We have previously shown that acylethanolamide oleoylethanolamide (OEA) and its metabolically stable analog AM3102 induce apoptosis in OV2008 ovarian adenocarcinoma cells independent of PPAR-α signaling pathway. This cytotoxicity is reversed in the presence of α-tocopherol, indicative of reactive oxygen species involvement in cell death. We have also shown that palmityl trifluoromethyl ketone (PTK), independent of its inhibitory effect on phospholipase A2, enhances the toxicity of OEA (ASCB 2012, Abstract #888). The enzyme neutral cholesterol ester hydrolase 1 (NCEH1) has also been shown to be a target of trifluoromethylketones (Nat Biotechnol 21:687, 2003). Using Chlorpyrifos-Oxon (CPO), a potent inhibitor of the enzyme NCEH1, we explored the relationship between OEA and NCEH1 in the OV2008 cell line. Cytotoxicity was observed in response to OEA (IC50: 14-21 µM) and CPO (IC50: 48-50 µM). When the compounds were administered together, cytotoxicity was enhanced (OEA IC50: 8-9 µM; CPO IC50: 15-19 µM) with evidence of weak synergism. However, Western blot analysis of OEA and CPO treated cells indicated no change in NCEH1 expression. Live cell labeling with dihydroethidium showed an increase superoxide by about 10% in the presence of OEA. CPO itself did not increase ROS in cells. Live cell fluorescence microscopy using MitoSOX Red also revealed an increase in superoxide in the mitochondria in the presence of OEA. The results of our study suggest that one mechanism by which OEA induces cytotoxicity in OV2008 cells is via superoxide generation. It is possible that OEA-mediated increase in ROS makes the OV2008 cells further susceptible to CPO.
Dr. P. Bagavandoss