Safingol: phosphorylation and effect on endogenous sphingolipid formation in human ALL leukemia and neuroblastoma cell lines

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Safingol (L-threo-dihydrosphingosine) synergizes the cytotoxicity of the dihydroceramide-generating retinoid, N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) in human cancer cell lines. The mechanism by which safingol synergizes 4-HPR cytotoxicity is not well defined. Determining safingol metabolism in human cancer cells is essentialto understanding the mechanism of 4-HPR + safingol cytotoxicsynergy, and to optimize clinical strategies for 4-HPR + safingol administration.Mass spectroscopy, and [³H]safingol and [¹⁴C]serine radiolabeling, were employed to determine the phosphorylation and metabolism of safingol in human ALL leukemia cell line, MOLT-4, and human neuroblastoma cell line, CHLA-90. Mass spectrometry analysis showed that in MOLT-4 and CHLA-90 cells, safingol (3 micromolar, for 16 hours) was acylated to L-threo-dihydroceramides, with an increase predominantly of C14-, C18-, C24- and C24:1- dihydroceramide species (P<0.01); also observed was a ~60-fold (P<0.01) increase of dihydrosphingosine-1-phosphates (stereochemistry not resolved). Thin layer chromatography (TLC) results showed that ³H-safingol (1 - 5 micromolar, for 6 hours) was phosphorylated to L-threo-dihydrosphingosine-1-phosphate (to ~0.2% of added safingol) in MOLT-4 cells. Additionally, L-threo-dihydroceramide, a metabolite of safingol, was phosphorylated to L-t-dihydroceramide-1-phosphate (to ~2% of added safingol), but was not glucosylated. Assay showed that exposure of whole MOLT-4 cells to safingol (5 micromolar) for 6 hours inhibited sphingosine kinase activity by ~60% (P<0.01). Interestingly, by [¹⁴C]serine labeling, MOLT-4 cells treated with safingol (5 micromolar) for 24 hours increased formation of endogenous D-erythro-ceramides by 70%, while decreasing formation of endogenous D-erythro-glucosylceramides and D-erythro-sphingomyelins by 40%, and 35%, respectively (p<0.01).

It is concluded that safingol was metabolized to safingol-1-phosphate, L-threo-dihydroceramides, and L-threo-dihydroceramide-1-phosphates; safingol treatment also increased levels of endogenous D-erythro-ceramides, likely by inhibiting the formation of D-erythro-glucoceramides, D-erythro-sphingomyelins, and sphingosine-1-phosphate. Results also suggest that phosphorylated safingol metabolites have the potential to interfere with the biological functions of sphinganine-1-phosphate, sphingosine-1-phosphate and ceramide-1-phosphates. Thus, safingol exposure impacts normal sphingolipid metabolism at multiple steps; investigations on the mechanism(s) of fenretinide + safingol cytotoxic synergy continue.