Introduction: N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) is a clinical-stage synthetic retinoid cytotoxic to many cancer cell lines in a dose- and time-dependent manner in vitro by mechanisms that include reactive oxygen species (ROS) generation and increase of D-erythro-dihydroceramides via de novo synthesis. The cytotoxicity of 4-HPR is synergistically increased by low, minimally cytotoxic concentrations (1 - 4 µM) of the stereochemical sphinganine analog, safingol (C18-L-threo-sphinganine). Currently reported activities of safingol include inhibition of sphingosine kinase 1 (SPHK1) and/or protein kinase C's (PKCs) at higher concentrations (20 - 50 µM) in vitro. However, previous work done in our lab has shown that parent safingol is substantially metabolized (90%) by +24 hrs, and as a result, activities dependent on the presence of parental safingol would not be expected to be maintained over time. Given these apparent discrepancies, we hypothesized that safingol does not synergize 4-HPR cytotoxicity through parental inhibition of SPHK1 or PKC's, but rather through a mechanism dependent on the concurrent increase of normal and variant, D-erythro- and L-threo-dihydroceramides. Methods: Human neuroblastoma (NB) cell lines CHLA-15, CHLA-90, and SK-N-RA, which display synergistic cytotoxicity to 4-HPR + safingol (H+S) were selected as a model system. In our washout experiments cells were treated with 4-HPR (0 - 6 µM) either concurrently with a 4-hr safingol (4 µM) exposure, or at later times after safingol exposure (+4_ +48 hours), to allow for safingol metabolism. Cytotoxicity was measured using a semi-automated, fluorescence-based assay system (DIMSCAN). Flow cytometry experiments to analyze ROS levels in response to H+S treatment have also been conducted. Subsequently, liquid chromatography tandem mass spectroscopy (LC/MS/MS) is being utilized to quantitate various ceramides, L-threo-, and D-erythro-dihydroceramides in order to determine any possible correlation between any particular species and cytotoxicity. Results and Conclusions: Comparable cytotoxicity (P<.05) was observed whether 4-HPR was administered concurrently with safingol or at various later time points. These data support that cytotoxic synergy was not dependent on the presence of parental safingol. Experiments quantitating ROS by flow cytometry do not show changes in the levels of these species in H vs. H+S groups, ruling out ROS generation as a mechanism of synergy between H+S. Preliminary LC/MS/MS experiments paired with DIMSCAN cytotoxicity assays indicate that certain sphingolipid species correlate with cytotoxicity in our NB models. Alternative hypotheses include that L-threo-dihydroceramides inhibit SPHK1 or PKC's activity. This information will help inform the clinical delivery schedules of H+S to decrease toxicities and maximize antitumor efficacy.
Citation Format: Mohammad S. Abedi. Fenretinide and safingol synergistic cytotoxicity in neuroblastoma is, in part, mediated by increase of dihydroceramides. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5516. doi:10.1158/1538-7445.AM2014-5516