Melatonin partially protects 661W cells from H2O2-induced death by inhibiting Fas/FasL-caspase-3

Mol Vis. 2017 Dec 3:23:844-852. eCollection 2017.


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 (MT1 and MT2) in 661W cells, then whether MEL can prevent H2O2-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 H2O2 for 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in H2O2-mediated cell death, a Fas/FasL antagonist was used before the exposure to H2O2. Finally, Fas/FasL and caspase-3 mRNA was analyzed with q-PCR and immunocytochemistry in cells treated with H2O2 and/or MEL. Cell viability was analyzed by using Trypan Blue.

Results: Both MEL receptors (MT1 and MT2) were detected at the mRNA and protein levels in 661W cells. MEL partially prevented H2O2-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 H2O2 similarly to what was observed for MEL. Fas, FasL, and caspase-3 expression was increased in cells treated with H2O2, and this effect was prevented by MEL. Finally, MEL treatment partially prevented the activation of caspase-3 caused by H2O2.

Conclusions: The results demonstrate that MEL receptors are present and functional in 661W cells. MEL can prevent photoreceptor cell death induced by H2O2 via the inhibition of the proapoptotic pathway Fas/FasL-caspase-3.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antioxidants / pharmacology*
  • Caspase 3 / genetics
  • Caspase 3 / metabolism*
  • Cell Line
  • Cell Survival / drug effects
  • Dose-Response Relationship, Drug
  • Fas Ligand Protein / antagonists & inhibitors*
  • Fas Ligand Protein / genetics
  • Fas Ligand Protein / metabolism
  • Hydrogen Peroxide / toxicity*
  • Immunohistochemistry
  • Melatonin / pharmacology*
  • Mice
  • Microscopy, Confocal
  • Oxidants / toxicity
  • RNA, Messenger / genetics
  • Real-Time Polymerase Chain Reaction
  • Receptor, Melatonin, MT1 / genetics
  • Receptor, Melatonin, MT1 / metabolism
  • Receptor, Melatonin, MT2 / genetics
  • Receptor, Melatonin, MT2 / metabolism
  • Retinal Cone Photoreceptor Cells / drug effects*
  • Retinal Cone Photoreceptor Cells / metabolism
  • Retinal Cone Photoreceptor Cells / pathology
  • fas Receptor / antagonists & inhibitors*
  • fas Receptor / genetics
  • fas Receptor / metabolism


  • Antioxidants
  • Fas Ligand Protein
  • Fasl protein, mouse
  • Oxidants
  • RNA, Messenger
  • Receptor, Melatonin, MT1
  • Receptor, Melatonin, MT2
  • fas Receptor
  • Hydrogen Peroxide
  • Casp3 protein, mouse
  • Caspase 3
  • Melatonin