Ceramides which arise in part from the breakdown of sphingomyelin comprise a class of antiproliferative lipids and have been implicated in the regulation of programmed cell death better known as apoptosis. In the present study, two new synthetic ceramide analogues, N‐thioacetylsphingosine and FS‐5, were used in Molt 4 cells to induce cell death. Besides their cytotoxic effects at concentrations ≥14 μM the data obtained clearly show that both analogues induced apoptosis at concentrations below this critical concentration as assessed by trypan blue exclusion and cleavage of the death substrate poly‐(ADP‐ribose) polymerase (PARP). Additional experiments in bcl‐2‐transfected Molt 4 cells revealed that the apoptotic but not the lytic effects of the analogues were antagonized by the apoptosis inhibitor Bcl‐2. Furthermore, neither N‐thio‐acetylsphingosine nor FS‐5 induced PARP cleavage in bcl‐2‐transfected Molt 4 cells indicating that the induction of apoptotic cell death by cell permeable ceramides is not due to unspecific disturbance of the cell membrane.

In contrast to extracellular, long chain ceramides which comprise a structural component of the epidermal water barrier, intracellular ceramides originating from sphingomyelin hydrolysis have been shown to inhibit proliferation and to induce apoptosis in different cell populations. To further elucidate the possible role of intracellular ceramides in human epidermis, two new cell-permeable ceramide analogues, N-thioacetylsphingosine (C2-Cer=S) and 4-dodecanoylamino-decan-5-ol (FS-5), were synthesized and tested for their ability to suppress cell growth and to induce apoptosis in immortalized human keratinocytes. It was shown that the well-investigated ceramide analogue N-acetylsphingosine (C2-Cer=O), as well as the new compound C2-Cer=S inhibited proliferation of HaCaT cells with half-inhibitory concentrations (IC50) of 20 microg/ml and 10 microg/ml, respectively, whereas FS-5 has been potent with an IC50>40 microg/ml. Overall, all three ceramide analogues induced apoptosis in HaCaT cells as assessed by DNA-fragmentation using ELISA technique and in situ nick end labelling, thereby confirming the importance of ceramide signalling in keratinocytes.

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