ACS6, a Hydrogen sulfide-donating derivative of sildenafil, inhibits homocysteine-induced apoptosis by preservation of mitochondrial function

Cell viability was determined by Cell Counting Kit-8 assay. Cell apoptosis was observed using the chromatin dye Hoechst 33258 and analyzed by Flow Cytometry after propidium iodide staining. Mitochondrial membrane potential was monitored using the fluorescent dye Rh123. Intracellular reactive oxygen species were determined by oxidative conversion of cell permeable 2',7'-dichlorfluorescein-diacetate to fluorescent 2',7'-dichlorfluorescein. The expression of cleaved caspase-3 and bcl-2 and the accumulation of cytosolic cytochrome c were analyzed by Western blot.We show that ACS6 protects PC12 cells against cytotoxicity and apoptosis induced by homocysteine and blocks homocysteine-triggered cytochrome c release and caspase-3 activation. ACS6 treatment results in not only prevention of homocysteine-caused mitochondrial membrane potential (Δψ) loss and reactive oxygen species (ROS) overproduction but also reversal of Bcl-2 down-expression.These results indicate that ACS6 protects PC12 cells against homocysteine-induced cytotoxicity and apoptosis by preservation of mitochondrial function though inhibiting both loss of Δψ and accumulation of ROS as well as modulating the expression of Bcl-2. Our study provides evidence both for a neuroprotective effect of ACS6 and for further evaluation of ACS6 as novel neuroprotectants for Alzheimer's disease associated with homocysteine.Homocysteine, a thiol-containing amino acid, is a key metabolic intermediate in sulfuramino acid metabolism [1,2]. Homocysteine not only can be remethylated to methionine by enzymes that require folic acid but also can be catabolized to form cysteine by cystathionine-β-synthetase (CBS). Both in vitro and in vivo studies have shown that homocysteine is toxic to neuronal cells [3-9]. One explanation for the mechanism of homocysteine neurotoxicity is that auto-oxidation of homocysteine leads to the formation of superoxide and hydrogen peroxide [10]. The causative link between hyperhomocysteinemia and neurodeg