Fenretinide cytotoxicity for Ewing's sarcoma and primitive neuroectodermal tumor cell lines is decreased by hypoxia and synergistically enhanced by ceramide modulators

Patients with disseminated Ewing's family of tumors (ESFT) often experience drug-resistant relapse. We hypothesize that targeting minimal residual disease with the cytotoxic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR; fenretinide) may decrease relapse. We determined the following: (a) 4-HPR cytotoxicity against 12 ESFT cell lines in vitro; (b) whether 4-HPR increased ceramide species (saturated and desaturated ceramides); (c) whether physiological hypoxia (2% O-2) affected cytotoxicity, mitochondrial membrane potential (DeltaPsi(m)) change, or ceramide species or reactive oxygen species levels; (d) whether cytotoxicity was enhanced by L-threo-dihydrosphingosine (safingol); (e) whether physiological hypoxia increased acid ceramidase (AC) expression; and (f) the effect of the AC inhibitor N-oleoyl-ethanolamine (NOE) on cytotoxicity and ceramide species. Ceramide species were quantified by thin-layer chromatography and scintillography. Cytotoxicity was measured by a fluorescence-based assay using digital imaging microscopy (DIMSCAN). Gene expression profiling was performed by oligonucleotide array analysis. We observed, in 12 cell lines tested in normoxia (20% O-2), that the mean 4-HPR LC99 (the drug concentration lethal to 99% of cells) = 6.1 +/- 5.4 mum (range, 1.7-21.8 mum); safingol (1-3 muM) synergistically increased 4-HPR cytotoxicity and reduced the mean 4-HPR LC99 to 3.2 +/- 1.7 mum (range, 2.0-8.0 muM; combination index < 1). 4-HPR increased ceramide species in the three cell lines tested (up to 9-fold; P < 0.05). Hypoxia (2% O-2) reduced ceramide species increase, DeltaPsi(m) loss, reactive oxygen species increase (P < 0.05), and 4-HPR cytotoxicity (P = 0.05; 4-HPR LC99, 19.7 +/- 23.9 mu M; range, 2.3-91.4). However, hypoxia affected 4-HPR + safingol cytotoxicity to a lesser extent (P = 0.04; 4-HPR LC99, 4.9 +/- 2.3 mu M; range, 2.0-8.2). Hypoxia increased AC RNA expression; the AC inhibitor NOE enhanced 4-HPR-induced ceramide species increase and cytotoxicity. The antioxidant N-acetyl-L-cysteine somewhat reduced 4-HPR cytotoxicity but did not affect ceramide species increase. We conclude the following: (a) 4-HPR was active against ESFT cell lines in vitro at concentrations achievable clinically, but activity was decreased in hypoxia; and (b) combining 4-HPR with ceramide modulators synergized 4-HPR cytotoxicity in normoxia and hypoxia.