Effects of C2-ceramide on the Malme-3M melanoma cell line.

Ceramide is implicated in the regulation of various signaling pathways leading to proliferation, differentiation or apoptotic cell death, but there have been few investigations about the effects of ceramide on the cell growth and the melanogenesis of melanocytes. In the present study, we investigated the effects of cell-permeable ceramide on Malme-3M human melanoma cell line. MTT proliferation assay showed that C2-ceramide inhibited the growth of Malme-3M cells in a dose-dependent manner. Cell cycle analysis confirmed the inhibition of DNA synthesis by a reduction in the S phase and an increase in the G0/G1 phase. Flow cytometric analysis for apoptotic cells and morphological observations indicated that the antiproliferative effect of C2-ceramide was not due to apoptosis. We next investigated the effects of C2-ceramide on the pigmentation of Malme-3M melanoma cells. The results showed that C2-ceramide induced only a slight decrease of tyrosinase activity and melanin synthesis. To investigate the ceramide signaling pathway, we studied the influence of C2-ceramide on extracellular signal-regulated kinase (ERK) and Akt activation by Western blot. We demonstrated that the amount of phosphorylated Akt was decreased by C2-ceramide, whereas ERK was activated transiently. Because of a well-known involvement of ceramide in apoptosis, we further investigated the level of caspase-3 and HSP70 after treatment of C2-ceramide. We found that the caspase-3 was not activated and the expression of HSP70 increased moderately. In conclusion, C2-ceramide inhibited the cell growth of Malme-3M cells without the induction of apoptosis. We suggest that increased HSP70 may be related to the resistance against apoptosis.