Gangliosides Link the Acidic Sphingomyelinase-Mediated Induction of Ceramide to 12-Lipoxygenase-Dependent Apoptosis of Neuroblastoma in Response to Fenretinide

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Abstract
Background: The lipid second messenger ceramide, which is generated by acidic and neutral sphingomyelinases or ceramide synthases, is a common intermediate of many apoptotic pathways. Metabolism of ceramide involves several enzymes, including glucosylceramide synthase and GD3 synthase, and results in the formation of gangliosides (GM3, GD3, and GT3), which in turn promote the generation of reactive oxygen species (ROS) and apoptosis. Fenretinide, a retinoic acid derivative, is thought to induce apoptosis via increases in ceramide levels, but the link between ceramide and subsequent apoptosis in neuroblastoma cells is unclear. Methods: SH-SY5Y and HTLA230 neuroblastoma cells were treated with fenretinide in the presence or absence of inhibitors of enzymes important in ceramide metabolism (fumonisin B 1 , inhibitor of ceramide synthase; desipramine, inhibitor of acidic and neutral sphingomyelinases; and PDMP, inhibitor of glucosylceramide). Small interfering RNAs were used to specifically block acidic sphingomyelinase or GD3 synthase activities. Apoptosis, ROS, and GD3 expression were measured by flow cytometry. Results: In neuroblastoma cells, ROS generation and apoptosis were associated with fenretinide-induced increased levels of ceramide, glucosylceramide synthase activity, GD3 synthase activity, and GD3. Fenretinide also induced increased levels of GD2, a ganglioside derived from GD3. Inhibition of acidic sphingomyelinase but not of neutral sphingomyelinase or ceramide synthase, blocked fenretinide-induced increases in ceramide, ROS, and apoptosis. Exogenous GD3 induced ROS and apoptosis in SH-SY5Y cells but not in SH-SY5Y cells treated with baicalein, a specific 12-lipoxygenase inhibitor. Exogenous GD2 did not induce apoptosis. Conclusions: A novel pathway of fenretinide-induced apoptosis is mediated by acidic sphingomyelinase, glucosylceramide synthase, and GD3 synthase, which may represent targets for future drug development. GD3 may be a key signaling intermediate leading to apoptosis via the activation of 12-lipoxygenase.