title: Fas-mediated apoptosis in neuroblastoma requires mitochondrial activation and is inhibited by FLICE inhibitor protein and Bcl-2.
abstract: Fas-mediated apoptosis proceeds though mitochondria-dependent or -independent pathways and is deficient in drug-resistant cells. Neuroblastoma, a common pediatric malignancy, often develops drug-resistance and has a silenced caspase 8 (FLICE) gene, which has been associated with Fas- and drug-resistance. We report that besides caspase 8, which was absent in approximately one-third of 26 neuroblastoma cases in this study, other proteins such as bcl-2 and FLICE-inhibitory protein (FLIP), are equally important in conferring Fas-resistance to neuroblastoma cells. Both bcl-2 and FLIP were frequently expressed in neuroblastoma tissues. Our in vitro studies showed that FLIP was recruited to the death-inducing signaling complex and interfered with the recruitment of caspase 8 in neuroblastoma cells. bcl-2 inhibited the activation of the mitochondria; but it also lowered the free cytoplasmic levels of caspase 8 by binding and sequestering it, thus acting through a novel antiapoptotic mechanism upstream of the mitochondria. In vitro down-regulation of bcl-2 with antisense oligonucleotides allowed the release of cytochrome c from mitochondria and the activation of caspases 8 and 3 upon Fas activation as well as sensitized neuroblastoma cells to Fas-mediated apoptosis. Down-regulation of FLIP had only a modest apoptotic effect because of the coexistent mitochondrial block. However, combined treatment with bcl-2 and FLIP antisense oligonucleotides had a statistically significant synergistic effect reversing Fas-resistance in neuroblastoma cells in vitro. These data indicate that Fas-mediated apoptosis in neuroblastoma cells is mitochondria-dependent and inhibited both at the mitochondrial level and at the level of caspase 8 activation. Thus, gene-targeting therapies for bcl-2 and FLIP may reverse Fas-resistance and prove useful in the treatment of drug-resistant neuroblastomas.
authors.affiliation: Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, Maryland 20892.
authors.forename: V
authors.initials: V
authors.lastname: Poulaki
authors.forename: N
authors.initials: N
authors.lastname: Mitsiades
authors.forename: M E
authors.initials: ME
authors.lastname: Romero
authors.forename: M
authors.initials: M
authors.lastname: Tsokos
mesh_terms: D000293:Adolescent; D017209:Apoptosis / Q000502:physiology*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000096:biosynthesis / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D047908:Intracellular Signaling Peptides and Proteins; D008928:Mitochondria / Q000502:physiology*; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D019014:fas Receptor / Q000502:physiology*
chemical_list: D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D047908:Intracellular Signaling Peptides and Proteins; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D019014:fas Receptor; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases