Differential effects of glycolipid biosynthesis inhibitors on ceramide-induced cell death in neuroblastoma cells.
An in vitro model of Gaucher's disease in murine neuroblastoma x rat glioma NG108-15 cells was used to investigate the physiological effects of two specific inhibitors of glucosylceramide synthase, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d,l-PDMP) and N-butyldeoxynojirimycin (NB-DNJ), which have been suggested as agents for treatment of glycolipid storage disorders. Incubation of NG108-15 cells with conduritol-B-epoxide, a covalent inhibitor of glucosylceramidase, raised the intracellular concentration of glucosylceramide (GC) by more than fourfold, indicating a glycolipid composition equivalent to that of Gaucher's cells. The level of GC was decreased, and the cells were depleted of gangliosides by postincubation with d,l-PDMP or NB-DNJ. Treatment with d,l-PDMP, but not with NB-DNJ, resulted in a dose-dependent reduction of the growth rate and eventually caused cell death in NG108-15 cells on reaching confluency. An in situ detection assay using terminal nucleotidyltransferase indicated that cell degeneration was accompanied by apoptosis. Lipid analysis by high-performance TLC revealed that on incubation with d,l-PDMP, but not with NB-DNJ, the concentration of endogenous ceramide was elevated by threefold. Ceramide elevation and apoptosis were also observed when NG108-15 cells were incubated with daunorubicin, which was previously reported to induce programmed cell death by stimulation of ceramide synthesis. Structural characterization by HPLC and subsequent laser desorption mass spectrometry revealed that the endogenous ceramide contained fatty acids with chain lengths ranging from C14:0 to C24:0. The results indicate that elevation of levels of these ceramide species by incubation with d,l-PDMP or daunorubicin induces programmed cell death in NG108-15 cells. Because ceramide accumulation and cell death were not observed on incubation with NB-DNJ, its use is suggested to be less toxic than that of d,l-PDMP for treatment of Gaucher's disease and other sphingolipid storage disorders.
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia of Virginia Commonwealth University, Richmond 23298-0614, USA.
E
E
Bieberich
B
B
Freischütz
M
M
Suzuki
R K
RK
Yu
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology*; D016923:Cell Death / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism / Q000502:physiology* / Q000633:toxicity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005776:Gaucher Disease / Q000378:metabolism / Q000473:pathology*; D005963:Glucosylceramides / Q000037:antagonists & inhibitors; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors; D006017:Glycolipids / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology*; D051381:Rats; D014407:Tumor Cells, Cultured
D002518:Ceramides; D004791:Enzyme Inhibitors; D005963:Glucosylceramides; D006017:Glycolipids; D009025:Morpholines; D017485:1-Deoxynojirimycin; C033110:RV 538; C059896:miglustat; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase