Melatonin partially protects 661W cells from H(2)O(2)-induced death by inhibiting Fas/FasL-caspase-3

PURPOSE: Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photoreceptor viability during aging. Recent work by our laboratory suggested that MEL may protect cones by modulating the Fas/FasL-caspase-3 pathway. In this study, we first investigated the presence of MEL receptors (MT(1) and MT(2)) in 661W cells, then whether MEL can prevent H(2)O(2)-induced cell death, and last, through which pathway MEL confers protection. METHODS: The mRNA and proteins of the MEL receptors were detected with quantitative PCR (q-PCR) and immunocytochemistry, respectively. To test the protective effect of MEL, 661W cells were treated with H(2)O(2) for 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in H(2)O(2)–mediated cell death, a Fas/FasL antagonist was used before the exposure to H(2)O(2). Finally, Fas/FasL and caspase-3 mRNA was analyzed with q–PCR and immunocytochemistry in cells treated with H(2)O(2) and/or MEL. Cell viability was analyzed by using Trypan Blue. RESULTS: Both MEL receptors (MT(1) and MT(2)) were detected at the mRNA and protein levels in 661W cells. MEL partially prevented H(2)O(2)-mediated cell death (20–25%). This effect was replicated with IIK7 (a melatonin receptor agonist) when used at a concentration of 1 µM. Preincubation with luzindole (a melatonin receptor antagonist) blocked MEL protection. Kp7–6, an antagonist of Fas/FasL, blocked cell death caused by H(2)O(2) similarly to what was observed for MEL. Fas, FasL, and caspase-3 expression was increased in cells treated with H(2)O(2), and this effect was prevented by MEL. Finally, MEL treatment partially prevented the activation of caspase-3 caused by H(2)O(2). CONCLUSIONS: The results demonstrate that MEL receptors are present and functional in 661W cells. MEL can prevent photoreceptor cell death induced by H(2)O(2) via the inhibition of the proapoptotic pathway Fas/FasL-caspase-3.