Selected polyphenols potentiate the apoptotic efficacy of glycolytic inhibitors in human acute myeloid leukemia cell lines. Regulation by protein kinase activities.
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.
Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
Elena
E
de Blas
Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain ; Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain.
María Cristina
MC
Estañ
Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain ; Departamento de Biología de Sistemas, Unidad de Bioquímica y Biología Molecular, Facultad de Medicina y Ciencias de la Salud, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain.
María
M
Del Carmen Gómez de Frutos
Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain ; Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica, Madrid, Spain.
Javier
J
Ramos
Departamento de Biología de Sistemas, Unidad de Bioquímica y Biología Molecular, Facultad de Medicina y Ciencias de la Salud, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain.
María
M
Del Carmen Boyano-Adánez
Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
Patricio
0000-0003-4405-9541
P
Aller
2-Deoxy-d-glucose; Acute myeloid leukemia cells; Apoptosis; Lonidamine; Natural polyphenols; Oxidative stress; Protein kinases (Akt, ERKs)