title: Inhibition of autophagy with chloroquine is effective in melanoma.
abstract: BACKGROUND
  Cancer cells adapt to the stress resulting from accelerated cell growth and a lack of nutrients by activation of the autophagy pathway. Two proteins that allow cell growth in the face of metabolic stress and hypoxia are hypoxia-inducible factor-1α (HIF-1α) and heat shock protein 90 (Hsp 90). We hypothesize that chloroquine (CQ), an antimalarial drug that inhibits autophagosome function, in combination with either echinomycin, a HIF-1α inhibitor, or 17-dimethylaminoethylamino-17-dimethoxygeldanamycin (17-DMAG), an Hsp 90 inhibitor, will result in cytotoxicity in melanoma.
  
  
  METHODS
  Multiple human melanoma cell lines (BRAF wild-type and mutant) were tested in vitro with CQ in combination with echinomycin or 17-DMAG. These treatments were performed in hypoxic (5% O2) and normoxic (18% O2) conditions. Mechanism of action was determined through Western blot of autophagy-associated proteins HIF-1α and Hsp 90.
  
  
  RESULTS
  Chloroquine, echinomycin, and 17-DMAG each induced cytotoxicity in multiple human melanoma cell lines, in both normoxia and hypoxia. Chloroquine combined with echinomycin achieved synergistic cytotoxicity under hypoxic conditions in multiple melanoma cell lines (BRAF wild-type and mutant). Western blot analysis indicated that echinomycin reduced HIF-1α levels, both alone and in combination with CQ. Changes in LC3 flux indicated inhibition of autophagy at the level of the autophagosome by CQ therapy.
  
  
  CONCLUSIONS
  Targeting autophagy with the antimalarial drug CQ may be an effective cancer therapy in melanoma. Sensitivity to chloroquine is independent of BRAF mutational status. Combining CQ with the HIF-1α inhibitor echinomycin improves cytotoxicity in hypoxic conditions.
authors.affiliation: Hiram C. Polk Jr MD Department of Surgery, University of Louisville, Louisville, Kentucky 40202, USA.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Egger
authors.forename: Justin S
authors.initials: JS
authors.lastname: Huang
authors.forename: Wenyuan
authors.initials: W
authors.lastname: Yin
authors.forename: Kelly M
authors.initials: KM
authors.lastname: McMasters
authors.forename: Lacey R
authors.initials: LR
authors.lastname: McNally
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000962:Antimalarials / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects*; D016227:Benzoquinones / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002738:Chloroquine / Q000494:pharmacology*; D004359:Drug Therapy, Combination; D004448:Echinomycin / Q000494:pharmacology*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors; D006801:Humans; D000860:Hypoxia / Q000378:metabolism / Q000473:pathology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors; D047029:Lactams, Macrocyclic / Q000494:pharmacology*; D008545:Melanoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D012878:Skin Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D013312:Stress, Physiological / Q000187:drug effects
chemical_list: D000903:Antibiotics, Antineoplastic; D000962:Antimalarials; D016227:Benzoquinones; C497442:HIF1A protein, human; D018841:HSP90 Heat-Shock Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047029:Lactams, Macrocyclic; C448659:17-(dimethylaminoethylamino)-17-demethoxygeldanamycin; D004448:Echinomycin; D002738:Chloroquine
keywords: Autophagosome; Autophagy; Autophagy inhibition; Cancer hypoxia; Chloroquine; Echinomycin; Heat shock protein 90; Hypoxia-inducible factor-1; Melanoma