Abstract
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.
Highlights
Cytarabine is a chemotherapeutic drug used alone or in combination with other antineoplastic agents to treat different forms of leukemia
Were not increased by cytarabine treatment in any of leukemic cells tested. This basal expression of beclin-1 was apparently required for cytarabine-mediated induction of autophagy, as RNA interference with beclin-1 reduced the autophagic response to cytarabine treatment
The downregulation of mammalian target of rapamycin (mTOR)/p70S6 kinase (p70S6K) activation was almost complete, and autophagy induction was prevented by mTOR activator leucine, indicating that the release from mTOR suppression was the main mechanism for the autophagy induction by cytarabine
Summary
Cytarabine (cytosine arabinoside, arabinofuranosyl cytidine) is a chemotherapeutic drug used alone or in combination with other antineoplastic agents to treat different forms of leukemia. Autophagy depends on the hierarchically ordered activity of autophagy-related (Atg) proteins, controlled by the main autophagy repressor, mammalian target of rapamycin (mTOR) [4] This serine/threonine kinase is activated by phosphoinositide 3-kinase (PI3K)/Akt pathway and inhibited by the intracellular energy sensor AMP-activated protein kinase (AMPK) [4]. The activation of extracellular signal-regulated kinase (ERK) has been described as a non-canonical signal downstream of AMPK that contributes to mTOR-dependent induction of autophagy in certain conditions [16]. Under stress conditions such as nutrient deprivation, hypoxia, oxidative stress and DNA damage, autophagy could provide energy for maintaining essential cellular metabolism and/or directly interfere with apoptotic/necrotic cell death pathways [17]. To the best of our knowledge, no study so far has investigated the ability of cytarabine to induce autophagic response in cancer cells, or the possible role of cytarabine-triggered autophagy in regulation of cancer cell death
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