-dihydrosphingosine, in cultured cells

We studied the metabolism of radioactively labeled safingol ( L threodihydrosphingosine) in primary cultured neurons, B104 neuroblastoma cells, and Swiss 3T3 fibroblasts, and compared it to that of its natural stereoisomer D erythro -dihydrosphingosine. Both sphingoid bases are used as biosynthetic precursors for complex sphingolipids, albeit to different rates. Whereas a considerable amount of the natural sphingoid base is also directed to the catabolic pathway (20–66%, cell type dependent), only a minor amount of the nonnatural safingol is subjected to catabolic cleavage, most of it being N -acylated to the respective stereochemical variant of dihydroceramide. Interestingly, N -acylation of safingol to L threo -dihydroceramide is less sensitive to fumonisin B1 than the formation of the natural D erythro -dihydroceramide. In addition, safingol-derived L threodihydroceramide, unlike its physiologic counterpart, is not desaturated. Most of it either accumulates in the cells (up to 50%) or is used as a biosynthetic precursor of the respective dihydrosphingomyelin (up to 45%). About 5% is, however, glucosylated and channeled into the glycosphingolipid biosynthetic pathway. Our results demonstrate that, despite its nonnatural stereochemistry, safingol is recognized and metabolized preferentially by enzymes of the sphingolipid biosynthetic pathway. Furthermore, our data suggest that the cytotoxic potential of safingol is reduced rather than enhanced via its metabolic conversion. —Dragusin, M., C. Gurgui, G. Schwarzmann, J. Hoernschemeyer, and G. van Echten-Deckert. Metabolism of the unnatural anticancer lipid safingol, L threo -dihydrosphingosine, in cultured cells. J. Lipid Res. 2003. 44: 1772–1779. Supplementary key words primary cultured neurons • neuroblastoma cells B104 • Swiss 3T3 cells • l threo -sphinganine • ceramide Sphingolipids occur in all eukaryotic cells, where they are primarily components of the plasma membrane. Their ceramide backbone anchors them in the outer leaflet of the lipid bilayer. Their hydrophilic moiety, composed of carbohydrate chains or phosphorylcholine in the case of glycosphingolipids (GSLs) and sphingomyelin (SM), respectively, faces the extracellular space. Gangliosides are sialic acid-containing GSLs and are specifically abundant in the central nervous system, where they have been associated with development and maturation of the brain, neuritogenesis, synaptic transmission, memory formation, and synaptic aging (1). Ceramide is not only a key intermediate in the synthetic and degradative pathway of sphingolipid metabolism but also a key player in the antiproliferative cellular responses, including apoptosis, cellcycle arrest, differentiation, and senescence (2). In contrast, its catabolic intermediate sphingosine 1-phosphate (S1P) has been implicated as a second messenger in cellular proliferation and survival (3), and also in protection against ceramide-mediated apoptosis (4). Thus, the dynamic balance between intracellular S1P and ceramide, also known as sphingolipid rheostat, appears to be essential for the determination of whether cells survive or die (5). Safingol ( l threodihydrosphingosine) is a nonnatural isomer of dihydrosphingosine (sphinganine). Usually sphingoid bases contain two chiral centers, namely at carbon atoms 2 and 3. Natural sphingoid bases occur in the d erythro (2S, 3R) configuration, but three additional nonnatural stereoisomers exist. Among the unnatural sphingoid bases, l threo (2 S , 3 S )-dihydrosphingosine (safingol) is of particular interest due to its anticancer activity. It was shown to synergistically increase the toxicity of established chemotherapeutic agents in several cancer cells in vitro (6), as well as in preclinical animal studies (7) and in a phase I clinical trial (8). The anticancer properties of safingol can be explained by its inhibitory effect on the activity of either protein kinase C (PKC) or sphingosine kinase. The competitive interaction of safingol with the Abbreviations: FB1, fumonisin B1; GSL, glycosphingolipid; S1P, sphingosine 1-phosphate; SM, sphingomyelin. 1 Present address of C. Gurgui: Institut für Physiologie II, Wilhelmstrasse 31, 53111 Bonn, Germany. 2 To whom correspondence should be addressed. e-mail: [email protected] Manuscript received 17 April 2003 and in revised form 30 May 2003. Published, JLR Papers in Press, June 1, 2003. DOI 10.1194/jlr.M300160-JLR200 by gest, on Jauary 5, 2018 w w w .j.org D ow nladed fom