Sphingolipid metabolic changes during chiral C2-ceramides induced apoptosis in human leukemia cells

N-acetylsphingosine (C2-ceramide) is a synthetic water-soluble ceramide mim icking the activity of natural ceramides. By fixing chiral conformation on carbon numbers 2 and 3 in the ceramide structure, four chiral C2-ceramides naming d-erythro-, I-erythro-, d-threo- and I-three C2-ceramide were synthe sized. We have investigated the chiral effects of these C2-ceramides on the sphingolipid metabolism, particularly on both the sphingolipid biosyntheti c pathway and on the degradation pathway. In both HL-60 and U937 cells, the chiral C2-ceramide (10 muM) showed sphingosine accumulation monitored fluo romatrically by a high performance liquid chromatographic separation of the sphingoid bases. Most importantly, in HL-60 cells, 1-erythro C2-ceramide i nduced a 50 fold increase in sphingosine as compared to the control, while I-three C2-ceramide exhibited a minimal 7-fold increase. In contrast, sphin ganine, another sphingoid base, showed less accumulation by any chiral C2-c eramide tested under the same conditions. These results suggested that chir al C2-ceramide primarily acts on the sphingolipid degradation pathway rathe r than on the sphingolipid biosynthetic route. The strong G(0)/G(1) phase a rrest in the cell cycle by treatment of I-erythro C2-ceramide indicates tha t the blockade of the sphingolipid degradation pathway might be concomitant ly involved in the dysfunction of the cell cycle. On the other hand, the fa ct that all chiral C2-ceramides tested failed to inhibit the activity of sp hingosine kinase acting on the removal of sphingosine by producing sphingos ine-1-phosphate demonstrates that chiral C2- ceramides may increase sphingo sine by activating various ceramidases by which natural ceramides are divid ed into sphingosine and free fatty acids. However, the precise steps involv ed in this interaction are still unknown.