Cytochrome c translocation does not lead to caspase activation in maitotoxin-treated SH-SY5Y neuroblastoma cells

Abstract

Cytosolic cytochrome c elevation has been associated with activation of caspase-3-like proteases. In this study, we demonstrate that treatment with the neurotoxin and potent calcium channel opener maitotoxin (MTX) induces cytochrome c release from the mitochondria that is not accompanied by caspase activation. Cytochrome c translocation in MTX-treated SH-SY5Y cells was readily apparent after 30 min and peaked at 2.5 h. We assayed caspase activity by acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-AMC) hydrolysis and by immunoblotting for caspase-3 processing and proteolysis of αII-spectrin and PARP. In contrast, treatment with pro-apoptosis agent staurosporine (STS) induced both cytochrome c release and caspase-3 activation after 2 h. In addition, with MTX treatment, we found no evidence of caspase activation at any time point or MTX concentration used. Instead, we observed that caspase-9, Apaf-1 and caspase-3 were all partially truncated by calpain under these conditions. These combined effects likely contribute to the lack of caspase activation cascade in MTX-treated cells, despite the presence of cytochrome c in the cytosol.