Inhibition of mTOR-dependent autophagy sensitizes leukemic cells to cytarabine-induced apoptotic death.
The present study investigated the role of autophagy, a cellular self-digestion process, in the cytotoxicity of antileukemic drug cytarabine towards human leukemic cell lines (REH, HL-60, MOLT-4) and peripheral blood mononuclear cells from leukemic patients. The induction of autophagy was confirmed by acridine orange staining of intracellular acidic vesicles, electron microscopy visualization of autophagic vacuoles, as well as by the increase in autophagic proteolysis and autophagic flux, demonstrated by immunoblot analysis of p62 downregulation and LC3-I conversion to autophagosome-associated LC3-II in the presence of proteolysis inhibitors, respectively. Moreover, the expression of autophagy-related genes Atg4, Atg5 and Atg7 was stimulated by cytarabine in REH cells. Cytarabine reduced the phosphorylation of the major negative regulator of autophagy, mammalian target of rapamycin (mTOR), and its downstream target p70S6 kinase in REH cells, which was associated with downregulation of mTOR activator Akt and activation of extracellular signal- regulated kinase. Cytarabine had no effect on the activation of mTOR inhibitor AMP-activated protein kinase. Leucine, an mTOR activator, reduced both cytarabine-induced autophagy and cytotoxicity. Accordingly, pharmacological downregulation of autophagy with bafilomycin A1 and chloroquine, or RNA interference-mediated knockdown of LC3β or p62, markedly increased oxidative stress, mitochondrial depolarization, caspase activation and subsequent DNA fragmentation and apoptotic death in cytarabine-treated REH cells. Cytarabine also induced mTOR-dependent cytoprotective autophagy in HL-60 and MOLT-4 leukemic cell lines, as well as primary leukemic cells, but not normal leukocytes. These data suggest that the therapeutic efficiency of cytarabine in leukemic patients could be increased by the inhibition of the mTOR-dependent autophagic response.
Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia.
Mihajlo
M
Bosnjak
Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, Belgrade, Serbia.
Biljana
B
Ristic
Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, Belgrade, Serbia.
Katarina
K
Arsikin
Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia.
Aleksandar
A
Mircic
Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia.
Violeta
V
Suzin-Zivkovic
Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, Belgrade, Serbia.
Vladimir
V
Perovic
Clinic of Hematology, Clinical Center of Serbia, School of Medicine, University of Belgrade, Belgrade, Serbia.
Andrija
A
Bogdanovic
Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, Belgrade, Serbia.
Verica
V
Paunovic
Institute of Medical and Clinical Biochemistry, School of Medicine, University of Belgrade, Belgrade, Serbia.
Ivanka
I
Markovic
Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia.
Vladimir
V
Bumbasirevic
Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, Belgrade, Serbia.
Vladimir
V
Trajkovic
Institute for Biological Research, University of Belgrade, Belgrade, Serbia.
Ljubica
L
Harhaji-Trajkovic
D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics; D045744:Cell Line, Tumor; D003561:Cytarabine / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015973:Gene Expression Regulation, Leukemic; D006801:Humans; D007938:Leukemia / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000964:Antimetabolites, Antineoplastic; D003561:Cytarabine; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D055372:AMP-Activated Protein Kinases