Oxidative Stress and Cyclooxygenase-2 Induction Mediate Cyanide-Induced Apoptosis of Cortical Cells

Cyanide (KCN)-induced generation of reactive oxygen species (ROS) involves cyclooxygenase-2 (COX-2)-mediated reactions in some neurons. The present study examines the extent to which COX isoforms are involved in KCN-induced apoptotic cell death processes of cultured cortical cells. After treatment with KCN (10–300 μM), COX-2 was expressed in a time- and concentration-dependent manner increasing markedly over a 4-h period. However, no significant changes were observed in COX-1 levels at any cyanide concentration. Correlated with COX-2 up-regulation, KCN induced a time-dependent apoptotic death. TUNEL staining showed that the COX-2 inhibitor NS-398 (30 μM) blocked KCN-induced apoptosis, whereas the selective COX-1 inhibitor valeryl salicylate did not affect the level of apoptotic cell death. Exposure of cells to KCN (300 μM) for 24 h resulted in DNA fragmentation, which was also reduced by NS-398. Prostaglandin E 2 (PGE 2) accumulation in cell culture supernatants was increased by KCN and NS-398 blocked PGE 2 generation. PCR studies further confirmed that COX-2 expression was increased by KCN. Antioxidants phenyl- N- test-butylnitrone, superoxide dismutase, and catalase significantly inhibited KCN-induced COX-2 up-regulation and subsequent apoptosis. N G-nitro- l-arginine methylester an inhibitor of nitric oxide synthase, blocked KCN-induced PGE 2 production and apoptosis, but not COX-2 expression. Increased nitric oxide levels caused by cyanide may directly activate the COX-2 enzyme. These data show that cyanide treatment of cortical cells involves increased COX-2 expression, PGE 2 accumulation, and ROS generation, resulting in apoptotic cell death.