Increase of ceramide and induction of mixed apoptosis necrosis by N-(4-hydroxyphenyl)-retinamide in neuroblastoma cell lines

Background: The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR or fenretinide) is toxic to myeloid leukemia and cervical carcinoma cell lines , probably in part due to its ability to increase levels of reactive oxygen species (ROS), We have studied the effects of 4-HPR on neuroblastoma cell lines. Since neuroblastomas commonly relapse in bone marrow, a hypoxic tiss ue compartment, and many chemotherapeutic agents are antagonized by hypoxia , our purpose was to study in these cell lines several factors influencing 4-HPR-induced cytotoxicity, including induced levels of ROS, effects of phy siologic hypoxia and antioxidants, levels of ceramide, and the mechanism of cell death. Methods: ROS generation was measured by carboxy-dichlorofluore scein diacetate fluorescence. Ceramide was quantified by radiolabeling and thin-layer chromatography, Immunoblotting was used to assess p53 protein le vels. Apoptosis (programmed cell death) and necrosis were analyzed by nucle ar morphology and internucleosomal DNA fragmentation patterns, Cytotoxicity was measured by a fluorescence-based assay employing digital imaging micro scopy in the presence or absence of the pancaspase enzyme inhibitor BOC-d-f mk, Statistical tests were two-sided, Results/ Conclusions: In addition to increasing ROS, 4-HPR (2.5-10 mu M) statistically significantly increased t he level of intracellular ceramide (up to approximately 10-fold; P<.001) in a dose-dependent manner in two neuroblastoma cell. lines, one of which is highly resistant to alkylating agents and to etoposide. Cell death induced by 4-HPR was reduced but not abrogated by hypoxia in the presence or absenc e of an antioxidant, N-acetyl-L-cysteine, Expression of p53 protein was not affected by 4-HPR, Furthermore, the pan-caspase enzyme inhibitor BOC-d-fmk prevented apoptosis, but not necrosis, and only partially decreased cytoto xicity induced by 4-HPR, indicating that 4-HPR induced both apoptosis and n ecrosis in neuroblastoma cells, Implications: LC-HPR may form the basis for a novel, p53-independent chemotherapy that operates through increased intr acellular levels of ceramide and that retains cytotoxicity under reduced ox ygen conditions.