Inhibition of mTOR-Dependent Autophagy Sensitizes Leukemic Cells to Cytarabine-Induced Apoptotic Death

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.

Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy

“…Therefore, the rationale provided here to study AVO volume with the Acridine Orange R/GFIR solves long-standing perceived interferences, such as binding to nucleic acids, which increases red and green fluorescence, but leaves R/GFIR unaltered. With the pK a and R/GFIR curve, other potential interferences can be objectively assessed, such as whole-cell acidification, which has been assessed with Acridine Orange (Bosnjak et al, 2014). Acidification to pH 7.0, for example, would lead to an intracellular concentration of protonated Acridine Orange of 2.5 µg/ml, considering the external concentration of Acridine Orange to be 1 µg/ml.…”

Atg7 suppression enhances chemotherapeutic agent sensitivity and overcomes stroma-mediated chemoresistance in acute myeloid leukemia

“…32 Similarly, chemotherapy-induced autophagy in AML and acute lymphocytic leukemia cell lines 9 has been shown to be cytoprotective. 19 Atg7, being an E1 ligase, is potentially targetable, and targeting Atg7 largely disables the autophagic machinery in AML cells. 33 From a drug development perspective, it is exciting to see that recently several small molecules have been developed that specifically target components of autophagy network.…”

“…19 The purine analog Ara-C and anthracycline-like agent Ida are frontline therapies for AML. We confirmed by immunoblotting that both Ara-C and Ida individually induced autophagy in AML cells.…”

You eat what you are: autophagy inhibition as a therapeutic strategy in leukemia

“…71 Similar autophagy induction, as revealed by rapid conversion of LC3-I into LC3-II, a decrease in p62/SQSTM1, and an increase in AVs, has been demonstrated after cytarabine treatment of the human leukemia cell lines, REH and HL-60, as well as peripheral blood mononuclear cells from patients with chronic myelogenous leukemia (CML). 72 …”