Ceramide in rafts (detergent‐insoluble fraction) mediates cell death in neurotumor cell lines

Abstract

Detergent-resistant lipid microdomains (Rafts) were isolated from human oligodendroglioma (HOG), human neuroblastoma (LA-N-5), and immortalized dorsal root ganglion (F-11) cell lines by sucrose-density gradient ultracentrifugation and shown to be enriched in cholesterol, sphingomyelin, and ceramide. [(3)H]palmitate labeling allowed the Raft fraction to be easily identified as a sharp peak of (3)H radioactivity in the 5-30% sucrose interphase. Treatment of [(3)H]palmitate-labeled cells with staurosporine (to activate caspase 8 and induce apoptosis) or exogenous sphingomyelinase specifically increased the [(3)H]ceramide content of the Raft fraction. Depletion of cholesterol with beta-methylcyclodextran decreased Raft formation and partially blocked staurosporine-induced apoptosis. Similarly, treatment of cells with Fumonisin B1 to inhibit de novo sphingolipid synthesis by 50% reduced the labeling of the Raft fraction and partially blocked staurosporine-induced apoptosis. Staurosporine treatment activated neutral sphingomyelinase but had no effect on acid sphingomyelinase activity or on other lysosomal hydrolases, such as alpha-L-fucosidase. Most of the neutral sphingomyelinase activity is in the Raft fraction, suggesting that the conversion of sphingomyelin to ceramide in Rafts is an important event in neural cell apoptosis.

Critical role of cPLA2 in Aβ oligomer-induced neurodegeneration and memory deficit

“…The lysates were sonicated for 30 seconds in ice-cold bath, and protein concentrations were determined by bicinchoninic acid (BCA) Protein Assay Kit (Pierce, Illkirch, France). Acidic and neutral sphingomyelinase (A-SMase and NSMase) activities were measured at pH 4.5 and 7.4, respectively, with the fluorescent substrate 6-hexadecanoylamino-4-methylumbelliferyl phosphocholine (HMU-P-Chol; Moscerdam Substrates, Oegstgeest, the Netherlands) as previously described (Kilkus et al, 2003). Twenty-five microgram proteins, buffered either in 150 mM sodium acetate solution, containing 0.1% Triton X-100, 0.2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) (pH 4.5) (A-SMase activity), or in PBS, 0.1% Triton X-100, 0.2% CHAPS, 0.5 mM MgCl 2 (pH 7.4) (N-SMase activity), were incubated for up to 10 hours with 0.6 mM HMU-P-Chol.…”

Thematic Review Series: Sphingolipids. Role of ganglioside metabolism in the pathogenesis of Alzheimer's disease—a review

“…Fucosylated gangliosides, such as afucosyl and a-galactosyl-GM1, have been found in lipid rafts of PC12 cells (105). Ceramide (126) and ceramidase (127) are reported as components of lipid rafts. Neutral ceramidase was found to be enriched in the raft microdomains with cholesterol and GM1 (127).…”

Cancer-Selective Targeting and Cytotoxicity by Liposomal-Coupled Lysosomal Saposin C Protein

“…Several lines of evidence have suggested a linkage between cellular membrane abnormalities and ceramide-mediated induction of apoptosis in tumors (26)(27)(28)(29). Based on these observations, agents that interfere with cellular membranes have been developed to modulate membrane organization, fluidity, metabolism, and signal transduction (24,25).…”