title: Histone deacetylase inhibitors strongly sensitise neuroblastoma cells to TRAIL-induced apoptosis by a caspases-dependent increase of the pro- to anti-apoptotic proteins ratio.
abstract: BACKGROUND
  Neuroblastoma (NB) is the second most common solid childhood tumour, an aggressive disease for which new therapeutic strategies are strongly needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumour cells, but not in normal tissues and therefore represents a valuable candidate in apoptosis-inducing therapies. Caspase-8 is silenced in a subset of highly malignant NB cells, which results in full TRAIL resistance. In addition, despite constitutive caspase-8 expression, or its possible restoration by different strategies, NB cells remain weakly sensitive to TRAIL indicating a need to develop strategies to sensitise NB cells to TRAIL. Histone deacetylase inhibitors (HDACIs) are a new class of anti-cancer agent inducing apoptosis or cell cycle arrest in tumour cells with very low toxicity toward normal cells. Although HDACIs were recently shown to increase death induced by TRAIL in weakly TRAIL-sensitive tumour cells, the precise involved sensitisation mechanisms have not been fully identified.
  
  
  METHODS
  NB cell lines were treated with various doses of HDACIs and TRAIL, then cytotoxicity was analysed by MTS/PMS proliferation assays, apoptosis was measured by the Propidium staining method, caspases activity by colorimetric protease assays, and (in)activation of apoptotic proteins by immunoblotting.
  
  
  RESULTS
  Sub-toxic doses of HDACIs strongly sensitised caspase-8 positive NB cell lines to TRAIL induced apoptosis in a caspases dependent manner. Combined treatments increased the activation of caspases and Bid, and the inactivation of the anti-apoptotic proteins XIAP, Bcl-x, RIP, and survivin, thereby increasing the pro- to anti-apoptotic protein ratio. It also enhanced the activation of the mitochondrial pathway. Interestingly, the kinetics of caspases activation and inactivation of anti-apoptotic proteins is accelerated by combined treatment with TRAIL and HDACIs compared to TRAIL alone. In contrast, cell surface expression of TRAIL-receptors or TRAIL is not affected by sub-toxic doses of HDACIs.
  
  
  CONCLUSIONS
  HDACIs were shown to activate the mitochondrial pathway and to sensitise NB cells to TRAIL by enhancing the amplitude of the apoptotic cascade and by restoring an apoptosis-prone ratio of pro- to anti-apoptotic proteins. Combining HDACIs and TRAIL could therefore represent a weakly toxic and promising strategy to target TRAIL-resistant tumours such as neuroblastomas.
authors.affiliation: Paediatric Oncology Research, Paediatric Department, University Hospital CHUV, CH-1011 Lausanne, Switzerland. Annick.Muhlethaler@chuv.ch
authors.forename: Annick
authors.initials: A
authors.lastname: Mühlethaler-Mottet
authors.forename: Marjorie
authors.initials: M
authors.lastname: Flahaut
authors.forename: Katia Balmas
authors.initials: KB
authors.lastname: Bourloud
authors.forename: Katya
authors.initials: K
authors.lastname: Auderset
authors.forename: Roland
authors.initials: R
authors.lastname: Meier
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Joseph
authors.forename: Nicole
authors.initials: N
authors.lastname: Gross
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism* / Q000494:pharmacology; D002087:Butyrates / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism*; D002470:Cell Survival; D004791:Enzyme Inhibitors / Q000494:pharmacology / Q000627:therapeutic use*; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D051034:Inhibitor of Apoptosis Proteins; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D009447:Neuroblastoma / Q000188:drug therapy*; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D000077022:Survivin; D053221:TNF-Related Apoptosis-Inducing Ligand; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D000077337:Vorinostat
chemical_list: D051017:Apoptosis Regulatory Proteins; C107489:BIRC5 protein, human; D002087:Butyrates; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D051034:Inhibitor of Apoptosis Proteins; D008562:Membrane Glycoproteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; D000077022:Survivin; D053221:TNF-Related Apoptosis-Inducing Ligand; C105190:TNFRSF10A protein, human; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; C012589:trichostatin A; D000077337:Vorinostat; D020169:Caspases