We have previously shown that both the acylethanolamide oleoylethanolamide (OEA) and its metabolically stable analog AM3102 (N-[(1R)-2-hydroxy-1-methylethyl-9Z-octadecenamide) induces apoptosis in OV2008 ovarian adenocarcinoma cells independent of PPAR-α receptor signaling pathway. This cytotoxicity is reversed in the presence of α-tocopherol, indicative of reactive oxygen species (ROS) 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). Furthermore, inhibition of NCEH1 leads to reduced migration of SKOV3 ovarian cancer cells in vitro and their growth in vivo (Chem Biol 13: 1041, 2006). Therefore, in this study, using Chlorpyrifos-Oxon (CPO), a potent inhibitor of the enzyme NCEH1, we explored the relationship between OEA and NCEH1 in the OV2008 ovarian cancer cell line. Cytotoxicity was observed in response to both 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, when compared with control, no change in the expression of NCEH1. Live cell labeling with dihydroethidium showed an increase superoxide by about 10% in the presence of OEA. CPO itself did not increase the ROS in cells. Live cell fluorescence microscopy using MitoSOX Red also revealed an increase in superoxide anion 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 (This undergraduate student research was supported by funds from the University Research Council).
Justin Ricker is a senior at Kent State Stark. He is majoring in integrated health studies with a concentration in health sciences and a minor in health care ethics. After graduation and before graduate school, he plans to attend a post-baccalaureate program through the National Institute of Health. Justin’s dream job is to one day become a principal investigator at a leading biomedical research institution.