We have reported that fenretinide (4-HPR) increased generation of ceramides via de novo synthesis in human cancer cell lines. Cells can potentially reduce ceramide-induced cytotoxicity by converting ceramides to nontoxic glucosylceramides (GC), 1-O-acylceramides (AC), and sphingomyelin (SM). We hypothesized that inhibitors that block ceramide-shunting pathways may synergize fenretinide cytotoxicity. We determined the effects of various isomers of 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP) on fenretinide-induced sphingolipid metabolism and cyototoxicity in human neuroblastoma cell line, SK-N-RA, and breast cancer cell line, MCF-7, using high performance thin-layer chromatography (HPTLC) and phosphoimager scanning. We observed that: In SK-N-RA cells, 4-HPR (10 μM) alone increased formation of total ceramides (saturated and unsaturated) by ∼7-fold, and increased AC, GC, and SM formation by 1.3, 1.6 and 2.3 fold at +24 hours, respectively. D-threo-PPMP (10 μM) alone inhibited baseline GC formation by ∼25%, increased ceramides by 1.9 fold, increased AC formation by 4.4 fold, but had no effect on SM formation, compared to controls, at +24 hours. D-threo-PPMP + 4-HPR increased ceramides by ∼13 fold, prevented the increase of SM induced by 4-HPR-alone, and more strongly inhibited GC formation (64% reduction) compared to controls. AC formation was 20% lower compared D-threo-PPMP-alone. Compared to controls, D,L-erythro-PPMP (10 μM) alone increased ceramides by 1.9 fold, increased AC formation by 2.4 fold, but had no effect on GC and SM formation. D,L-erythro-PPMP + 4-HPR increased total ceramides by 11 fold compared with control, decreased SM formation by ∼20% compared to 4-HPR-alone, and decreased AC formation by 30% compared DL-erythro-PPMP-alone. GC formation was the same as 4-HPR-alone. DL-erythro-PPMP also did not inhibit total ganglioside generation down stream of GC. Overall, similar results were found in MCF-7 cells. We conclude: that both PPMP isomers (L-threo > D,L-erythro) increased 4-HPR-induced ceramides, and synergized 4-HPR cytotoxicity, but apparently through differing effects on sphingolipid metabolism. L-threo-PPMP prevented both 4-HPR-induced GC and SM formation. In contrast, D,L-erythro-PPMP did not prevent 4-HPR-induced GC increase and only somewhat decreased 4-HPR-induced SM increase. Unexpectedly, 4-HPR appeared to inhibit PPMP isomer-induced AC increase. These results also demonstrate the complex stereochemical specificities of such inhibitors. SiRNA experiments designed to further elucidate the relative importance of these sphingolipid pathways to fenretinide cytotoxicity are in progress.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]