title: Combination of vorinostat and flavopiridol is selectively cytotoxic to multidrug-resistant neuroblastoma cell lines with mutant TP53.
abstract: As p53 loss of function (LOF) confers high-level drug resistance in neuroblastoma, p53-independent therapies might have superior activity in recurrent neuroblastoma. We tested the activity of vorinostat, a histone deacetylase inhibitor, and flavopiridol, a pan-Cdk inhibitor, in a panel of multidrug-resistant neuroblastoma cell lines that included lines with wild-type (wt) and transcriptionally active TP53 (n = 3), mutated (mt), and LOF TP53 (n = 4) or p14(ARF) deletion (n = 1). The combination of vorinostat and flavopiridol was synergistic and significantly more cytotoxic (P < 0.001) in cell lines with p53-LOF and in the clones stably transfected with dominant-negative p53 plasmids. Cell cycle analysis by flow cytometry showed prominent cell-cycle arrest in G(2)/M (37%) for a cell line with wt TP53 (SK-N-RA) at 16 to 20 hours, while cells with mt TP53 (CHLA-90) slipped into sub-G(1) at 6 to 24 hours (25%-40% specific cell death). The morphological hallmarks of mitotic cell death, including defective spindle formation and abnormal cytokinesis, were detected by confocal microscopy after the treatment with vorinostat + flavopiridol combination in CHLA-90. The combination caused reduction in the expression of G(2)/M proteins (cyclin B1, Mad2, MPM2) in 2 cell lines with mt TP53 but not in those with wt TP53. Plk1 expression was reduced in all treated lines. Small interfering RNA knockdown of Mad2 and cyclin B1 or Plk1 synergistically reduced the clonogenicity of CHLA-90 cells. The combination of HDAC inhibitor and flavopiridol may be a unique approach to treating neuroblastomas with p53 LOF, one that evokes induction of mitotic failure.
authors.affiliation: Division of Hematology-Oncology, Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA.
authors.forename: Jen-Ming
authors.initials: JM
authors.lastname: Huang
authors.forename: Michael A
authors.initials: MA
authors.lastname: Sheard
authors.forename: Lingyun
authors.initials: L
authors.lastname: Ji
authors.forename: Richard
authors.initials: R
authors.lastname: Sposto
authors.forename: Nino
authors.initials: N
authors.lastname: Keshelava
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D018432:Drug Resistance, Multiple / Q000187:drug effects*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D004357:Drug Synergism; D005419:Flavonoids / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D016195:G2 Phase / Q000187:drug effects; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006801:Humans; D006877:Hydroxamic Acids / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D008938:Mitosis / Q000187:drug effects; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009447:Neuroblastoma / Q000188:drug therapy* / Q000473:pathology*; D010880:Piperidines / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D000077337:Vorinostat
chemical_list: D014408:Biomarkers, Tumor; D005419:Flavonoids; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D050505:Mutant Proteins; D010880:Piperidines; D047428:Protein Kinase Inhibitors; D016159:Tumor Suppressor Protein p53; C077990:alvocidib; D000077337:Vorinostat