Impact of autophagy inhibition at different stages on cytotoxic effect of autophagy inducer in glioblastoma cells

Cell Physiol Biochem. 2015;35(4):1303-16. doi: 10.1159/000373952. Epub 2015 Feb 11.

Abstract

Background/aims: 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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenine / analogs & derivatives*
  • Adenine / pharmacology
  • Antineoplastic Agents / pharmacology*
  • Apoptosis / drug effects
  • Apoptosis Regulatory Proteins / antagonists & inhibitors
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism
  • Arsenic Trioxide
  • Arsenicals / pharmacology*
  • Autophagy / drug effects*
  • Beclin-1
  • Blotting, Western
  • Brain Neoplasms / metabolism
  • Brain Neoplasms / pathology
  • Caspase 3 / metabolism
  • Cell Line, Tumor
  • Chloroquine / pharmacology
  • Drug Synergism
  • Flow Cytometry
  • Glioblastoma / metabolism
  • Glioblastoma / pathology
  • Humans
  • Membrane Proteins / antagonists & inhibitors
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Microscopy, Electron, Transmission
  • Oxides / pharmacology*
  • RNA Interference
  • RNA, Small Interfering / metabolism
  • rab GTP-Binding Proteins / antagonists & inhibitors
  • rab GTP-Binding Proteins / genetics
  • rab GTP-Binding Proteins / metabolism

Substances

  • Antineoplastic Agents
  • Apoptosis Regulatory Proteins
  • Arsenicals
  • BECN1 protein, human
  • Beclin-1
  • Membrane Proteins
  • Oxides
  • RNA, Small Interfering
  • rab7 protein
  • 3-methyladenine
  • Chloroquine
  • Caspase 3
  • rab GTP-Binding Proteins
  • Adenine
  • Arsenic Trioxide