title: Induction of autophagic cell death and radiosensitization by the pharmacological inhibition of nuclear factor-kappa B activation in human glioma cell lines.
abstract: OBJECTIVE
  The intrinsic radioresistance of certain cancer cells may be closely associated with the constitutive activation of nuclear factor-kappa B (NF-kappaB) activity, which may lead to protection from apoptosis. Recently, nonapoptotic cell death, or autophagy, has been revealed as a novel response of cancer cells to ionizing radiation. In the present study, the authors analyzed the effect of pitavastatin as a potential inhibitor of NF-kappaB activation on the radiosensitivity of A172, U87, and U251 human glioma cell lines.
  
  
  METHODS
  The pharmacological inhibition of NF-kappaB activation was achieved using pitavastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Growth and radiosensitivity assays were performed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Hoechst 33258 staining, supravital acridine orange staining, and electron microscopy were performed utilizing 3 glioma cell lines with or without pitavastatin pretreatment to identify apoptosis or autophagy after irradiation.
  
  
  RESULTS
  The growth of these 3 glioma cell lines was not significantly inhibited by pitavastatin at a concentration of up to 1 microM. Treatment with 0.1 microM of pitavastatin enhanced radiation-induced cell death in all glioma cell lines, with different sensitivity. Apoptosis did not occur in any pretreated or untreated (no pitavastatin) cell line following irradiation. Instead, autophagic cell changes were observed regardless of the radiosensitivity of the cell line. An inhibitor of autophagy, 3-methyladenine suppressed the cytotoxic effect of irradiation with pitavastatin, indicating that autophagy is a result of an antitumor mechanism. Using the most radiosensitive A172 cell line, the intracellular localization of p50, a representative subunit of NF-kappaB, was evaluated through immunoblotting and immunofluorescence studies. The NF-kappaB of A172 cells was immediately activated and translocated from the cytosol to the nucleus in response to irradiation. Pitavastatin inhibited this activation and translocation of NF-kappaB.
  
  
  CONCLUSIONS
  Autophagic cell death rather than apoptosis is a possible mechanism of radiation-induced and pitavastatin-enhanced cell damage, and radiosensitization by the pharmacological inhibition of NF-kappaB activation may be a novel therapeutic strategy for malignant gliomas.
authors.affiliation: Departments of Neurosurgery, Faculty of Medicine, University of Toyama, Sugitani, Toyama, Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Tsuboi
authors.forename: Masanori
authors.initials: M
authors.lastname: Kurimoto
authors.forename: Shoichi
authors.initials: S
authors.lastname: Nagai
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Hayakawa
authors.forename: Hironaga
authors.initials: H
authors.lastname: Kamiyama
authors.forename: Nakamasa
authors.initials: N
authors.lastname: Hayashi
authors.forename: Isao
authors.initials: I
authors.lastname: Kitajima
authors.forename: Shunro
authors.initials: S
authors.lastname: Endo
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology; D001343:Autophagy / Q000502:physiology*; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005910:Glioma / Q000473:pathology*; D006801:Humans; D015151:Immunoblotting; D016328:NF-kappa B / Q000032:analysis / Q000187:drug effects*; D011804:Quinolines / Q000494:pharmacology*; D011836:Radiation Tolerance
chemical_list: D004791:Enzyme Inhibitors; D016328:NF-kappa B; D011804:Quinolines; C025946:3-methyladenine; D000225:Adenine; C108475:pitavastatin