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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-a receptor signaling pathway (ASCB 2012, Abstract #888). This cytotoxicity is reversed in the presence of a-tocopherol, indicative of reactive oxygen species (ROS) involvement in cell death. Also, 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). 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) and JW480, potent inhibitors of the enzyme NCEH1, I explored the relationship between OEA and NCEH1 in the OV2008 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. Western blot analysis and immunofluorescent localization of OEA, CPO, and JW480 treated cells showed that CPO appears to increase the expression of NCEH1. Furthermore, Western blot analysis of the antioxidant enzymes GST-µ, GPX4, Mn-SOD and Zn/Cu-SOD, and the autophagic marker, light chain 3 A/B shows that OEA, CPO, or JW480 do not appear to affect the expression of these proteins. Live cell labeling with dihydroethidium showed that these compounds have no statistically significant effect on overall superoxide production. Live cell fluorescence microscopy using MitoSOX Red, however, revealed an increase in superoxide anion in the mitochondria in the presence of OEA. The results of this 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 and JW480. I also demonstrate an uncharacterized exaggerated pH gradient across the cellular membrane of cells treated with OEA and JW480, with the effects being more pronounced after incubation in JW480. The cause of this exaggerated gradient remains to be elucidated. |
