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 D3 inducible sphingomyelin cycle generates ceramide in keratinocytes. Although it has been suggested that exogenous ceramide induces apoptosis of keratinocytes, which is similar to their effect on other cell types, such as leukaemia cells, only a few studies have reported ceramide-induced apoptosis of keratinocytes.

Objective: To determine whether ceramide induces apoptosis of keratinocytes, we used the synthetic ceramide analogue, C2-ceramide (N-acetylsphingosine) and a human squamous cell carcinoma cell line, HSC-I.

Methods: We treated HSC-I cells with C2-ceramide, followed by a viability assay, morphological observations, nick end-labelling (TUNEL), DNA electrophoresis, and electron microscopy.

Results: In the viability assay, C2-ceramide was toxic to HSC-I cells in a dose-dependent manner. Manifestations of apoptotic morphology occurred in the ceramide-treated cells, whereas these morphological changes did not occur in cells treated with dihydroceramide (N-acetylsphinganine). TUNEL revealed that many of the ceramide-treated cells showed positive reactivity. DNA electrophoresis demonstrated that C2-ceramide caused internucleosomal fragmentation in a dose- and time-dependent manner. Electron microscopy revealed that the ceramide-treated cells manifested morphological characteristics typical of apoptosis.

Conclusions: The present results demonstrate that C2-ceramide induces apoptosis of transformed human keratinocytes, whereas C2-dihydroceramide does not have such an effect. The fact that ceramide induces apoptosis of keratinocyctes raises the possibility that intracellular ceramide, which is increased with differentiation of the epidermis, might be involved in terminal differentiation, a specialized form of apoptosis of keratinocytes.