C2-ceramide induces apoptosis in a human squamous cell carcinoma cell line

Background Previous studies have demonstrated that synthetic cell-permeable analogues of ceramide promote differentiation and inhibit proliferation of keratinocytes, and that the vitamin D-3 inducible sphingomyelin cycle gene rates ceramide in keratinocytes. Although it has been suggested that exogen ous ceramide induces apoptosis of keratinocytes, which is similar to their effect on other cell types, such as leukaemia cells, only a few studies hav e reported ceramide-induced apoptosis of keratinocytes. Objective To determine whether ceramide induces apoptosis of keratinocytes, we used the synthetic ceramide analogue, C-2-ceramide (N-acetylsphingosine ) and a human squamous cell carcinoma cell line. HSC-I. Methods We treated HSC-I cells with C-2-ceramide, followed by a viability a ssay, morphological observations, nick end-labelling (TUNEL), DNA electroph oresis, and electron microscopy. Results In the viability assay, C-2-ceramide was toxic to HSC-I cells in a dose-dependent manner. Manifestations of apoptotic morphology occurred in t he ceramide-treated cells, whereas these morphological changes did not occu r in cells treated with dihydroceramide (N-acetylsphinganine). TUNEL reveal ed that many of the ceramide-treated cells showed positive reactivity. DNA electrophoresis demonstrated that C-2-ceramide caused internucleosomal frag mentation in a dose- and time-dependent manner. Electron microscopy reveale d that the ceramide-treated cells manifested morphological characteristics typical of apoptosis. Conclusions The present results demonstrate that C-2-ceramide induces apopt osis of transformed human keratinocytes, whereas C-2-dihydroceramide does n ot have such an effect. The fact that ceramide induces apoptosis of keratin ocyctes raises the possibility that intracellular ceramide, which is increa sed with differentiation of the epidermis, might be involved in terminal di fferentiation, a specialized form of apoptosis of keratinocytes.