Myeloperoxidase is a key regulator of oxidative stress mediated apoptosis in myeloid leukemic cells.
OBJECTIVE
We reported previously that reactive oxygen species (ROS) are key mediators of apoptosis induced by a polyphenol, (-)-epigallocatechin-3-gallate (EGCG), in myeloid leukemic cells. This study aimed to further examine the mechanism of ROS-mediated apoptosis induced by EGCG and its relationship to the heme enzyme myeloperoxidase (MPO).


METHODS
We established stably transfected K562 cells expressing wild-type and mutant MPO. Then, sensitivity against EGCG and other ROS-inducing agent was examined and further investigated the detailed molecular mechanism of ROS-inducing apoptosis in MPO-positive leukemic cells.


RESULTS
EGCG rapidly induced apoptosis in MPO-positive leukemia cells. Preincubation of myeloid leukemic cells with the MPO-specific inhibitor, 4-aminobenzoic acid hydrazide, and the heme biosynthesis inhibitor, succinylacetone, resulted in inhibition of the intracellular MPO activity, ROS production, and induction of apoptosis following addition of EGCG. Overexpression of MPO sensitized EGCG-resistant K562 cells to apoptosis induced by EGCG. In contrast, an enzymatically inactive MPO mutant-expressing K562 cell could not respond to EGCG, suggesting that MPO is important for determining the sensitivity to EGCG-induced oxidative stress. Hypochlorous acid scavengers and the hydroxyl radical (.OH) scavenger inhibited EGCG-induced apoptosis in myeloid leukemic cells. The fluorescence intensity of both aminophenyl fluorescein- and hydroxyphenyl fluorescein-loaded myeloid leukemic cells significantly increased on stimulation with EGCG, indicating that EGCG generated highly toxic ROS in myeloid leukemic cells.


CONCLUSIONS
These results indicated that highly toxic ROS such as .OH generated via the hydrogen peroxide/MPO/halide system induce apoptosis and that ROS may be the direct mediators of EGCG-induced apoptosis in MPO-positive leukemic cells.
Nakazato
Tomonori
T
Division of Hematology, Department of Internal Medicine, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.
Sagawa
Morihiko
M
Yamato
Kenji
K
Xian
Mingji
M
Yamamoto
Takehiro
T
Suematsu
Makoto
M
Ikeda
Yasuo
Y
Kizaki
Masahiro
M
D000814:Aniline Compounds / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006418:Heme / Q000037:antagonists & inhibitors; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology*; D018384:Oxidative Stress; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism
D000814:Aniline Compounds; D000975:Antioxidants; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D013481:Superoxides; D006418:Heme; C034343:4-aminobenzhydrazide; D002392:Catechin; C045651:epigallocatechin gallate; D009195:Peroxidase