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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.


Effect of quercetin and 2-deoxy-d-glucose on cell viability and apoptosis generation. HL60 cells were either kept untreated (Cont) or treated with quercetin (Quer) and 2-deoxy-d-glucose (2-DG), alone and in combination. In the combined treatments the cells were pre-treated with Quer for 2 h, followed by addition of 2-DG for 24 h more, and with the occasional presence of the pan-caspase inhibitor z-VAD-fmk (50 μM). Drug concentrations are indicated as subheadings (Quer, μM; 2-DG, mM). When nothing is indicated, Quer was used at 20 μM and 2-DG at 5 mM. a Changes in cell viability, as evidenced by the MTT assay. Absorption values are expressed in relation to the control. b Frequency of apoptosis, represented by the sub-population of cells with sub-G1 DNA content obtained by flow cytometry. Examples of histograms showing cell cycle phases (G1, S and G2/M) and sub-G1 (Ap) are presented in c. d Frequency of early and late apoptotic cells, measured by cell surface annexin V binding and propidium iodide (PI) exclusion or uptake, respectively. e Flow cytometry histograms showing the frequency of PI-permeable cells, indicating plasma membrane disruption. The bar charts in (a–b) represent the mean ± S.D. of at least three determinations, measured by duplicate. The histograms in (d, e) are representative of one out of three determinations with similar results. Symbols mean: (*) significant differences between single treatments; (#) significant differences between the combined treatment and the sum of values in the corresponding individual treatments (e.g., co-incubation with 20 μM Quer and 5 mM 2-DG, in relation to the sum of 20 μM Quer alone plus 5 mM 2-DG alone) (n.s. non-significant). To better discern differences, in this case the sum of values in individual treatments is indicated by a thick black bar within the bar corresponding to the combined treatment. Single symbol, p < 0.05; double symbol, p < 0.01; triple symbol, p < 0.001
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Fig1: Effect of quercetin and 2-deoxy-d-glucose on cell viability and apoptosis generation. HL60 cells were either kept untreated (Cont) or treated with quercetin (Quer) and 2-deoxy-d-glucose (2-DG), alone and in combination. In the combined treatments the cells were pre-treated with Quer for 2 h, followed by addition of 2-DG for 24 h more, and with the occasional presence of the pan-caspase inhibitor z-VAD-fmk (50 μM). Drug concentrations are indicated as subheadings (Quer, μM; 2-DG, mM). When nothing is indicated, Quer was used at 20 μM and 2-DG at 5 mM. a Changes in cell viability, as evidenced by the MTT assay. Absorption values are expressed in relation to the control. b Frequency of apoptosis, represented by the sub-population of cells with sub-G1 DNA content obtained by flow cytometry. Examples of histograms showing cell cycle phases (G1, S and G2/M) and sub-G1 (Ap) are presented in c. d Frequency of early and late apoptotic cells, measured by cell surface annexin V binding and propidium iodide (PI) exclusion or uptake, respectively. e Flow cytometry histograms showing the frequency of PI-permeable cells, indicating plasma membrane disruption. The bar charts in (a–b) represent the mean ± S.D. of at least three determinations, measured by duplicate. The histograms in (d, e) are representative of one out of three determinations with similar results. Symbols mean: (*) significant differences between single treatments; (#) significant differences between the combined treatment and the sum of values in the corresponding individual treatments (e.g., co-incubation with 20 μM Quer and 5 mM 2-DG, in relation to the sum of 20 μM Quer alone plus 5 mM 2-DG alone) (n.s. non-significant). To better discern differences, in this case the sum of values in individual treatments is indicated by a thick black bar within the bar corresponding to the combined treatment. Single symbol, p < 0.05; double symbol, p < 0.01; triple symbol, p < 0.001

Mentions: 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. Because of the hypothesis advanced in the ‘‘Background’’ section, namely that polyphenols might prevent early regulatory gene responses elicited by metabolic inhibitors, in the combined treatments Quer was applied 2 h before 2-DG (and except when otherwise indicated, this procedure will be also followed in all experiments along the whole work). As shown in Fig. 1a, treatment with 5–20 μM Quer or 1–5 mM 2-DG separately caused a concentration-dependent decrease in viable cell number, as determined by the MTT assay, and the response was augmented when the drugs were used in combination. In spite of the evident effect on proliferation, treatment with either 5–20 µM Quer or 1-5 mM 2-DG separately caused very low (less than 10 %) apoptotic effect, as measured by the frequency of cells with sub-G1 DNA content in flow cytometry assays. Nonetheless, apoptosis was greatly potentiated in more than additive manner when the drugs were used in combination (Fig. 1b, c). On the ground of the obtained results, the concentrations of 20 µM Quer and 5 mM 2-DG were selected for the following experiments, except when otherwise indicated. The cooperative apoptotic action between Quer and 2-DG was confirmed using the annexin V/PI assay (Fig. 1d). Moreover, the pan-caspase inhibitor z-VAD-fmk almost totally abrogated the formation of apoptotic cells (Fig. 1b–d), corroborating that cell death represents genuine caspase-dependent apoptosis. Of note, treatment with Quer plus 2-DG resulted in free PI uptake by a high proportion of cells. Nevertheless, this effect was also suppressed by z-VAD-fmk (Fig. 1e), indicating that these cells represent late apoptosis (or “secondary necrosis”) instead of a genuine necrotic response. Finally, a time-course study (3–24 h) revealed that significant more than additive drug cooperation was firstly detectable at 6 h of treatment (approx. 15 % apoptosis in the combined treatment), and increased thereafter (see Additional file 1: Fig. S1). Thus, except when otherwise indicated, 6 h was the maximum time period used for further investigation of early regulatory events.Fig. 1


Selected polyphenols potentiate the apoptotic efficacy of glycolytic inhibitors in human acute myeloid leukemia cell lines. Regulation by protein kinase activities
Effect of quercetin and 2-deoxy-d-glucose on cell viability and apoptosis generation. HL60 cells were either kept untreated (Cont) or treated with quercetin (Quer) and 2-deoxy-d-glucose (2-DG), alone and in combination. In the combined treatments the cells were pre-treated with Quer for 2 h, followed by addition of 2-DG for 24 h more, and with the occasional presence of the pan-caspase inhibitor z-VAD-fmk (50 μM). Drug concentrations are indicated as subheadings (Quer, μM; 2-DG, mM). When nothing is indicated, Quer was used at 20 μM and 2-DG at 5 mM. a Changes in cell viability, as evidenced by the MTT assay. Absorption values are expressed in relation to the control. b Frequency of apoptosis, represented by the sub-population of cells with sub-G1 DNA content obtained by flow cytometry. Examples of histograms showing cell cycle phases (G1, S and G2/M) and sub-G1 (Ap) are presented in c. d Frequency of early and late apoptotic cells, measured by cell surface annexin V binding and propidium iodide (PI) exclusion or uptake, respectively. e Flow cytometry histograms showing the frequency of PI-permeable cells, indicating plasma membrane disruption. The bar charts in (a–b) represent the mean ± S.D. of at least three determinations, measured by duplicate. The histograms in (d, e) are representative of one out of three determinations with similar results. Symbols mean: (*) significant differences between single treatments; (#) significant differences between the combined treatment and the sum of values in the corresponding individual treatments (e.g., co-incubation with 20 μM Quer and 5 mM 2-DG, in relation to the sum of 20 μM Quer alone plus 5 mM 2-DG alone) (n.s. non-significant). To better discern differences, in this case the sum of values in individual treatments is indicated by a thick black bar within the bar corresponding to the combined treatment. Single symbol, p < 0.05; double symbol, p < 0.01; triple symbol, p < 0.001
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Fig1: Effect of quercetin and 2-deoxy-d-glucose on cell viability and apoptosis generation. HL60 cells were either kept untreated (Cont) or treated with quercetin (Quer) and 2-deoxy-d-glucose (2-DG), alone and in combination. In the combined treatments the cells were pre-treated with Quer for 2 h, followed by addition of 2-DG for 24 h more, and with the occasional presence of the pan-caspase inhibitor z-VAD-fmk (50 μM). Drug concentrations are indicated as subheadings (Quer, μM; 2-DG, mM). When nothing is indicated, Quer was used at 20 μM and 2-DG at 5 mM. a Changes in cell viability, as evidenced by the MTT assay. Absorption values are expressed in relation to the control. b Frequency of apoptosis, represented by the sub-population of cells with sub-G1 DNA content obtained by flow cytometry. Examples of histograms showing cell cycle phases (G1, S and G2/M) and sub-G1 (Ap) are presented in c. d Frequency of early and late apoptotic cells, measured by cell surface annexin V binding and propidium iodide (PI) exclusion or uptake, respectively. e Flow cytometry histograms showing the frequency of PI-permeable cells, indicating plasma membrane disruption. The bar charts in (a–b) represent the mean ± S.D. of at least three determinations, measured by duplicate. The histograms in (d, e) are representative of one out of three determinations with similar results. Symbols mean: (*) significant differences between single treatments; (#) significant differences between the combined treatment and the sum of values in the corresponding individual treatments (e.g., co-incubation with 20 μM Quer and 5 mM 2-DG, in relation to the sum of 20 μM Quer alone plus 5 mM 2-DG alone) (n.s. non-significant). To better discern differences, in this case the sum of values in individual treatments is indicated by a thick black bar within the bar corresponding to the combined treatment. Single symbol, p < 0.05; double symbol, p < 0.01; triple symbol, p < 0.001
Mentions: 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. Because of the hypothesis advanced in the ‘‘Background’’ section, namely that polyphenols might prevent early regulatory gene responses elicited by metabolic inhibitors, in the combined treatments Quer was applied 2 h before 2-DG (and except when otherwise indicated, this procedure will be also followed in all experiments along the whole work). As shown in Fig. 1a, treatment with 5–20 μM Quer or 1–5 mM 2-DG separately caused a concentration-dependent decrease in viable cell number, as determined by the MTT assay, and the response was augmented when the drugs were used in combination. In spite of the evident effect on proliferation, treatment with either 5–20 µM Quer or 1-5 mM 2-DG separately caused very low (less than 10 %) apoptotic effect, as measured by the frequency of cells with sub-G1 DNA content in flow cytometry assays. Nonetheless, apoptosis was greatly potentiated in more than additive manner when the drugs were used in combination (Fig. 1b, c). On the ground of the obtained results, the concentrations of 20 µM Quer and 5 mM 2-DG were selected for the following experiments, except when otherwise indicated. The cooperative apoptotic action between Quer and 2-DG was confirmed using the annexin V/PI assay (Fig. 1d). Moreover, the pan-caspase inhibitor z-VAD-fmk almost totally abrogated the formation of apoptotic cells (Fig. 1b–d), corroborating that cell death represents genuine caspase-dependent apoptosis. Of note, treatment with Quer plus 2-DG resulted in free PI uptake by a high proportion of cells. Nevertheless, this effect was also suppressed by z-VAD-fmk (Fig. 1e), indicating that these cells represent late apoptosis (or “secondary necrosis”) instead of a genuine necrotic response. Finally, a time-course study (3–24 h) revealed that significant more than additive drug cooperation was firstly detectable at 6 h of treatment (approx. 15 % apoptosis in the combined treatment), and increased thereafter (see Additional file 1: Fig. S1). Thus, except when otherwise indicated, 6 h was the maximum time period used for further investigation of early regulatory events.Fig. 1

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-3&alpha;,&beta; 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.