title: Chloroquine-mediated lysosomal dysfunction enhances the anticancer effect of nutrient deprivation.
abstract: OBJECTIVE
  To investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation.
  
  
  METHODS
  Serum-deprived U251 glioma, B16 melanoma and L929 fibrosarcoma cells were treated with chloroquine in vitro. Cell viability was measured by crystal violet and MTT assay. Oxidative stress, apoptosis/necrosis and intracellular acidification were analyzed by flow cytometry. Cell morphology was examined by light and electron microscopy. Activation of AMP-activated protein kinase (AMPK) and autophagy were monitored by immunoblotting. RNA interference was used for AMPK and LC3b knockdown. The anticancer efficiency of intraperitoneal chloroquine in calorie-restricted mice was assessed using a B16 mouse melanoma model.
  
  
  RESULTS
  Chloroquine rapidly killed serum-starved cancer cells in vitro. This effect was not mimicked by autophagy inhibitors or LC3b shRNA, indicating autophagy-independent mechanism. Chloroquine-induced lysosomal accumulation and oxidative stress, leading to mitochondrial depolarization, caspase activation and mixed apoptotic/necrotic cell death, were prevented by lysosomal acidification inhibitor bafilomycin. AMPK downregulation participated in chloroquine action, as AMPK activation reduced, and AMPK shRNA mimicked chloroquine toxicity. Chloroquine inhibited melanoma growth in calorie-restricted mice, causing lysosomal accumulation, mitochondrial disintegration and selective necrosis of tumor cells.
  
  
  CONCLUSIONS
  Combined treatment with chloroquine and calorie restriction might be useful in cancer therapy.
authors.affiliation: Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia. buajk@yahoo.com
authors.forename: Ljubica
authors.initials: L
authors.lastname: Harhaji-Trajkovic
authors.forename: Katarina
authors.initials: K
authors.lastname: Arsikin
authors.forename: Tamara
authors.initials: T
authors.lastname: Kravic-Stevovic
authors.forename: Sasa
authors.initials: S
authors.lastname: Petricevic
authors.forename: Gordana
authors.initials: G
authors.lastname: Tovilovic
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Pantovic
authors.forename: Nevena
authors.initials: N
authors.lastname: Zogovic
authors.forename: Biljana
authors.initials: B
authors.lastname: Ristic
authors.forename: Kristina
authors.initials: K
authors.lastname: Janjetovic
authors.forename: Vladimir
authors.initials: V
authors.lastname: Bumbasirevic
authors.forename: Vladimir
authors.initials: V
authors.lastname: Trajkovic
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology / Q000627:therapeutic use*; D031204:Caloric Restriction; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002738:Chloroquine / Q000494:pharmacology / Q000627:therapeutic use*; D005260:Female; D006801:Humans; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008545:Melanoma / Q000178:diet therapy / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D009369:Neoplasms / Q000178:diet therapy* / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects
chemical_list: D000962:Antimalarials; D002738:Chloroquine