title: NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, enhances the radiosensitivity of human glioma stem cells in vitro.
abstract: OBJECTIVE
  NVP-BEZ235 is a novel dual PI3K/mTOR inhibitor and shows dramatic effects on gliomas. The aim of this study was to investigate the effects of NVP-BEZ235 on the radiosensitivity and autophagy of glioma stem cells (GSCs) in vitro.
  
  
  METHODS
  Human GSCs (SU-2) were tested. The cell viability and survival from ionizing radiation (IR) were evaluated using MTT and clonogenic survival assay, respectively. Immunofluorescence assays were used to identify the formation of autophagosomes. The apoptotic cells were quantified with annexin V-FITC/PI staining and flow cytometry, and observed using Hoechst 33258 staining and fluorescence microscope. Western blot analysis was used to analyze the expression levels of proteins. Cell cycle status was determined by measuring DNA content after staining with PI. DNA repair in the cells was assessed using a comet assay.
  
  
  RESULTS
  Treatment of SU-2 cells with NVP-BEZ235 (10-320 nmol/L) alone suppressed the cell growth in a concentration-dependent manner. A low concentration of NVP-BEZ235 (10 nmol/L) significantly increased the radiation sensitivity of SU-2 cells, which could be blocked by co-treatment with 3-MA (50 μmol/L). In NVP-BEZ235-treated SU-2 cells, more punctate patterns of microtubule-associated protein LC3 immunoreactivity was observed, and the level of membrane-bound LC3-II was significantly increased. A combination of IR with NVP-BEZ235 significantly increased the apoptosis of SU-2 cells, as shown in the increased levels of BID, Bax, and active caspase-3, and decreased level of Bcl-2. Furthermore, the combination of IR with NVP-BEZ235 led to G1 cell cycle arrest. Moreover, NVP-BEZ235 significantly attenuated the repair of IR-induced DNA damage as reflected by the tail length of the comet.
  
  
  CONCLUSIONS
  NVP-BEZ235 increases the radiosensitivity of GSCs in vitro by activating autophagy that is associated with synergistic increase of apoptosis and cell-cycle arrest and decrease of DNA repair capacity.
authors.affiliation: Department of Pharmacology, Medical School of Soochow University, Suzhou 215123, China.
authors.forename: Wen-juan
authors.initials: WJ
authors.lastname: Wang
authors.forename: Lin-mei
authors.initials: LM
authors.lastname: Long
authors.forename: Neng
authors.initials: N
authors.lastname: Yang
authors.forename: Qing-qing
authors.initials: QQ
authors.lastname: Zhang
authors.forename: Wen-jun
authors.initials: WJ
authors.lastname: Ji
authors.forename: Jiang-hu
authors.initials: JH
authors.lastname: Zhao
authors.forename: Zheng-hong
authors.initials: ZH
authors.lastname: Qin
authors.forename: Zhong
authors.initials: Z
authors.lastname: Wang
authors.forename: Gang
authors.initials: G
authors.lastname: Chen
authors.forename: Zhong-qin
authors.initials: ZQ
authors.lastname: Liang
mesh_terms: D001343:Autophagy / Q000187:drug effects / Q000528:radiation effects; D001932:Brain Neoplasms / Q000188:drug therapy / Q000473:pathology / Q000532:radiotherapy*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects / Q000528:radiation effects; D005910:Glioma / Q000188:drug therapy / Q000473:pathology / Q000532:radiotherapy*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000473:pathology / Q000528:radiation effects; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011804:Quinolines / Q000494:pharmacology*; D011836:Radiation Tolerance / Q000187:drug effects; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors*
chemical_list: D007093:Imidazoles; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D011804:Quinolines; D011838:Radiation-Sensitizing Agents; D058570:TOR Serine-Threonine Kinases; C531198:dactolisib