Limits...
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

Apoptosis generation by other polyphenols, and by 2-deoxy-d-glucose and quercetin in other cell lines. (a, b) HL60 cells were incubated with the indicated concentrations of (a) curcumin (Cur, μM) or (b) genistein (Gen, μM) and 2-DG (mM), alone or in combination. c NB4 cells and d THP-1 cells were incubated with the indicated concentrations of Quer (μM) and 2-DG (mM), alone and in combination. The frequency of apoptosis (cells with sub-G1 DNA content) was estimated by flow cytometry. Other conditions, including pre-incubation with polyphenols in the combined treatments and symbols used in statistical analysis, were as in Fig. 1
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5015235&req=5

Fig2: Apoptosis generation by other polyphenols, and by 2-deoxy-d-glucose and quercetin in other cell lines. (a, b) HL60 cells were incubated with the indicated concentrations of (a) curcumin (Cur, μM) or (b) genistein (Gen, μM) and 2-DG (mM), alone or in combination. c NB4 cells and d THP-1 cells were incubated with the indicated concentrations of Quer (μM) and 2-DG (mM), alone and in combination. The frequency of apoptosis (cells with sub-G1 DNA content) was estimated by flow cytometry. Other conditions, including pre-incubation with polyphenols in the combined treatments and symbols used in statistical analysis, were as in Fig. 1

Mentions: In a new set of experiments, 2-DG was combined with Cur (8 μM) and Gen (50 μM) instead of Quer. The suitability of these concentrations for combinatory studies in leukemia cell models was established in earlier publications [21, 29]. Some of the obtained results are presented in Fig. 2a, b. Cur alone caused negligible apoptosis but cooperated with 2-DG with similar efficacy as Quer (Fig. 2a). On the other hand, the efficacy of cooperation using 50 μM Gen was very low, and the concentration had to be increased to 100 μM (which is per se moderately lethal) to obtain a more satisfactory response (Fig. 2b). The results with both Cur and Gen were corroborated using annexin V/PI analysis (see Additional file 2: Fig. S2).Fig. 2


Selected polyphenols potentiate the apoptotic efficacy of glycolytic inhibitors in human acute myeloid leukemia cell lines. Regulation by protein kinase activities
Apoptosis generation by other polyphenols, and by 2-deoxy-d-glucose and quercetin in other cell lines. (a, b) HL60 cells were incubated with the indicated concentrations of (a) curcumin (Cur, μM) or (b) genistein (Gen, μM) and 2-DG (mM), alone or in combination. c NB4 cells and d THP-1 cells were incubated with the indicated concentrations of Quer (μM) and 2-DG (mM), alone and in combination. The frequency of apoptosis (cells with sub-G1 DNA content) was estimated by flow cytometry. Other conditions, including pre-incubation with polyphenols in the combined treatments and symbols used in statistical analysis, were as in Fig. 1
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5015235&req=5

Fig2: Apoptosis generation by other polyphenols, and by 2-deoxy-d-glucose and quercetin in other cell lines. (a, b) HL60 cells were incubated with the indicated concentrations of (a) curcumin (Cur, μM) or (b) genistein (Gen, μM) and 2-DG (mM), alone or in combination. c NB4 cells and d THP-1 cells were incubated with the indicated concentrations of Quer (μM) and 2-DG (mM), alone and in combination. The frequency of apoptosis (cells with sub-G1 DNA content) was estimated by flow cytometry. Other conditions, including pre-incubation with polyphenols in the combined treatments and symbols used in statistical analysis, were as in Fig. 1
Mentions: In a new set of experiments, 2-DG was combined with Cur (8 μM) and Gen (50 μM) instead of Quer. The suitability of these concentrations for combinatory studies in leukemia cell models was established in earlier publications [21, 29]. Some of the obtained results are presented in Fig. 2a, b. Cur alone caused negligible apoptosis but cooperated with 2-DG with similar efficacy as Quer (Fig. 2a). On the other hand, the efficacy of cooperation using 50 μM Gen was very low, and the concentration had to be increased to 100 μM (which is per se moderately lethal) to obtain a more satisfactory response (Fig. 2b). The results with both Cur and Gen were corroborated using annexin V/PI analysis (see Additional file 2: Fig. S2).Fig. 2

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