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Selected polyphenols potentiate the apoptotic efficacy of glycolytic inhibitors in human acute myeloid leukemia cell lines. Regulation by protein kinase activities

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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. 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.

Methods: Cell 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.

Conclusions: The 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 material: The online version of this article (doi:10.1186/s12935-016-0345-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Effect of 2-deoxy-d-glucose and quercetin on protein kinase activities. The figure shows the relative levels of phosphorylated (P) and total (T) Akt, ERKs, S6 ribosomal protein (rpS6), GSK-3α,β, and p38-MAPK, and β-actin (assessed as a control of sample loading). HL60 cells were kept untreated, incubated for 1 or 6 h with 5 mM 2-DG alone, or incubated for 2 h with 20 μM Quer and then for 1 or 6 h more with or without addition of 2-DG. Whenever possible, band intensities of phosphorylated forms were measured, normalized with to the corresponding total form, and expressed in relation the Cont (arbitrary value of 1.0) (see values within the blots: nd not determined). ERK1/ERK2 were measured together, while GSK-3α and GSK-3β were separately analyzed. The blots are representative of one of at least three independent determinations, with qualitatively similar results. For other conditions see legend of Fig. 1
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Fig6: Effect of 2-deoxy-d-glucose and quercetin on protein kinase activities. The figure shows the relative levels of phosphorylated (P) and total (T) Akt, ERKs, S6 ribosomal protein (rpS6), GSK-3α,β, and p38-MAPK, and β-actin (assessed as a control of sample loading). HL60 cells were kept untreated, incubated for 1 or 6 h with 5 mM 2-DG alone, or incubated for 2 h with 20 μM Quer and then for 1 or 6 h more with or without addition of 2-DG. Whenever possible, band intensities of phosphorylated forms were measured, normalized with to the corresponding total form, and expressed in relation the Cont (arbitrary value of 1.0) (see values within the blots: nd not determined). ERK1/ERK2 were measured together, while GSK-3α and GSK-3β were separately analyzed. The blots are representative of one of at least three independent determinations, with qualitatively similar results. For other conditions see legend of Fig. 1

Mentions: As commented above (see ‘‘Background’’ section), we hypothesized that polyphenols might potentiate the apoptotic efficacy of glycolytic inhibitors by preventing the activation of defensive kinase pathways, either PI3K/Akt and/or MEK/ERK. To examine this hypothesis, in a first set of experiments we examined Akt and ERK phosphorylation/activation upon 1 and 6 h treatment of HL60 cells with Quer and 2-DG, alone and in combination (where, as previously indicated, Quer was applied 2 h before 2-DG). We also checked the response of S6-ribosomal protein (rpS6), which is downstream Akt; of GSK3α/β, which are phosphorylated by Akt and ERK [40, 41]; and of p38-MAPK, also described as a target of quercetin or quercetin-derived analogs in leukemia cells [42, 43]. The results, presented in Fig. 6, were as follows: (i) 2-DG (5 mM) stimulated Akt and rpS6 phosphorylation/activation, and the stimulation was abrogated or greatly attenuated by Quer. (ii) By contrast, ERK phosphorylation/activation was stimulated by both Quer and 2-DG, alone and in combination. (iii) Quer and 2-DG, alone and in combination, stimulated GSK3α/β phosphorylation (Ser21/9)/inactivation. Quer alone exerted higher effect on the α isoform, while 2-DG alone stimulated both isoforms. (iv) Quer, alone or with 2-DG, stimulated p38-MAPK phosphorylation/activation, while the effect of 2-DG alone was negligible.Fig. 6


Selected polyphenols potentiate the apoptotic efficacy of glycolytic inhibitors in human acute myeloid leukemia cell lines. Regulation by protein kinase activities
Effect of 2-deoxy-d-glucose and quercetin on protein kinase activities. The figure shows the relative levels of phosphorylated (P) and total (T) Akt, ERKs, S6 ribosomal protein (rpS6), GSK-3α,β, and p38-MAPK, and β-actin (assessed as a control of sample loading). HL60 cells were kept untreated, incubated for 1 or 6 h with 5 mM 2-DG alone, or incubated for 2 h with 20 μM Quer and then for 1 or 6 h more with or without addition of 2-DG. Whenever possible, band intensities of phosphorylated forms were measured, normalized with to the corresponding total form, and expressed in relation the Cont (arbitrary value of 1.0) (see values within the blots: nd not determined). ERK1/ERK2 were measured together, while GSK-3α and GSK-3β were separately analyzed. The blots are representative of one of at least three independent determinations, with qualitatively similar results. For other conditions see legend of Fig. 1
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Fig6: Effect of 2-deoxy-d-glucose and quercetin on protein kinase activities. The figure shows the relative levels of phosphorylated (P) and total (T) Akt, ERKs, S6 ribosomal protein (rpS6), GSK-3α,β, and p38-MAPK, and β-actin (assessed as a control of sample loading). HL60 cells were kept untreated, incubated for 1 or 6 h with 5 mM 2-DG alone, or incubated for 2 h with 20 μM Quer and then for 1 or 6 h more with or without addition of 2-DG. Whenever possible, band intensities of phosphorylated forms were measured, normalized with to the corresponding total form, and expressed in relation the Cont (arbitrary value of 1.0) (see values within the blots: nd not determined). ERK1/ERK2 were measured together, while GSK-3α and GSK-3β were separately analyzed. The blots are representative of one of at least three independent determinations, with qualitatively similar results. For other conditions see legend of Fig. 1
Mentions: As commented above (see ‘‘Background’’ section), we hypothesized that polyphenols might potentiate the apoptotic efficacy of glycolytic inhibitors by preventing the activation of defensive kinase pathways, either PI3K/Akt and/or MEK/ERK. To examine this hypothesis, in a first set of experiments we examined Akt and ERK phosphorylation/activation upon 1 and 6 h treatment of HL60 cells with Quer and 2-DG, alone and in combination (where, as previously indicated, Quer was applied 2 h before 2-DG). We also checked the response of S6-ribosomal protein (rpS6), which is downstream Akt; of GSK3α/β, which are phosphorylated by Akt and ERK [40, 41]; and of p38-MAPK, also described as a target of quercetin or quercetin-derived analogs in leukemia cells [42, 43]. The results, presented in Fig. 6, were as follows: (i) 2-DG (5 mM) stimulated Akt and rpS6 phosphorylation/activation, and the stimulation was abrogated or greatly attenuated by Quer. (ii) By contrast, ERK phosphorylation/activation was stimulated by both Quer and 2-DG, alone and in combination. (iii) Quer and 2-DG, alone and in combination, stimulated GSK3α/β phosphorylation (Ser21/9)/inactivation. Quer alone exerted higher effect on the α isoform, while 2-DG alone stimulated both isoforms. (iv) Quer, alone or with 2-DG, stimulated p38-MAPK phosphorylation/activation, while the effect of 2-DG alone was negligible.Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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. 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.

Methods: Cell 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.

Conclusions: The 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 material: The online version of this article (doi:10.1186/s12935-016-0345-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus