Differential Effects of Glycolipid Biosynthesis Inhibitors on Ceramide‐Induced Cell Death in Neuroblastoma Cells

Abstract

An in vitro model of Gaucher's disease in murine neuroblastoma ϫ 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. Key Words: Apoptosis-Gaucher's disease-Neuroblastoma cells-Cell death-Glycolipids. J. Neurochem. 72, 1040Neurochem. 72, -1049Neurochem. 72, (1999.Sphingolipids are known to be involved in various biological functions, including modulation of cell proliferation, differentiation, and apoptosis (Schwarz et al., 1995;Ariga et al., 1998). The main active compounds are ceramide and its glycosylated, e.g., glycosphingolipids, or phosphorylated, e.g., sphingomyelins, derivatives (Schwarz et al., 1995;Ballou et al., 1996). Ceramide, mainly generated from signal-induced cleavage of sphingomyelin by sphingomyelinase, has been shown to arrest cell cycle and to stimulate apoptosis (Jarvis et al., 1994;Ji et al., 1995;Obeid and Hannun, 1995;Witty et al., 1996;Ariga et al., 1998). Recent studies have demonstrated that induction of apoptosis can also be achieved by activation of de novo synthesis of ceramide (Bose et al., 1995). Hydrolysis of ceramide by ceramidase releases sphingosine, which has been found to be ...

Inhibition of GSK3β by pharmacological modulation of sphingolipid metabolism occurs independently of ganglioside disturbance in a cellular model of Alzheimer's disease

“…However, as our results show, both PDMP enantiomers strongly and equally reduced the ceramide content, specifically the long-chain ceramide species (≤18C), while NB-DNJ did not. At first sight, this could be surprising because D-PDMP is usually described as a ceramide-accumulating agent (Bieberich et al, 1999, Huang et al, 2011, Radin et al, 1993, Takasugi et al, 2015, Yu et al, 2012. Indeed, when blocking the conversion of ceramides to GlcCer, it could be expected that an accumulation of ceramides may have occurred.…”

“…*p b 0.05, **p b 0.01, ***p b 0.001 compared to the control cells. (Bieberich et al, 1999;Guerrera and Ladisch, 2003;Platt et al, 1994). Treatment with 50 μM NB-DNJ for 3 and 5 days induced a significant drop in the rate of gangliosides, comparable to that observed with D-PDMP (Fig.…”

Decreased Ceramide Transport Protein (CERT) Function Alters Sphingomyelin Production following UVB Irradiation

“…In addition, analogous prior studies showed that blockade of GlcCer synthase by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) or 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), specific inhibitors of GlcCer synthase, can lead to Cer accumulation in cells (47), whereas other GlcCer synthase inhibitors, e.g. N-butyldeoxynojirimycin, decrease GlcCer production without accumulation of Cer (48). These differences are thought to reflect 1-phenyl-2-decanoylamino-3-morpholino-1-propanol or 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, but not N-butyldeoxynojirimycin, inhibition of the conversion of Cer to 1-O-acylCer, resulting in Cer accumulation (49).…”

Gaucher Disease: Clinical, Biological and Therapeutic Aspects

“…Sphingolipids have been implicated in inflammatory and apoptotic processes [5] and glucosylceramide might have direct activating or enhancing effects on macrophage function [6], possibly mediated through selective calcium channel dysregulation. Indeed, several indicators of macrophage activation, including chitotriosidase, CCL18 , angiotensin-converting enzyme [7] and cathepsin S, have been identified in excess in the plasma of patients with GD.…”