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 LC(99) (the
drug concentration lethal to 99% of cells) = 6.1 +/- 5.4 microm (range, 1.7-21.8 microm);
safingol (1-3 microm) synergistically increased
4-HPR cytotoxicity and reduced the mean
4-HPR LC(99) to 3.2 +/- 1.7 microm (range, 2.0-8.0 microm; 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 LC(99), 19.7 +/- 23.9 microm; range, 2.3-91.4). However,
hypoxia affected
4-HPR +
safingol cytotoxicity to a lesser extent (P = 0.04; 4-HPR LC(99), 4.9 +/- 2.3 microm; 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.