title: Impact of autophagy inhibition at different stages on cytotoxic effect of autophagy inducer in glioblastoma cells.
abstract: OBJECTIVE
  Glioblastoma multiforme (GBM) is the most malignant primary brain tumor with a poor prognosis. Combination treatment of autophagy inducer and autophagy inhibitor may be a feasible solution to improve the therapeutic effects. However, the correlation between them is unclear. The purpose of this study was to investigate the effect of autophagy inhibition at different stages on cytotoxicity of autophagy inducers in glioblastoma cells.
  
  
  METHODS
  Autophagy inhibition at early stage was achieved by 3-methyladenine (3-MA) or Beclin 1 shRNA. Autophagy inhibition at late stage was achieved by chloroquine (CQ) or Rab7 shRNA. Cell viability was assessed by MTT assay. Autophagy was measured using transmission electron microscopy and western blot. Apoptosis was measured using western blot and flow-cytometry.
  
  
  RESULTS
  Inhibition of early steps of autophagy by 3-MA or Beclin 1 knockdown decreased the toxic effect of arsenic trioxide (ATO) in GBM cell lines. In contrast, blockade of autophagy flux at late stage by CQ or Rab7 knockdown enhanced the cytotoxicity of ATO, and caused accumulation of degradative autophagic vacuoles and robust apoptosis. Moreover, depletion of Beclin 1 abolished the synergistic effect of ATO and CQ by reducing autophagy and apoptosis. Combination of CQ with other autophagy inducers also induced synergistic apoptotic cell death.
  
  
  CONCLUSIONS
  These results suggest that inhibition of late process of autophagy, not initial step, increases the cytotoxic effect of autophagy inducers via autophagy and apoptosis, which may contribute to GBM chemotherapy.
authors.affiliation: Department of Neurosurgery, The First Affiliated Hospital of Harbin Medical University, Harbin, The People's Republic of China.
authors.forename: Chenguang
authors.initials: C
authors.lastname: Li
authors.forename: Yaohua
authors.initials: Y
authors.lastname: Liu
authors.forename: Huailei
authors.initials: H
authors.lastname: Liu
authors.forename: Weiguang
authors.initials: W
authors.lastname: Zhang
authors.forename: Chen
authors.initials: C
authors.lastname: Shen
authors.forename: KenKa
authors.initials: K
authors.lastname: Cho
authors.forename: Xin
authors.initials: X
authors.lastname: Chen
authors.forename: Fei
authors.initials: F
authors.lastname: Peng
authors.forename: Yunke
authors.initials: Y
authors.lastname: Bi
authors.forename: Xu
authors.initials: X
authors.lastname: Hou
authors.forename: Zhuowen
authors.initials: Z
authors.lastname: Yang
authors.forename: Zhixing
authors.initials: Z
authors.lastname: Zheng
authors.forename: Kaikai
authors.initials: K
authors.lastname: Wang
authors.forename: Xiaoxiong
authors.initials: X
authors.lastname: Wang
authors.forename: Jiakang
authors.initials: J
authors.lastname: Zhang
authors.forename: Chen
authors.initials: C
authors.lastname: Zhong
authors.forename: Huichao
authors.initials: H
authors.lastname: Zou
authors.forename: Xinjian
authors.initials: X
authors.lastname: Zhang
authors.forename: Shiguang
authors.initials: S
authors.lastname: Zhao
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002738:Chloroquine / Q000494:pharmacology; D004357:Drug Synergism; D005434:Flow Cytometry; D005909:Glioblastoma / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D010087:Oxides / Q000494:pharmacology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D001152:Arsenicals; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D010087:Oxides; D034741:RNA, Small Interfering; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; C025946:3-methyladenine; D002738:Chloroquine; D053148:Caspase 3; D020691:rab GTP-Binding Proteins; D000225:Adenine; D000077237:Arsenic Trioxide