Activation of Akt/PKB at serine 473 by N-acetylphytosphingosine (NAPS) and C-2-ceramide reduces melanin synthesis in B16F10 mouse melanoma cells

Abstract

Sphingolipid metabolites regulate many aspects of cell proliferation, differentiation, and apoptosis. In the present study, we have assessed the effects of the novel phytosphingosine derivative, N-acetylphytospingosine (NAPS), on the depigmentation of murine B16F10 melanoma cells, and have also attempted to identify the possible signaling pathway involved, in comparison with C-2-ceramide. NAPS and C-2-ceramide both inhibited the growth of the B16F10 cells in a dose-dependent manner. Melanin content and tyrosinase activity were significantly reduced in response to treatment with NAPS and C2-ceramide at concentrations in a range between 1-5 mu M. However, the levels of tyrosinase mRNA, as well as the levels of tyrosinase related protein-1 (TRP-1) and tyrosinase related protein-2 (TRP-2) genes and the level of tyrosinase protein remained unaffected by treatment with either NAPS or C2-ceramide. We also attempted to determine the signaling pathway exploited by NAPS and C2-ceramide. Interestingly, the phosphorylation of Akt/PKB at serine 473 by NAPS was reduced at the 5 minute mark, whereas C2-ceramide induced the phosphorylation of Akt/PKB at serine 473. Finally, Akt/PKB activity in the NAPS-treated cells was elevated in comparison with the untreated cells. LY294002, a specific PI3-K inhibitor which is located upstream of Akt/PKB, inhibited the phosphorylation of Akt/PKB, but induced an increase in melanin synthesis. These results suggest that the activation of Akt/PKB at serine 473 is related with the suppression of melanin production in the B16F10 mouse melanoma cells. Therefore, the mechanisms exploited by NAPS and C-2-ceramide responsible for the depigmentation of B16F10 cells were concluded to involve the inhibition of melanosomal tyrosinase activity.