Fenretinide (4-HPR) is a cytotoxic retinoid active in many cancer cell lines in vitro. 4-HPR-induced death occurs in association with an increase of ceramide species (dihydroceramides > ceramides) in many cell lines. Modulators of sphingolipid metabolism can increase 4-HPR-induced cytotoxicity. We reported that 4-HPR increased ceramides by increasing the activities of serine palmitoyltransferase (SPT), the rate-limiting step in de novo ceramide synthesis, and of (dihydro)ceramide synthase. However, recently it was reported that 4-HPR increased ceramides in neuroblastoma cells by sphingomyelin (SM) hydrolysis. Therefore, we further investigated the mechanisms of 4-HPR-induced ceramide(s) increase. METHODS. Sphingomyelin (SM) was pre-labeled using [14C]palmitic acid (PA) followed by washout, labeled continuously during 4-HPR exposure, or pulse-labeled at the end of 4-HPR treatment; de novo synthesized sphingolipids were labeled using [14C]PA or [14C]serine. Sphingolipids were extracted, separated by thin-layer chromatography (TLC), and quantitated by phosphoimager scanning. Cell cytotoxicity was determined using a fluoresence-based assay system (DIMSCAN). RESULTS. In the SK-N-RA neuroblastoma cell line, in cells with pre-labeled SM pools, 4-HPR (10 μM) treatment decreased ceramides by ∼40% over controls at +6 hr (P<0.01) and SM pools did not decrease compared to controls (P=0.09). In contrast, 4-HPR treatment increased new SM formation (125% at +6 hr (P<0.001) and 19% at +24 hr (P=0.016), respectively). [14C]SM labeled at the end of 4-HPR treatment increased ∼23% at +6 hr (P=0.004) but labeling returned to baseline by +24 hr (P>0.05). In contrast, 4-HPR treatment increased de novo labeled ceramides (dihydroceramides >> ceramides) by ∼130% over controls at +6 hours. Together, these results suggest that, in SK-N-RA cells, 4-HPR increased ceramides mainly through de novo synthesis but with a contribution from inhibition of SM formation.To further investigate the role of sphingomyelin metabolism in 4-HPR-treated cells, D609, a SM synthase inhibitor, was employed. A 24 hr treatment of D609 (375 μM) inhibited SM formation by 65% (P<0.001), increased total ceramides by ∼60% (P=0.003), and, in combination with 4-HPR, D609 may have increased total ceramides a further 20% compared to 4-HPR-only treated cells (P=0.13). Further, D609 synergized 4-HPR cytotoxicity (4-HPR-alone, LD99 = ∼9.5 μM; D609+4-HPR, LD99 = 6.5 μM 4-HPR; survival fraction = 0.08 vs 0.0007 at 9 μM 4-HPR, C.I. < 0.4). CONCLUSIONS. In SK-N-RA neuroblastoma cells, 4-HPR increased ceramides primarily by de novo synthesis with a contribution from inhibition of SM synthesis. Further, inhibition of SM formation may constitute a novel method of synergizing 4-HPR cytotoxicity for neuroblastoma treatment.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]