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Aida Sanchez-Bretano, Uzair Janjua, ilaria Piano, Glaudia Gargini, Kenkichi Baba, Gianluca Tosini; Melatonin protects 661W cells from cell death induced by H2O2 via inhibition of the Fas/FasL-Caspase 3 pathway. Invest. Ophthalmol. Vis. Sci. 2017;58(8):354.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photoreceptor viability during aging. Recent work from our laboratory has suggested that MEL may protect cones by modulating the Fas/FasL-Caspase 3 pathway. We have also reported that 661W cells - a photoreceptor-like cell line – express MEL receptors (MT1 and MT2) mRNAs. In this study, we first investigated the signaling pathway activated by MEL in 661W cells and then whether MEL can protect these cells from H2O2 induced cell death.
MEL receptors presence in 661W was studied by immunohistochemistry (IHC). 661W were treated with MEL and MEL agonist and antagonist to identify the intracellular pathways activated by MEL in these cells. To test the protective effect, 661W cells were subjected to a 2-h treatment with H2O2 and/or MEL. To analyze the pathways involved in H2O2–mediated cell death, Fas/FasL and caspase 3 were analyzed at the level or mRNA expression and protein abundance by western blot. Cell viability was analyzed by using trypan blue.
MEL receptors immunoreactivity was observed in 661W cells by IHC. The increase in cAMP production induced by forskolin was inhibited by MEL and IIK7 (a melatonin agonist) in a dose-dependent manner (p<0.05). These results suggest that in 661W cells MEL receptors are functional and - also in these cells - MT1 and MT2 receptors may heterodimerize. MEL partially prevented the H2O2-mediated cell death (20-25%; p<0.05). This effect was replicate with IIK7 when used at 1 µM (p<0.05). Pre-incubation with luzindole, a MEL antagonist, blocked MEL protection (p<0.05). Fas, FasL and caspase-3 gene expression was increased in cells treated with H2O2 and this effect was prevented by MEL (p<0.05). Finally, Kp7-6, an antagonist of Fas/FasL, blocked the cell death caused by H2O2 (p<0.05). Melatonin treatment also partially prevented the activation of Caspase 3 caused by H2O2.
Our results demonstrate that MEL receptors are present and functional in 661W. MEL can prevent photoreceptor cell death induced by H2O2 via the inhibition of the pro-apoptotic pathway Fas/FasL-Caspase 3. Finally, our data indicate that 661W cells may represent a new useful model to study melatonin signaling in a photoreceptor-like cell line.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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