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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.


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Effect of polyphenols and glycolytic inhibitors on intracellular ROS and GSH levels. a Intracellular accumulation of ROS in HL60 cells, as determined by flow cytometry using H2DCFDA, upon treatment with the indicated concentrations of Quer, Cur, Gen and Lon (μM) or 2-DG (mM), alone and in combination. The results in the bar chart are expressed in relation to untreated (Cont) cells. Cytometry histograms corresponding to untreated cells and cells incubated for 3 h with Quer plus 2-DG are presented as examples. b Intracellular GSH levels in HL60 cells, as determined at 6 h of treatment by monochlorobimane derivatization in luminometric assays. 3-BrP (60 μM) was used as a positive control (see Ref. [44]). The results are expressed in relation to untreated (Cont) cells (approximate GSH content, 8.5 nmol/106 cells). c Frequency of apoptosis inHL60 cells upon incubation for 24 h with 1 mM BSO, alone and in combination with 2-DG or Lon. The combinations Quer plus 2-DG and Quer plus Lon are included as positive controls. Symbols in (a, b) indicate significant differences in relation to Cont, and in (c) between the indicated pairs of values (n.s. non significant). Other conditions, including pre-incubation with Quer in the combined treatments (a, b) and symbols used in statistical analysis, were as in Figs. 1 and 3
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Fig5: Effect of polyphenols and glycolytic inhibitors on intracellular ROS and GSH levels. a Intracellular accumulation of ROS in HL60 cells, as determined by flow cytometry using H2DCFDA, upon treatment with the indicated concentrations of Quer, Cur, Gen and Lon (μM) or 2-DG (mM), alone and in combination. The results in the bar chart are expressed in relation to untreated (Cont) cells. Cytometry histograms corresponding to untreated cells and cells incubated for 3 h with Quer plus 2-DG are presented as examples. b Intracellular GSH levels in HL60 cells, as determined at 6 h of treatment by monochlorobimane derivatization in luminometric assays. 3-BrP (60 μM) was used as a positive control (see Ref. [44]). The results are expressed in relation to untreated (Cont) cells (approximate GSH content, 8.5 nmol/106 cells). c Frequency of apoptosis inHL60 cells upon incubation for 24 h with 1 mM BSO, alone and in combination with 2-DG or Lon. The combinations Quer plus 2-DG and Quer plus Lon are included as positive controls. Symbols in (a, b) indicate significant differences in relation to Cont, and in (c) between the indicated pairs of values (n.s. non significant). Other conditions, including pre-incubation with Quer in the combined treatments (a, b) and symbols used in statistical analysis, were as in Figs. 1 and 3

Mentions: Although dietary polyphenols are normally considered as anti-oxidant, protective agents, there is ample evidence indicating that they may exert both anti-oxidant and pro-oxidant effects, depending on the chemical structure and treatment conditions. For instance, it was reported that Quer may either decrease [36] or increase [37, 38] ROS production in HL60 cells, and ROS increase mediated apoptosis induction [38]. For these reasons, we evaluated possible alterations in intracellular ROS accumulation after short treatments (3 h) with Quer and 2-DG, alone and in combination, using the ROS-sensitive fluorescent probe H2DCFDA. Some of the obtained results are presented in Fig. 5a. It was observed that 2-DG (5 mM) and Quer (10–40 μM, in a concentration-dependent manner) reduced the basal intracellular ROS content in HL60 cells, and the reduction was higher in the combined treatment. By contrast to Quer, Gen (50 μM) increased ROS levels (as previously reported [29]), but the increase was attenuated by combination with 2-DG. In a similar manner, and by contrast to 2-DG, Lon (100 μM) increased ROS content (as previously reported [11]), but this increase was totally abrogated by combination with Quer, reaching similar levels as with Quer alone. In summary, these results evidence large discrepancies in ROS production depending on the used treatment, which do not match and hence may not explain apoptosis potentiation in the combined treatments (see Figs. 1–3).Fig. 5


Selected polyphenols potentiate the apoptotic efficacy of glycolytic inhibitors in human acute myeloid leukemia cell lines. Regulation by protein kinase activities
Effect of polyphenols and glycolytic inhibitors on intracellular ROS and GSH levels. a Intracellular accumulation of ROS in HL60 cells, as determined by flow cytometry using H2DCFDA, upon treatment with the indicated concentrations of Quer, Cur, Gen and Lon (μM) or 2-DG (mM), alone and in combination. The results in the bar chart are expressed in relation to untreated (Cont) cells. Cytometry histograms corresponding to untreated cells and cells incubated for 3 h with Quer plus 2-DG are presented as examples. b Intracellular GSH levels in HL60 cells, as determined at 6 h of treatment by monochlorobimane derivatization in luminometric assays. 3-BrP (60 μM) was used as a positive control (see Ref. [44]). The results are expressed in relation to untreated (Cont) cells (approximate GSH content, 8.5 nmol/106 cells). c Frequency of apoptosis inHL60 cells upon incubation for 24 h with 1 mM BSO, alone and in combination with 2-DG or Lon. The combinations Quer plus 2-DG and Quer plus Lon are included as positive controls. Symbols in (a, b) indicate significant differences in relation to Cont, and in (c) between the indicated pairs of values (n.s. non significant). Other conditions, including pre-incubation with Quer in the combined treatments (a, b) and symbols used in statistical analysis, were as in Figs. 1 and 3
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Fig5: Effect of polyphenols and glycolytic inhibitors on intracellular ROS and GSH levels. a Intracellular accumulation of ROS in HL60 cells, as determined by flow cytometry using H2DCFDA, upon treatment with the indicated concentrations of Quer, Cur, Gen and Lon (μM) or 2-DG (mM), alone and in combination. The results in the bar chart are expressed in relation to untreated (Cont) cells. Cytometry histograms corresponding to untreated cells and cells incubated for 3 h with Quer plus 2-DG are presented as examples. b Intracellular GSH levels in HL60 cells, as determined at 6 h of treatment by monochlorobimane derivatization in luminometric assays. 3-BrP (60 μM) was used as a positive control (see Ref. [44]). The results are expressed in relation to untreated (Cont) cells (approximate GSH content, 8.5 nmol/106 cells). c Frequency of apoptosis inHL60 cells upon incubation for 24 h with 1 mM BSO, alone and in combination with 2-DG or Lon. The combinations Quer plus 2-DG and Quer plus Lon are included as positive controls. Symbols in (a, b) indicate significant differences in relation to Cont, and in (c) between the indicated pairs of values (n.s. non significant). Other conditions, including pre-incubation with Quer in the combined treatments (a, b) and symbols used in statistical analysis, were as in Figs. 1 and 3
Mentions: Although dietary polyphenols are normally considered as anti-oxidant, protective agents, there is ample evidence indicating that they may exert both anti-oxidant and pro-oxidant effects, depending on the chemical structure and treatment conditions. For instance, it was reported that Quer may either decrease [36] or increase [37, 38] ROS production in HL60 cells, and ROS increase mediated apoptosis induction [38]. For these reasons, we evaluated possible alterations in intracellular ROS accumulation after short treatments (3 h) with Quer and 2-DG, alone and in combination, using the ROS-sensitive fluorescent probe H2DCFDA. Some of the obtained results are presented in Fig. 5a. It was observed that 2-DG (5 mM) and Quer (10–40 μM, in a concentration-dependent manner) reduced the basal intracellular ROS content in HL60 cells, and the reduction was higher in the combined treatment. By contrast to Quer, Gen (50 μM) increased ROS levels (as previously reported [29]), but the increase was attenuated by combination with 2-DG. In a similar manner, and by contrast to 2-DG, Lon (100 μM) increased ROS content (as previously reported [11]), but this increase was totally abrogated by combination with Quer, reaching similar levels as with Quer alone. In summary, these results evidence large discrepancies in ROS production depending on the used treatment, which do not match and hence may not explain apoptosis potentiation in the combined treatments (see Figs. 1–3).Fig. 5

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