title: Inhibition of histone deacetylases sensitizes glioblastoma cells to lomustine.
abstract: OBJECTIVE
  Glioblastoma (GBM) ranks among the deadliest solid cancers worldwide and its prognosis has remained dismal, despite the use of aggressive chemo-irradiation treatment regimens. Limited drug delivery into the brain parenchyma and frequent resistance to currently available therapies are problems that call for a prompt development of novel therapeutic strategies. While only displaying modest efficacies as mono-therapy in pre-clinical settings, histone deacetylase inhibitors (HDACi) have shown promising sensitizing effects to a number of cytotoxic agents. Here, we sought to investigate the sensitizing effect of the HDACi trichostatin A (TSA) to the alkylating agent lomustine (CCNU), which is used in the clinic for the treatment of GBM.
  
  
  METHODS
  Twelve primary GBM cell cultures grown as neurospheres were used in this study, as well as one established GBM-derived cell line (U87 MG). Histone deacetylase (HDAC) expression levels were determined using quantitative real-time PCR and Western blotting. The efficacy of either CCNU alone or its combination with TSA was assessed using various assays, i.e., cell viability assays (MTT), cell cycle assays (flow cytometry, FACS), double-strand DNA break (DSB) quantification assays (microscopy/immunofluorescence) and expression profiling assays of proteins involved in apoptosis and cell stress (Western blotting and protein array).
  
  
  RESULTS
  We found that the HDAC1, 3 and 6 expression levels were significantly increased in GBM samples compared to non-neoplastic brain control samples. Additionally, we found that pre-treatment of GBM cells with TSA resulted in an enhancement of their sensitivity to CCNU, possibly via the accumulation of DSBs, decreased cell proliferation and viability rates, and an increased apoptotic rate.
  
  
  CONCLUSIONS
  From our data we conclude that the combined administration of TSA and CCNU eradicates GBM cells with a higher efficacy than either drug alone, thereby opening a novel avenue for the treatment of GBM.
authors.affiliation: Department of Radiation Biology, The Finsen Center, Rigshospitalet, Copenhagen, Denmark. mikkel.staberg@regionh.dk.
authors.forename: Mikkel
authors.identifier: http://orcid.org/0000-0001-9068-6939
authors.initials: M
authors.lastname: Staberg
authors.affiliation: Department of Radiation Biology, The Finsen Center, Rigshospitalet, Copenhagen, Denmark.
authors.forename: Signe Regner
authors.initials: SR
authors.lastname: Michaelsen
authors.affiliation: Brain Tumor Biology Group, Danish Cancer Society Research Center, Copenhagen, Denmark.
authors.forename: Rikke Darling
authors.initials: RD
authors.lastname: Rasmussen
authors.affiliation: Department of Radiation Biology, The Finsen Center, Rigshospitalet, Copenhagen, Denmark.
authors.forename: Mette
authors.initials: M
authors.lastname: Villingshøj
authors.affiliation: Department of Radiation Biology, The Finsen Center, Rigshospitalet, Copenhagen, Denmark.
authors.forename: Hans Skovgaard
authors.initials: HS
authors.lastname: Poulsen
authors.affiliation: Brain Tumor Biology Group, Danish Cancer Society Research Center, Copenhagen, Denmark. pkn@cancer.dk.
authors.forename: Petra
authors.initials: P
authors.lastname: Hamerlik
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000008:administration & dosage; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000201:enzymology*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005909:Glioblastoma / Q000201:enzymology*; D056572:Histone Deacetylase Inhibitors / Q000008:administration & dosage*; D006801:Humans; D006877:Hydroxamic Acids / Q000008:administration & dosage*; D008130:Lomustine / Q000008:administration & dosage*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D018906:Antineoplastic Agents, Alkylating; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; C012589:trichostatin A; D008130:Lomustine
keywords: Epigenetics; Glioblastoma; HDACi; Lomustine; Sensitization; Trichostatin A