ceramide following treatment with 0 or 100 µM of DHA with or without MS-PPOH 50 µM, EDPs mix 1 µM (1:1:1:1:1; 7,8-, 10,11-, 13,14-, 16,17- and 19,20-EDP) or 1 µM 19,20-EDP with or without GSK 3787 1 µM and assessed at 24 h. Values are expressed as mean ± SEM; N = 3; *p < 0.05 EDP 1 µM vs. control, #p < 0.05 EDPs 1 µM vs. EDPs 1 µM and GSK 3787 1 µM, #p < 0.05 19,20-EDP 1 µM vs. 19,20-EDP 1 µM and GSK 3787 1 µM.

Fig. 4

DHA-induced cytotoxicity is attenuated by pharmacological inhibition on de novo ceramide synthesis. H9c2 cells were treated with 0 or 100 µM of DHA with or without myriocin 1 µM, an inhibitor of SPT, and assessed at 24 h. (A) Cell viability, (B) caspase-3 and (C) total proteasomal activities were measured. Co-treatment of DHA with myriocin significantly abolished DHA-induced cytotoxity. Values are expressed as mean ± SEM; N = 3; *p < 0.05 DHA 100 µM vs. control, #p < 0.05 DHA 100 µM vs. DHA 100 µM and myriocin 1 µM. (D) LC/MS analysis was employed to measure the accumulation of ceramide following treatment with 0 or 100 µM of DHA with or without GSK 3787 1 µM, a PPARδ antagonist, and assessed at 24 h. Values are expressed as mean ± SEM; N = 3; *p < 0.05 DHA 100 µM vs. control, #p < 0.05 DHA 100 µM vs. DHA 100 µM and GSK 3787 1 µM.

Fig. 5

EDPs demonstrate cytotoxicity toward H9c2 cells. H9c2 cells were treated with 0 or 100 µM of DHA with or without MS-PPOH 50 µM and assessed at 24 h. (A) Cell viability was assessed using CCK-8, and (B) cellular metabolic activity was assessed via MTT. Values are expressed as mean ± SEM; N = 3; *p < 0.05 DHA 100 µM vs. control, # p < 0.05 DHA 100 µM vs. DHA 100 µM and MS-PPOH 50 µM. H9c2 cells were treated with 0, 1 µM of EDP mix (1:1:1:1:1; 7,8-, 10,11-, 13,14-, 16,17- and 19,20-EDP) or 1 µM