title: TRAIL-mediated apoptosis requires NF-kappaB inhibition and the mitochondrial permeability transition in human hepatoma cells.
abstract: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide range of malignant cells. However, several cancers, including human hepatoma, are resistant to TRAIL. In this study, we analyzed TRAIL-induced pro- and antiapoptotic signaling pathways in human hepatoma cells. Nuclear factor kappa B (NF-kappaB) was found to be a critical TRAIL-induced antiapoptotic factor in the PLC/PRF/5, HepG2, and Hep3B cell lines. TRAIL-induced NF-kappaB activation was preceded by IkappaBalpha kinase (IKK) activation and IkappaBalpha degradation and depended on TRAF2, NF-kappaB-inducing kinase (NIK), IKK1, and IKK2. Accordingly, inhibition of NF-kappaB by adenoviral dominant negative (dn) TRAF2, NIKdn, IKK1dn, IKK2dn, or IkappaBsr sensitized PLC/PRF/5 cells to rhTRAIL, resulting in 40% to 50% cell death after 48 hours as compared with <10% with rhTRAIL alone. Agonistic anti-TRAIL receptor 1 and anti-TRAIL receptor 2 antibodies or combinations of both were equally efficient in inducing apoptosis as rhTRAIL, indicating that decoy receptors did not contribute to resistance toward TRAIL under the conditions of our study. TRAIL-mediated apoptosis depended on FADD, caspase 8 and 3 as demonstrated by the ability of FADDdn, CrmA, and pharmacologic caspase inhibitors to prevent apoptosis. Confocal microscopy showed the onset of the mitochondrial permeability transition (MPT) 5 hours after rhTRAIL plus actinomycin D, which was followed by cytochrome c release. The MPT was critical for TRAIL-induced apoptosis as demonstrated by the ability of pharmacologic MPT inhibitors to completely protect PLC/PRF/5 cells. In conclusion, NF-kappaB prevents TRAIL-induced apoptosis in human hepatoma through a TRAIL-activated TRAF2-NIK-IKK pathway. Inhibition of NF-kappaB unmasks a TRAIL-induced apoptotic signaling cascade that involves FADD, caspase 8, the MPT, and caspase 3.
authors.affiliation: Department of Medicine, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Young-Soo
authors.initials: YS
authors.lastname: Kim
authors.forename: Robert F
authors.initials: RF
authors.lastname: Schwabe
authors.forename: Ting
authors.initials: T
authors.lastname: Qian
authors.forename: John J
authors.initials: JJ
authors.lastname: Lemasters
authors.forename: David A
authors.initials: DA
authors.lastname: Brenner
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D006528:Carcinoma, Hepatocellular; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000502:physiology; D003609:Dactinomycin / Q000494:pharmacology; D053200:Fas-Associated Death Domain Protein; D006801:Humans; D051550:I-kappa B Kinase; D008113:Liver Neoplasms; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D047992:TNF Receptor-Associated Factor 2; D053221:TNF-Related Apoptosis-Inducing Ligand; D051996:Transcription Factor RelA; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*; D000097782:NF-kappaB-Inducing Kinase
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000903:Antibiotics, Antineoplastic; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D003609:Dactinomycin; D053200:Fas-Associated Death Domain Protein; D051550:I-kappa B Kinase; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D047992:TNF Receptor-Associated Factor 2; D053221:TNF-Related Apoptosis-Inducing Ligand; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D000097782:NF-kappaB-Inducing Kinase; C505533:FADD protein, human; C105190:TNFRSF10A protein, human; C505639:TNFRSF10B protein, human; C505653:TNFSF10 protein, human; C496559:CHUK protein, human; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human