Abstract 2616: Comparison of the cytotoxicity and increase of reactive oxygen species and dihydroceramides of fenretinide to its major metabolites (4-oxo- and 4-methoxyphenyl fenretinide) in T-cell lymphoid malignancy, neuroblastoma, and ovarian cancer cell lines
Authors: Song Michael M. , Makena Monish R. , Hindle Ashly , Koneru Balakrishna , Nguyen Thinh H. , Cho Hwangeui , Maurer Barry J. , Kang Min H. , Reynolds C. Patrick 
Published: August 2015

Introduction: Early-phase clinical trials of fenretinide (4-HPR, a synthetic retinoid) have demonstrated durable complete responses in T-cell lymphoma (TCL), neuroblastoma (NB), and signals of activity in ovarian cancer (OV). Cytotoxic mechanisms of 4-HPR include increase of reactive oxygen species (ROS) and dihydroceramides (DHCs). Major 4-HPR metabolites are N-(4-methoxyphenyl)retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (oxo-HPR). MPR is more abundant in human plasma; oxo-HPR is present in lower concentrations. We assessed the relative cytotoxicity and increase of ROS and DHCs at equimolar concentrations of 4-HPR, MPR, and oxo-HPR in TCL, NB, and OV cell lines.

Methods: A panel of twelve TCL, NB, and OV cell lines was studied in room air (20% O2) and physiologic (5% O2) cultured conditions. Cytotoxicity (0-10 μM) was assessed by DIMSCAN fluorescence cytotoxicity assay. ROS was measured via DCFDA with flow cytometry and sphingolipids (including DHCs, ceramides, sphingoid bases, sphingomyelins, and glycosylated ceramides) by quantitative tandem mass spectrometry.

Results: 4-HPR (10 μM) demonstrated &gt;3 logs of cell kill in 6 of 12 cell lines. Oxo-HPR demonstrated comparable cytotoxicity to that of 4-HPR in all cell lines. MPR lacked cytotoxicity and when combined with 4-HPR, did not increase cytotoxicity or induce antagonism. There was no observed difference in cytotoxicity between 4-HPR and oxo-HPR in either oxygen condition. Both 4-HPR and oxo-HPR significantly increased ROS compared to controls (p&lt;0.05) in three cell lines tested (1 each of TCL, NB, and OV). Relative to 4-HPR, the ROS increased by oxo-HPR was equal in two cell lines (COG-LL-317h and TX-OV-186h), and higher in one (FU-NB-2006h). There was a significant (35 to 75-fold) increase of various DHC species (C16-, C18-, C22-, C24-, or C24:1-DHC) with both 4-HPR and oxo-HPR in the four cell lines tested (p&lt;0.05). Two of the four cell lines (Jurkat and TX-OV-186h) demonstrated a greater increase in DHCs with 4-HPR compared to oxo-HPR (p&lt;0.05), while the increase was not statistically different in the other two (COG-LL-317h and FU-NB-2006h).

Conclusions: In contrast to the higher cytotoxicity of oxo-HPR compared with 4-HPR reported by Villani et al (Cancer Res, 15:3238, 2006), we observed relatively comparable cytotoxicity and increases of ROS and DHCs with both 4-HPR and oxo-HPR. MPR was not cytotoxic nor did it antagonize 4-HPR. These results support that the cytotoxicity and pharmacodynamics of 4-HPR and oxo-HPR in in vitro culture systems are equivalent.

Citation Format: Michael M. Song, Monish R. Makena, Ashly Hindle, Balakrishna Koneru, Thinh H. Nguyen, Hwangeui Cho, Barry J. Maurer, Min H. Kang, C. Patrick Reynolds. Comparison of the cytotoxicity and increase of reactive oxygen species and dihydroceramides of fenretinide to its major metabolites (4-oxo- and 4-methoxyphenyl fenretinide) in T-cell lymphoid malignancy, neuroblastoma, and ovarian cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2616. doi:10.1158/1538-7445.AM2015-2616