title: Comparative study of autophagy inhibition by 3MA and CQ  on Cytarabine‑induced death of leukaemia cells.
abstract: BACKGROUND
  As the molecular mechanisms of Cytarabine,one of the most important drugs used in the leukaemia’s treatment, are only partially understood and the role of autophagy on leukaemia development and treatment is only recently being investigated, in this study, by using Chloroquine (CQ) and 3-methyladenine (3MA) as autophagy inhibitors, we aim to evaluate the contribution of an autophagic mechanism to Cytarabine (AraC)-induced death of HL60 leukaemia cells.
  
  
  METHODS
  Trypan blue exclusion and AnnexinV/PI assays were used to evaluate HL60 cell death under AraC treatment in the presence or absence of 3MA and CQ. Western blotting and immunofluorescence experiments were performed to show the involvement of apoptosis and autophagy protein expressions. Phenotypic characterization of HL60-treated cells was performed by using immunophenotyping. Clonogenic assays were applied to analyse clonal function of HL60-treated cells.
  
  
  RESULTS
  We observed that although autophagy inhibition by 3MA, but not CQ, increased the death of HL60 AraC cells after 24 h of treatment, no significant differences between AraC and AraC + 3MA-treated groups were observed by using clonogenic assay. In addition, increased number of immature (CD34(+)/CD38(−)Lin(−/low)) HL60 cells was found in AraC and AraC-3MA groups when compared with control untreated cells.
  
  
  CONCLUSIONS
  Although AraC anti-leukaemia effects could be potentiated by 3MA autophagy inhibition after 24 h of exposure, leukaemia cell resistance, the main causes of treatment failure, is also promoted by autophagy initial stage impairment by 3MA, denoting the complex role of autophagy in leukaemia cells’ response to chemotherapy.
authors.forename: Caroline
authors.initials: C
authors.lastname: Palmeira dos Santos
authors.forename: Gustavo J S
authors.initials: GJ
authors.lastname: Pereira
authors.forename: Christiano M V
authors.initials: CM
authors.lastname: Barbosa
authors.forename: Aron
authors.initials: A
authors.lastname: Jurkiewicz
authors.forename: Soraya S
authors.initials: SS
authors.lastname: Smaili
authors.forename: Claudia
authors.initials: C
authors.lastname: Bincoletto
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000962:Antimalarials / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002738:Chloroquine / Q000494:pharmacology*; D003561:Cytarabine / Q000494:pharmacology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D016130:Immunophenotyping; D007938:Leukemia / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D018168:Receptors, Retinoic Acid / Q000037:antagonists & inhibitors / Q000378:metabolism; D000072482:Retinoic Acid Receptor alpha; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay
chemical_list: D000962:Antimalarials; D000964:Antimetabolites, Antineoplastic; C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D003561:Cytarabine; C025946:3-methyladenine; D002738:Chloroquine; D053148:Caspase 3; D000225:Adenine