title: Radiosensitizing effect of the novel Hsp90 inhibitor NVP-AUY922 in human tumour cell lines silenced for Hsp90α.
abstract: BACKGROUND
  Hsp90 inhibitors can enhance the tumour sensitivity to ionising radiation (IR). However, Hsp90 inhibition leads to the up-regulation of anti-apoptotic Hsp90 and Hsp70, which might diminish the radiosensitizing effects of the inhibitors. Therefore, inhibition of the up-regulation of Hsp90 by siRNA might be a promising strategy to enhance drug-mediated radiosensitization.
  
  
  METHODS
  The expression of Hsp90α was silenced in A549 and GaMG tumour cell lines by siRNA treatment. Pre-silenced for Hsp90α cells were treated with NVP-AUY922, a novel Hsp90 inhibitor, for 24 h and then irradiated. Radiation response was determined by colony-forming ability. The expression of several marker proteins was analysed by Western blot. DNA damage and repair were assessed by histone γH2AX measurements.
  
  
  RESULTS
  We found that transfection with siRNA against Hsp90α reduced Hsp90α at mRNA and protein levels. Pre-silencing of Hsp90α reduced NVP-AUY922-mediated up-regulation of Hsp90α but it did not increase drug-mediated radiosensitization in both tumour cell lines. As revealed by Western blot, pre-silencing of Hsp90α followed by NVP-AUY922 did not change the expression of Hsp90 client proteins (Akt, Raf-1, Cdk1 and Cdk4) compared with drug treatment alone, suggesting unchanged chaperone function in transfected cells.
  
  
  CONCLUSIONS
  Pre-silencing of Hsp90α followed by Hsp90 inhibition did not enhance the radiosensitizing effect of NVP-AUY922 in both tested tumour cell lines. Future work will be done on stable transfection with shRNA against Hsp90α or simultaneous silencing of both Hsp90 isoforms, Hsp90α and Hsp90β, in order to optimize tumour cell killing.
authors.affiliation: Department of Radiation Oncology, University of Würzburg, Würzburg, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Stingl
authors.forename: N
authors.initials: N
authors.lastname: Niewidok
authors.forename: N
authors.initials: N
authors.lastname: Müller
authors.forename: M
authors.initials: M
authors.lastname: Selle
authors.forename: C S
authors.initials: CS
authors.lastname: Djuzenova
authors.forename: M
authors.initials: M
authors.lastname: Flentje
mesh_terms: D017209:Apoptosis / Q000528:radiation effects; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics / Q000528:radiation effects*; D004249:DNA Damage / Q000235:genetics / Q000528:radiation effects; D004260:DNA Repair / Q000235:genetics / Q000528:radiation effects; D020868:Gene Silencing / Q000528:radiation effects; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D012118:Resorcinols / Q000494:pharmacology*; D014410:Tumor Stem Cell Assay; D015854:Up-Regulation / Q000235:genetics / Q000528:radiation effects
chemical_list: C528044:5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-morpholin-4-ylmethylphenyl)isoxazole-3-carboxylic acid ethylamide; D018841:HSP90 Heat-Shock Proteins; D007555:Isoxazoles; D034741:RNA, Small Interfering; D011838:Radiation-Sensitizing Agents; D012118:Resorcinols