Synergistic protection of a general caspase inhibitor and MK-801 in bilirubin-induced cell death in human NT2-N neurons

Unconjugated bilirubin (UCB) induces both apoptosis and necrosis in neurons. To investigate the role of caspases and excitotoxicity in UCB-induced cell death, we exposed NT2-N neurons to 5 AM UCB (a concentration known to induce apoptosis) or 2 mu M staurosporine (positive apoptosis control) and investigated the effects of treatments with the specific caspase-3 inhibitor, zDEVD.FMK (20 and 100 mu M), or the general caspase inhibitor, zVAD.FMK (20 and 100 mu M), and/or the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (10 mu M) during a 24- or 48-h exposure. UCB increased caspase-3 activity 2.3-fold after 6 h. Despite this, treatment with zDEVD.FMK did not prevent cell death. zVAD.FMK (100 mu M only) reduced apoptotic morphologies induced by UCB after 48 h, however, this effect was not reflected in MTT assays. MK-801 attenuated UCB-induced cell death after 24 h, but not significantly after 48 h. Similar effects were seen in the MTT assay. Combined treatment with MK-801 and zVAD.FMK synergistically reduced apoptotic morphologies after 24 and 48 h. MTT assays showed comparable effects after 24 h but did not show significant differences after 48 h. We conclude that NMDA receptor-mediated pathways and caspase-mediated pathways are involved in UCB-induced cell death in human NT2-N neurons. Concomitant inhibition of both pathways results in synergistic protection.