Journal of Cellular Biochemistry 89:698–708 (2003) Mechanisms of Free-Radical Induction in Relation to Fenretinide-Induced Apoptosis of Neuroblastoma Penny E. Lovat, 1 Marco Ranalli, 2 Marco Corazzari, 2 Lizzia Raffaghello, 3 Andy D.J. Pearson, 1 Mirco Ponzoni, 3 Mauro Piacentini, 4 Gerry Melino, 2 and Christopher P.F. Redfern 1 * 1 Northern Institute for Cancer Research, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom 2 IDI-IRCCS Laboratory, Department of Experimental Medicine, University of Rome, Tor Vergata, Rome 00133, Italy 3 Differentiation Therapy Unit, Laboratory of Oncology, G. Gaslini Children’s Hospital, Genoa 16148, Italy 4 Department of Biology, University of Rome, Tor Vergata, Rome 00133, Italy Abstract The mechanisms of fenretinide-induced cell death of neuroblastoma cells are complex, involving signaling pathways mediated by free radicals or reactive oxygen species (ROS). The aim of this study was to identify mechanisms generating ROS and apoptosis of neuroblastoma cells in response to fenretinide. Fenretinide-induced ROS or apoptosis of SH-SY5Y or HTLA 230 neuroblastoma cells were not blocked by Nitro L-argenine methyl ester (L-NAME), an inhibitor of nitric oxide synthase. Flavoprotein-dependent superoxide-producing enzymes such as NADPH oxidase were also not involved in fenretinide-induced apoptosis or ROS generation. Similarly, ketoconazole, a cytochrome P450 inhibitor, and inhibitors of cyclooxygenase (COX) were also ineffective. In contrast, inhibition of phospholipase A 2 or lipoxygenases (LOX) blocked the induction of ROS and apoptosis in response to fenretinide. Using specific inhibitors of LOX, blocking 12-LOX but not 5- or 15-LOX inhibited both fenretinide-induced ROS and apoptosis. The effects of eicosatriynoic acid, a specific 12-LOX inhibitor, were reversed by the addition of the 12-LOX products, 12 (S)- hydroperoxyeicosatetraenoic acid and 12 (S)-hydroxyeicosatetraenoic acid. The targeting of 12-LOX in neuroblastoma cells may thus be a novel pathway for the development of drugs inducing apoptosis of neuroblastoma with improved tumor specificity. J. Cell. Biochem. 89: 698 – 708, 2003. ß 2003 Wiley-Liss, Inc. Key words: lipoxygenase; fenretinide; neuroblastoma; free radicals; apoptosis Retinoic acid induces neuroblastoma cells to differentiate [Sidell et al., 1983]. In contrast, fenretinide, a synthetic derivative of retinoic acid in which the carboxyl end has been modi- fied by the addition of an N-4-(hydroxyphenyl) group, induces apoptosis of neuroblastoma cells rather than differentiation [Ponzoni et al., 1995]. The inhibition of apoptosis by retinoic acid receptor (RAR) antagonists and antioxidants suggests that signaling pathways involving RARs and reactive oxygen species (ROS) are both required for the fenretinide-induced apoptosis of neuroblastoma cells [Lovat et al., 2000a], with the latter also involving a ROS- dependent induction of the stress-response transcription factor GADD153 [Lovat et al., 2002]. Recent studies have also suggested that a p53-independent pathway of fenreti- nide-induced apoptosis of neuroblastoma may operate through increased intracellular levels of the lipid secondary-messenger ceramide [Maurer et al., 1999, 2000]. Since fenret- inide synergizes with chemotherapeutic drugs to induce apoptosis in vitro [Lovat et al., 2000b], defining the mechanism of free radical ß 2003 Wiley-Liss, Inc. Grant sponsor: North of England Children’s Cancer Research Fund (UK); Grant sponsor: CLIC (UK); Grant sponsor: Telethon E872 (Italy); Grant sponsor: AICR (Italy); Grant sponsor: MURST-cofin (Italy); Grant sponsor: EU (QLG1-1999-00739); Grant sponsor: CNR (Italy); Grant sponsor: The Italian Neuroblastoma Society (Italy). *Correspondence to: Dr. Chris Redfern, 4th Floor Cookson Building, Northern Institute for Cancer Research, Medical School, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK. E-mail: chris.redfern@ncl.ac.uk Received 10 February 2003; Accepted 21 March 2003 DOI 10.1002/jcb.10551.