Bieberich E, Freischütz B, Suzuki M, Yu R K
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia of Virginia Commonwealth University, Richmond 23298-0614, USA.
J Neurochem. 1999 Mar;72(3):1040-9. doi: 10.1046/j.1471-4159.1999.0721040.x.
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.
利用鼠神经母细胞瘤x大鼠胶质瘤NG108-15细胞构建的戈谢病体外模型，研究了两种葡糖神经酰胺合酶特异性抑制剂——d，l-苏式-1-苯基-2-癸酰氨基-3-吗啉代-1-丙醇（d，l-PDMP）和N-丁基脱氧野尻霉素（NB-DNJ）的生理效应，这两种抑制剂已被提议作为治疗糖脂贮积症的药物。用葡糖神经酰胺酶的共价抑制剂环氧硬脂醇-B-环氧化物孵育NG108-15细胞，使细胞内葡糖神经酰胺（GC）浓度提高了四倍多，表明其糖脂组成与戈谢细胞相当。用d，l-PDMP或NB-DNJ孵育后，GC水平降低，神经节苷脂减少。用d，l-PDMP处理而非NB-DNJ处理，导致NG108-15细胞生长速率呈剂量依赖性降低，最终在细胞达到汇合状态时导致细胞死亡。使用末端核苷酸转移酶的原位检测分析表明，细胞变性伴有细胞凋亡。高效薄层层析脂质分析显示，用d，l-PDMP孵育而非NB-DNJ孵育时，内源性神经酰胺浓度提高了三倍。当NG108-15细胞与柔红霉素孵育时，也观察到神经酰胺升高和细胞凋亡，此前报道柔红霉素通过刺激神经酰胺合成诱导程序性细胞死亡。高效液相色谱和随后的激光解吸质谱结构表征显示，内源性神经酰胺含有链长范围为C14:0至C24:0的脂肪酸。结果表明，用d，l-PDMP或柔红霉素孵育使这些神经酰胺种类水平升高，可诱导NG108-15细胞程序性死亡。由于用NB-DNJ孵育未观察到神经酰胺积累和细胞死亡，因此建议其在治疗戈谢病和其他鞘脂贮积症方面的毒性低于d，l-PDMP。
