title: Gefitinib radiosensitizes stem-like glioma cells: inhibition of epidermal growth factor receptor-Akt-DNA-PK signaling, accompanied by inhibition of DNA double-strand break repair.
abstract: OBJECTIVE
  We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair.
  
  
  METHODS
  Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, γ-H(2)AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, γ-H(2)AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt.
  
  
  RESULTS
  Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G(2)/M arrest and increased γ-H(2)AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased γ-H(2)AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation.
  
  
  CONCLUSIONS
  Stem-like gliomaspheres are resistant to irradiation-induced cytotoxicity, G(2)/M arrest, and DNA DSBs, compared with nonstem glioma cells. Gefitinib differentially enhances radiosensitivity of stem-like gliomaspheres by reducing EGFR-Akt activation and DNA-PKcs expression, accompanied by enhanced irradiation-induced DNA DSBs and inhibition of DSB repair.
authors.affiliation: Brain Tumour Research Laboratory, Division of Medical Sciences, National Cancer Centre Singapore, Singapore.
authors.forename: Khong Bee
authors.initials: KB
authors.lastname: Kang
authors.forename: Congju
authors.initials: C
authors.lastname: Zhu
authors.forename: Yin Ling
authors.initials: YL
authors.lastname: Wong
authors.forename: Qiuhan
authors.initials: Q
authors.lastname: Gao
authors.forename: Albert
authors.initials: A
authors.lastname: Ty
authors.forename: Meng Cheong
authors.initials: MC
authors.lastname: Wong
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001932:Brain Neoplasms / Q000473:pathology / Q000532:radiotherapy*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D051747:DNA-Activated Protein Kinase / Q000187:drug effects / Q000378:metabolism; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000077156:Gefitinib; D005910:Glioma / Q000473:pathology / Q000532:radiotherapy*; D006657:Histones / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014411:Neoplastic Stem Cells / Q000473:pathology / Q000528:radiation effects*; D009457:Neuroglia / Q000473:pathology / Q000528:radiation effects; D010766:Phosphorylation / Q000187:drug effects; D011799:Quinazolines / Q000494:pharmacology*; D011836:Radiation Tolerance / Q000187:drug effects*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D014410:Tumor Stem Cell Assay / Q000379:methods
chemical_list: D000970:Antineoplastic Agents; D006657:Histones; D011799:Quinazolines; D011838:Radiation-Sensitizing Agents; C475961:gamma-H2AX protein, mouse; D066246:ErbB Receptors; D051747:DNA-Activated Protein Kinase; D000077156:Gefitinib