Abstract

Green tea polyphenol, (−)-epigallocatechin-3-gallate (EGCG), has a potent chemopreventive effects against various tumors, and epidemiologic studies have suggested that green tea consumption might be effective for reducing the incidence of certain cancers. We have reported that EGCG rapidly induces apoptosis of myeloid leukemic cells via modulation of reactive oxygen species (ROS) production in vitro and in vivo (

Haematologica
2005
;
90
:
317
). In this study, we further examined the precise mechanism of EGCG-induced apoptosis and its relationship to the heme enzyme myeloperoxidase (MPO). EGCG inhibited cellular growth of various myeloid leukemic cells via induction of apoptosis in dose- and time-dependent manners. Interestingly, EGCG rapidly induced apoptosis in MPO-positive myeloid leukemic cell lines (HL-60, UF-1, NB4, Kasumi-1) and fresh myeloid leukemic cells from patients, whereas EGCG failed to induce apoptosis in MPO-negative leukemic cells (U937, THP-1, KG-1, K562). Pre-incubation of MPO-positive myeloid leukemic cells with the MPO-specific inhibitor, 4-aminobenzoic acid hydrazide (50 μM), and the heme biosynthesis inhibitor, succinylacetone (0.5 mM), resulted in significant inhibition of intracellular MPO activity, ROS production, and induction of apoptosis after treatment with 50 μM EGCG. Pre-incubation of MPO-positive myeloid leukemic cells with anti-oxidant, catalase, completely suppressed EGCG-induced ROS production and apoptosis. These results indicate that EGCG-induced apoptosis is mediated through the generation of hydrogen peroxide (H2O2). To investigate the role of MPO in EGCG-induced apoptosis of leukemic cells, MPO-negative K562 cells were stably transfected with full length of MPO cDNA (K562/MPO cells). Marked interest, K562/MPO cells induced MPO activity, intracellular ROS production, and enhanced susceptibility of cells to EGCG-induced apoptosis compared to wild-type K562 cells. These results suggested that MPO positivity may be important to determine the sensitivity to EGCG-induced apoptosis, and MPO-derived ROS are involved in apoptosis in myeloid leukemic cells. MPO catalyzes the formation of hypochlorous acid (HOCl), a powerful oxidant formed from Cl and H2O2. Therefore, we next examined the relationship between EGCG-induced apoptosis and H2O2/MPO/halide system in MPO-positive HL-60 cells. Addition of HOCl scavengers, methionine (10 mM) and taurine (25 mM), inhibited EGCG-induced apoptosis in HL-60 and K562/MPO cells, but not wild-type K562 cells, suggesting that HOCl is the mediator of EGCG-induced apoptosis. Interestingly, hydroxyl radical (•OH) scavenger, thiourea, also inhibited EGCG-induced apoptosis in HL-60 cells. To determine which reactive oxygen species play a key role in EGCG-induced apoptosis mediated through MPO, we used novel fluorescence probes APF and HPF, which can detect selectively highly ROS (hROS). It is noteworthy that the fluorescence intensity of both APF- and HPF-loaded HL-60 cells significantly increased upon stimulation with EGCG, suggesting that EGCG generated hROS (•OH, ONOO) and OCl, but not other ROS (H2O2, NO, O2, 1O2) in HL-60 cells. Taken together, these results indicated that highly toxic ROS such as hydroxyl radical generated via H2O2/MPO/halide system induces apoptosis, and that it may be the direct mediator of EGCG-induced apoptosis in MPO-positive myeloid leukemic cells.

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