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: V Poulaki, N Mitsiades, M E Romero, M Tsokos
• Journal: Cancer research (Cancer Res) Vol. 61 Issue 12 Pg. 4864-72 (Jun 15 2001) ISSN: 0008-5472 [Print] United States
• PMID: 11406564 (Publication Type: Journal Article)

Chemical References

• CASP8 and FADD-Like Apoptosis Regulating Protein
• CFLAR protein, human
• Carrier Proteins
• Caspase Inhibitors
• Cytochrome c Group
• Intracellular Signaling Peptides and Proteins
• Oligonucleotides, Antisense
• Proto-Oncogene Proteins c-bcl-2
• fas Receptor
• CASP8 protein, human
• CASP9 protein, human
• Caspase 8
• Caspase 9
• Caspases

Topics

• Adolescent
• Apoptosis (physiology)
• CASP8 and FADD-Like Apoptosis Regulating Protein
• Carrier Proteins (genetics, physiology)
• Caspase 8
• Caspase 9
• Caspase Inhibitors
• Caspases (biosynthesis, metabolism)
• Child
• Child, Preschool
• Cytochrome c Group (metabolism)
• Enzyme Activation
• Humans
• Infant
• Infant, Newborn
• Intracellular Signaling Peptides and Proteins
• Mitochondria (physiology)
• Neuroblastoma (metabolism, pathology)
• Oligonucleotides, Antisense (genetics, pharmacology)
• Proto-Oncogene Proteins c-bcl-2 (genetics, metabolism, physiology)
• fas Receptor (physiology)