title: Inhibition of AMPK-dependent autophagy enhances in vitro antiglioma effect of simvastatin.
abstract: The role of autophagy, a process in which the cell self-digests its own components, was investigated in glioma cell death induced by the hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase-inhibiting drug simvastatin. Induction of autophagy and activation of autophagy-regulating signalling pathways were analyzed by immunoblotting. Flow cytometry/fluorescent microscopy was used to assess autophagy-associated intracellular acidification and apoptotic markers (phosphatidylserine exposure, DNA fragmentation and caspase activation). Cell viability was determined by crystal violet, MTT or LDH release assay. Simvastatin treatment of U251 and C6 glioma cell lines caused the appearance of autophagolysosome-like intracytoplasmic acidic vesicles. The induction of autophagy in U251 cells was confirmed by the upregulation of autophagosome-associated LC3-II and pro-autophagic beclin-1, as well as by the downregulation of the selective autophagic target p62. Simvastatin induced the activation of AMP-activated protein kinase (AMPK) and its target Raptor, while simultaneously downregulating activation of Akt. Mammalian target of rapamycin (mTOR), a major AMPK/Akt downstream target and a major negative autophagy regulator, and its substrate p70 S6 kinase 1 were also inhibited by simvastatin. Mevalonate, the product of HMG-CoA reductase enzymatic activity, AMPK siRNA or pharmacological inactivation of AMPK with compound C suppressed, while the inhibitors of Akt (10-DEBC hydrochloride) and mTOR (rapamycin) mimicked autophagy induction by simvastatin. Inhibition of autophagy with bafilomycin A1, 3-methyladenine and LC3β shRNA, as well as AMPK inhibition with compound C or AMPK siRNA, markedly increased apoptotic death of simvastatin-treated U251 cells. These data suggest that inhibition of AMPK-dependent autophagic response might sensitize glioma cells to statin-induced apoptotic death.
authors.affiliation: Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr Subotica 1, 11000 Belgrade, Serbia.
authors.forename: Maja
authors.initials: M
authors.lastname: Misirkic
authors.forename: Kristina
authors.initials: K
authors.lastname: Janjetovic
authors.forename: Ljubica
authors.initials: L
authors.lastname: Vucicevic
authors.forename: Gordana
authors.initials: G
authors.lastname: Tovilovic
authors.forename: Biljana
authors.initials: B
authors.lastname: Ristic
authors.forename: Urosh
authors.initials: U
authors.lastname: Vilimanovich
authors.forename: Ljubica
authors.initials: L
authors.lastname: Harhaji-Trajkovic
authors.forename: Mirjana
authors.initials: M
authors.lastname: Sumarac-Dumanovic
authors.forename: Dragan
authors.initials: D
authors.lastname: Micic
authors.forename: Vladimir
authors.initials: V
authors.lastname: Bumbasirevic
authors.forename: Vladimir
authors.initials: V
authors.lastname: Trajkovic
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1; D001932:Brain Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005910:Glioma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D019821:Simvastatin / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D014162:Transfection
chemical_list: D055372:AMP-Activated Protein Kinases; D051017:Apoptosis Regulatory Proteins; D000071186:Beclin-1; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D019821:Simvastatin; D058570:TOR Serine-Threonine Kinases; C491997:BECN1 protein, human; C115010:Becn1 protein, mouse; C512829:Becn1 protein, rat; C526109:Anxa3 protein, rat; C477012:MAP1LC3A protein, human; C480860:Map1lc3b protein, mouse; C546842:MTOR protein, human; C546845:mTOR protein, rat