Abstract
BackgroundThe glycolysis inhibitor 2-deoxy-d-glucose (2-DG) is a safe, potentially useful anti-tumour drug, but its efficacy is normally low when used alone. Recent studies indicated that 2-DG stimulates the PI3K/Akt and MEK/ERK defensive pathways, which limits the apoptotic efficacy in tumour cell lines. We hypothesized that co-treatment with selected polyphenols could improve 2-DG-provoked apoptosis by preventing defensive kinase activation.MethodsCell proliferation was measured by cell counting or the MTT assay. Cell cycle, apoptosis and necrosis were determined by propidium iodide staining and/or annexin V labeling followed by flow cytometry. Mitochondria pore transition and depolarization were determined by calcein-ATM or rhodamine 123 labeling followed flow cytometry. Intracellular reactive oxygen species and GSH were determined by dichlorodihydrofluorescein diacetate or monochlorobimane labeling followed by flow cytometry or fluorimetry. Expression and phosphorylation of protein kinases were analyzed by the Western blot.Results(i) 2-DG-provoked apoptosis was greatly potentiated by co-treatment with the sub-lethal concentrations of the flavonoid quercetin in human HL60 acute myeloblastic leukemia cells. Allowing for quantitative differences, apoptosis potentiation was also obtained using NB4 promyelocytic and THP-1 promonocytic cells, using curcumin or genistein instead of quercetin, and using lonidamine instead of 2-DG, but not when 2-DG was substituted by incubation in glucose-free medium. (ii) Quercetin and 2-DG rapidly elicited the opening of mitochondria pore transition, which preceded the trigger of apoptosis. (iii) Treatments did not affect GSH levels, and caused disparate effects on reactive oxygen species generation, which did not match the changes in lethality. (iv) 2-DG and lonidamine stimulated defensive Akt and ERK phosphorylation/activation, while glucose starvation was ineffective. Polyphenols prevented the stimulation of Akt phosphorylation, and in some cases also ERK phosphorylation. In addition, quercetin and 2-DG stimulated GSK-3α,β phosphorylation/inactivation, although with different isoform specificity. The use of pharmacologic inhibitors confirmed the importance of these kinase modifications for apoptosis.ConclusionsThe present in vitro observations suggest that co-treatment with low concentrations of selected polyphenols might represent a manner of improving the poor anti-tumour efficacy of some glycolytic inhibitors, and that apoptosis potentiation may be at least in part explained by the regulation of defensive protein kinase activities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0345-y) contains supplementary material, which is available to authorized users.
Highlights
The glycolysis inhibitor 2-deoxy-d-glucose (2-DG) is a safe, potentially useful anti-tumour drug, but its efficacy is normally low when used alone
Using combinatory assays with the anti-leukemic agent arsenic trioxide (Trisenox), we recently demonstrated that a common effect of the anti-glycolytic drugs is the stimulation of protein kinase B (Akt)/mTOR and MEK/extracellular signal-regulated kinase (ERK) defensive pathways in several human acute myeloid leukemia (AML) cell lines, and that this stimulation restrains the apoptotic efficacy of 2-DG and Lon when used as single agents [11, 12]
Cell proliferation and cell death Firstly, we examined the capacity of Quer and 2-DG, alone and in combination, to affect proliferation activity and induce apoptosis at 24 h of treatment in HL60 cells
Summary
The glycolysis inhibitor 2-deoxy-d-glucose (2-DG) is a safe, potentially useful anti-tumour drug, but its efficacy is normally low when used alone. One of the best known modifications is the increased dependence on glucose metabolism instead of oxidative phosphorylation, even under aerobic conditions (a property known as “aerobic glycolysis” or “Warburg’’ effect). This peculiarity made possible the development of glycolysis-targeting drugs as potential anti-cancer agents. Using combinatory assays with the anti-leukemic agent arsenic trioxide (Trisenox), we recently demonstrated that a common effect of the anti-glycolytic drugs is the stimulation (albeit with different kinetics and intensity) of Akt/mTOR and MEK/ERK defensive pathways in several human acute myeloid leukemia (AML) cell lines, and that this stimulation restrains the apoptotic efficacy of 2-DG and Lon when used as single agents [11, 12]. Akt and/or ERK activation by 2-DG was observed in other tumour cell models [13,14,15]
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