2-Deoxy-d-glucose cooperates with arsenic trioxide to induce apoptosis in leukemia cells: Involvement of IGF-1R-regulated Akt/mTOR, MEK/ERK and LKB-1/AMPK signaling pathways

Abstract

While the anti-tumor efficacy of 2-deoxy-D-glucose (2-DG) is normally low in monotherapy, it may represent a valuable radio- and chemo-sensitizing agent. We here demonstrate that 2-10 mM 2-DG cooperates with arsenic trioxide (ATO) and other antitumor drugs to induce apoptosis in human myeloid leukemia cell lines. Using ATO and HL60 as drug and cell models, respectively, we observed that 2-DG/ATO combination activates the mitochondrial apoptotic pathway, as indicated by Bid-, and Bax-regulated cytochrome c and Omi/HtrA2 release, XIAP down-regulation, and caspase-9/-3 pathway activation. 2-DG neither causes oxidative stress nor increases ATO uptake, but causes inner mitochondria membrane permeabilization as well as moderate ATP depletion, which nevertheless do not satisfactorily explain the pro-apoptotic response. Surprisingly 2-DG causes cell line-specific decrease in LKB-1/AMPK phosphorylation/activation, and also causes Akt/mTOR/p70S6K and MEK/ERK activation, which is prevented by co-treatment with ATO. The use of kinase-specific pharmacologic inhibitors and/or siRNAs reveals that apoptosis is facilitated by AMPK inactivation and restrained by Akt and ERK activation, and that Akt and ERK activation mediates AMPK inhibition. Finally, 2-DG stimulates IGF-1R phosphorylation/activation, and co-treatment with IGF-1R inhibitor prevents 2-DG effects on Akt, ERK and AMPK, and facilitates 2-DG-provoked apoptosis. In summary 2-DG elicits IGF-1R-mediated AMPK inactivation and Akt and ERK activation, which facilitates or restrain apoptosis, respectively. 2-DG-provoked AMPK inactivation increases the apoptotic efficacy of ATO, while in turn ATO-provoked Akt and ERK inactivation may increase the efficacy of 2-DG as anti-tumor drug.

Regulation of Death Induction and Chemosensitizing Action of 3-Bromopyruvate in Myeloid Leukemia Cells: Energy Depletion, Oxidative Stress, and Protein Kinase Activity Modulation

“…3B). Similar mitochondrial dysfunction was also observed with other glycolytic inhibitors (Calviño et al, 2011;Estañ et al, 2012) and could represent a determinant for the induction of cell death.…”

“…Conversely, 3-BrP caused a dose-dependent dephosphorylation-inactivation of AMPK, as well as that of the upstream LKB-1 and downstream ACC kinases. Of note, after 8 hours in the presence of 3-BrP (30 and 60 mM), some AMPK degradation was evident (see double bands in the blot), an effect that was also observed on long-term treatments with other glycolytic inhibitors (Estañ et al, 2012). With minor quantitative differences, similar modifications of protein kinases were obtained using NB4 instead of HL60 cells (data not shown).…”

“…The criterion used for necrosis (either genuine, "primary" necrosis or apoptosis-derived, "secondary" necrosis) was the loss of plasma membrane integrity, as determined by free PI uptake into nonpermeabilized cells and flow cytometry analysis. A detailed description of all of these techniques can be found in earlier works (Sánchez et al, 2010;Estañ et al, 2012, and references therein), and hence is omitted here. In addition, apoptosis and necrosis were determined simultaneously by double labeling with annexin V-FITC and PI and flow cytometry measurement using an ApoAlert annexin V-FITC kit (Clontech, Mountain View, CA) following the procedure indicated by the manufacturer.…”

“…3-BrP was found to provoke dose-dependent apoptosis or necrosis in human acute myeloid leukemia (AML) cells, and the severity of cell death correlated closely with the intensity of ATP depletion (Xu et al, 2005). However, we recently found that moderate ATP depletion neither caused a loss of cell viability, nor could it explain the proapoptotic (chemosensitizing) capacity of the glycolytic inhibitor 2-DG in leukemia cells (Estañ et al, 2012). With these precedents in mind, we set out to investigate how the induction of cell death is regulated by 3-BrP in HL60 cells and in other cell models of human AML.…”

Metabolic interplay between glycolysis and mitochondrial oxidation: The reverse Warburg effect and its therapeutic implication

“…An enhanced antioxidant capacity allows cancer cells to not a universal finding, and mitochondrial respiration impairment is not a fixed feature of cancer cells [41] . Although the glycolytic inhibitors targeting the Warburg effect have been investigated in various cancer types, the glycolytic inhibitors with the exception of 3-BP (a lactate analog) [18,19, , 3-BrOP (a 3-bromopyruvate derivative) [71][72][73][74] , and dichloroacetate (DCA) have demonstrated low efficacy in arresting tumor growth when used alone [99] ; these inhibitors include 2-deoxy-D-glucose (a glucose analog) [70,[100][101][102][103][104][105][106][107][108][109][110][111][112] , lonidamine (a derivative of indazole-3-carboxylic acid) [113][114][115][116][117][118][119][120][121][122][123][124][125][126][127][128][129][130][131][132] , methyl jasmonate on HK , 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one on PFK [162]…”