Synthetic 3-methyl ether of flavonoid-induced cell death in human leukemia cells is dependent on caspases and is attenuated by inhibition of JNK/SAPK signalling

E. Hernández1, F. León0,2, J. Quintana1, I. Brouard0,2, F. Estévez1. 1Universidad de las Palmas de Gran Canaria, Bioquímica y Biología Molecular, Spain, 2Consejo Superior de Investigación Científicas, Instituto de Productos Naturales y Agrobiología del CSIC, Spain

Flavonoids are naturally occurring polyphenolic compounds and are among the most promising anticancer agents. A series of flavonols and their 3-methyl ether derivatives containing different substituents on the A and B (2-phenyl group) rings were synthesized and assessed for cytotoxicity against HL-60 and Molt-3 leukemia cell lines following the 3-(4-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cells were cultured in RPMI 1640 medium containing 10% (v/v) fetal bovine serum and exposed to increasing concentrations of the compounds in 96-well plates for 72 h at 37 ºC and surviving cells were detected on the basis of their ability to metabolize MTT into formazan crystals. Among them, a 3-methyl ether derivative of flavonol containing a hydroxyl group in C-3´ and also a methoxy group in C-4´ of the B ring displayed strong cytotoxic properties in both cell lines. The IC50 value in HL-60 cells was 2.2 ± 0.3 μM, similar to the obtained value for Molt-3 cells. Flow cytometric studies revealed that cell growth inhibition induced by the selected synthetic 3-methyl ether derivative was associated with cell cycle arrest at the G2-M phase and also with a significant increase in the hypodiploid DNA content (subG1-fraction). The percentage of apoptotic cells increased 10-fold in HL-60 cells after 24 h exposure of flavonoid at a concentration of 3 μM. Apoptotic cell death was also determined by fluorescent microscopy following nuclei staining with bisbenzimide hydrochloride, DNA fragmentation analysis on agarose gel electrophoresis and translocation of phosphatidylserine to the cell surface. The immunobloting and caspase activity studies from leukemia cell lysates revealed activation of multiple caspases (caspase-8, caspase-9 and caspase-3). Moreover, cell death was completely prevented by the non-specific caspase inhibitor z-VAD-fmk. These results suggest that the selected synthetic 3-methyl ether derivative induces apoptosis through a caspase-dependent mechanism on human leukemia cells. The involvement of mitogen-activated protein kinases (MAPK) signalling on cell death triggered by this compound was also evaluated using specific inhibitors. To this end, cells were preincubated with the corresponding inhibitor for 1 h and then treated with the selected flavonoid (3 μM) or vehicle for 24 h and apoptotic cells were determined and quantified by flow cytometry. Pharmacological inhibition of c-jun NH2-terminal kinases/stress activated protein kinases (JNK/SAPK) by SP600125 partially protected 3-methyl ether derivative-mediated cell death from 40.7 ± 1.1 % of apoptotic cells to 26.6 ± 1.9% in the combination group (flavonoid + SP600125) (% reduction = 35%; p < 0.05). In contrast inhibition of p38 MAPK by SB 203580 and of mitogen-activated extracellular kinases 1/2 by U0126 had no influence on 3-methyl ether derivative-induced cell death. Our results suggest that the chemical synthesis of this kind of compounds might allow the development of new and highly specific antitumor agents against leukemia cells.

This work was supported by a grant from the Ministry of Science and Innovation of Spain and from the European Regional Development Fund (SAF2010-21380).