title: GADD153 and 12-lipoxygenase mediate fenretinide-induced apoptosis of neuroblastoma.
abstract: The synthetic retinoid fenretinide induces apoptosis of neuroblastoma cells and in vitro acts synergistically with chemotherapeutic drugs used to treat neuroblastoma. The mechanisms of fenretinide-induced cell death of neuroblastoma cells are complex, involving cellular signaling pathways as yet incompletely defined but, in part, involving the generation of reactive oxygen species (ROS). In an attempt to characterize the mechanism of action of fenretinide, cDNA array filters were screened to identify apoptotic genes regulated in response to treatment of SH-SY5Y cells with fenretinide. Expression of the stress-induced transcription factor, GADD153, was up-regulated at both the protein and mRNA levels in response to fenretinide. Overexpression of GADD153 increased apoptosis in the presence and absence of fenretinide, whereas reduced expression of GADD153 by expression of antisense DNA abrogated the response to fenretinide. Although fenretinide is a partial retinoic acid receptor (RAR)-beta/gamma agonist, RARbeta/gamma antagonists did not block the induction of GADD153 by fenretinide; conversely, the induction of GADD153 was blocked by antioxidants. Enzyme inhibitors were used to identify pathways mediating the ROS-dependent effects of fenretinide: inhibitors of phospholipase A(2) and lypoxygenases (LOX), and specific inhibitors of 12-LOX, but not 5-LOX or 15-LOX, inhibited the induction of ROS, apoptosis, and GADD153 in response to fenretinide. The inhibition of ROS and apoptosis was reversed by the addition of the 12-LOX products, 12 (S)-hydroperoxyeicosatetraenoic acid (12-HpETE) and 12 (S)-hydroxyeicosatetraenoic acid (12-HETE). Fenretinide did not increase free arachidonic acid levels, but increased LOX activity without a detectable increase in 12-LOX protein. These results suggest that fenretinide induces apoptosis via RAR-dependent and -independent pathways in which the RAR-independent pathway is characterized by a fenretinide-dependent increase in 12-LOX activity, leading to the induction of GADD153. The targeting of 12-LOX and/or GADD153 in neuroblastoma cells may thus present a novel pathway for the development of drugs inducing apoptosis of neuroblastoma with improved tumor specificity.
authors.affiliation: Department of Child Health, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United Kingdom.
authors.forename: Penny E
authors.initials: PE
authors.lastname: Lovat
authors.forename: Serafina
authors.initials: S
authors.lastname: Oliverio
authors.forename: Marco
authors.initials: M
authors.lastname: Ranalli
authors.forename: Marco
authors.initials: M
authors.lastname: Corazzari
authors.forename: Carlo
authors.initials: C
authors.lastname: Rodolfo
authors.forename: Francesca
authors.initials: F
authors.lastname: Bernassola
authors.forename: Karen
authors.initials: K
authors.lastname: Aughton
authors.forename: Mauro
authors.initials: M
authors.lastname: Maccarrone
authors.forename: Quentin D Campbell
authors.initials: QD
authors.lastname: Hewson
authors.forename: Andy D J
authors.initials: AD
authors.lastname: Pearson
authors.forename: Gerry
authors.initials: G
authors.lastname: Melino
authors.forename: Mauro
authors.initials: M
authors.lastname: Piacentini
authors.forename: Christopher P F
authors.initials: CP
authors.lastname: Redfern
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism*; D022762:CCAAT-Enhancer-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D004357:Drug Synergism; D017313:Fenretinide / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D020411:Oligonucleotide Array Sequence Analysis; D017382:Reactive Oxygen Species / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000037:antagonists & inhibitors; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000502:physiology; D000097768:Retinoic Acid Receptor gamma
chemical_list: D000970:Antineoplastic Agents; D001092:Arachidonate 12-Lipoxygenase; D022762:CCAAT-Enhancer-Binding Proteins; D017313:Fenretinide; D017382:Reactive Oxygen Species; D018168:Receptors, Retinoic Acid; D051742:Transcription Factor CHOP; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D000097768:Retinoic Acid Receptor gamma; C497249:DDIT3 protein, human; C082925:retinoic acid receptor beta