Stereospecific Induction of Apoptosis in U937 Cells by N‐Octanoyl‐Sphingosine Stereoisomers and N‐Octyl‐Sphingosine

We investigated the ability of N ‐octanoyl‐sphingosine (C 8 ‐Cer) stereoisomers, N ‐octanoyl‐DL‐ erythro ‐dihydrosphingosine (DL‐ e ‐DHC 8 ‐Cer), and a new ceramide derivative, N ‐octyl‐D‐ erythro ‐sphingosine (D‐ e ‐C 8 ‐Ceramine), to induce apoptosis in U937 cells. We found the C 8 ‐Cer stereoisomers to be stereo‐specific with the D‐ and L‐ threo stereoisomers being severalfold more potent than the erythro in inducing nucleosomal fragmentation. The order of potency was: D‐ t ‐C 8 ‐Cer = L‐ t ‐C 8 ‐Cer > L‐ e ‐C 8 ‐Cer > D‐ e ‐C 8 ‐Cer > DL‐ e ‐DHC 8 ‐Cer. The importance of the carbonyl group in apoptosis was investigated by using a new ceramide derivative, D‐ e ‐C 8 ‐Ceramine, in which the carbonyl group was replaced by a methylene group. The carbonyl group was not necessary for triggering apoptosis. In fact, replacement of the carbonyl group decreased substantially the time required for cells to die, with maximum DNA fragmentation occurring at 6 h as opposed to the 18 h required by D‐ e ‐C 8 ‐Cer. To explore possible mechanisms by which these compounds trigger the apoptotic pathway, we tested their ability to increase the endogenous levels of cellular ceramide and to differentially activate a ceramide‐activated protein kinase (CAPK). While the potent DNA fragmentation‐inducing compounds D‐ e ‐C 8 ‐Ceramine and L‐ t ‐C 8 ‐Cer failed to increase the cellular ceramide levels, D‐ e ‐C 8 ‐Cer, D‐ t ‐C 8 ‐Cer and D‐ e ‐C 8 ‐Ceramine activated the CAPK equally. These studies suggest that the DNA fragmentation‐inducing ability of the threo stereoisomers and D‐ e ‐C 8 ‐Ceramine cannot be attributed either to an increase in the activity of CAPK, or, as illustrated by D‐ e ‐C 8 ‐Ceramine and L‐ t ‐C 8 ‐Cer, to the differential elevation of endogenous ceramide. The phosphatase inhibitor okadaic acid failed to protect U937 cells from apoptosis induced by D‐ e ‐C 8 ‐Cer.