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We reported that safingol (L-threo-dihydrosphingosine), the isomer of dihydrosphingosine, synergized multi-log fenretinide(4-HPR) cytotoxicity in a variety of types of solid tumor and leukemia cell lines in vitro. Safingol has a short plasma half-life in vivo. The mechanism of safingol synergistically increasing 4-HPR cytotoxicity is unknown. It has been reported that safingol inhibits protein kinase C activity, and DL-threo-dihydrosphingosine inhibits sphingosine kinase. As it has been mainly assayed in platelets, or cell extracts, or using short-term (minutes) drug exposure, its significance is unclear. To clarify the uptake and metabolism of safingol in human cancer/leukemia cells not only help us to understand the mechanism of safingol cytotoxicity, but also is a crucial step for design of an effective safingol administration strategy. Using tritium-labeled safingol as tracer, we studied the uptake and metabolism of safingol in human neuroblastoma cell line CHLA-90 and leukemia cell line MOLT-4. Safingol and its metabolites were analyzed by (high performance) thin-layer chromatography and liquid scintillation counting, cell viability were measured using DIMSCAN analysis. After 4 hrs incubation with 1 μM or 3 μM safingol, CHLA-90 cells uptake 59% and 38%, MOLT-4 cells uptake 72% and 59% of safingol, respectively. For the washout experiments, CHLA-90 or MOLT-4 cells were preincubated with 6 μM safingol for 2 hrs, then free safingol was washed away and fresh medium was added. Four hrs after washout, there was 50% and 70% free cellular safingol left in CHLA-90 cells and MOLT-4 cells, respectively. Twenty-four hrs after washout, only 15% and 21% free cellular safingol left in CHLA-90 cells and MOLT-4 cells, respectively. Lipids analysis showed that Safingol was N-acylated to form unsaturated L-threo-dihydroceramide and L-threo-dihydrosphingomyelin. No safingol-derived glucosylceramide was detected. Most of the cellular safingol was converted to L-threo-dihydrosphingomylelin in CHLA-90 cells, and to L-threo-dihydroceramide in MOLT-4 cells at 24 hrs after washout. Short chain L-threo-dihydroceramide (C2, C6 and C8) did not synergistically increase 4-HPR cytotoxicity in CHLA-90 and MOLT-4 cells. We conclude that 1). Safingol is uptoken very quickly by CHLA-90 and MOLT-4 cells; 2). High levels of free safingol exist in human cancer/leukemia cell for up to 4 hrs; 3). Most of safingol uptoken is converted to L-threo-sphingomyelin by CHLA-90 cells, and to L-threo-dihydroceramide by MOLT-4 cells; 4). No detectable glucosylceramide is formed from safingol; 5). Safingol cytotoxicity is decreased after converted to L-threo-dighydroceramide. This study has provided crucial information for design an effective strategy to administrate safingol for in vivo study, and for clarification of the mechanism of safingol synergizing 4-HPR cytotoxicity in human cancer/leukemia cells.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]