Myeloperoxidase (MPO) Is a Key Regulator of Oxidative Stress-Mediated Apoptosis in Myeloid Leukemic Cells.
Authors: Kizaki Masahiro , Nakazato Tomonori , Sagawa Morihiko , Yamato Kenji , Xian Mingji , Ikeda Yasuo 
Published: October 2019

Abstract
The therapeutic approach to acute myelogenous leukemia (AML) is usually chemotherapy, but severe side effects and complications, including infection and bleeding induced by the anticancer drugs, are major problems in the clinical setting. Recently, more specifically targeted agents have been developed for the treatment of AML; however, most candidate agents for targeted therapy have yet to be translated into clinical application. In the future, it will be important to identify other highly specific therapeutic agents based on our evolving understanding of the biology of AML. We have reported that reactive oxygen species (ROS) are key mediators of apoptosis induced by a green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), and other ROS-producing agents, in myeloid leukemic cells (Haematologica2005;90:317–325). In this study, we further examined the mechanism of oxidative stress-induced apoptosis and its relationship to the heme enzyme myeloperoxidase (MPO). EGCG rapidly induced apoptosis in MPO-positive (HL-60, UF-1, NB4, Kasumi-1), but not MPO-negative (U937, THP-1, KG-1, K562) leukemia cell lines as well as fresh samples from AML. Pre-incubation of MPO-positive 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. To investigate the role of MPO in EGCG-induced apoptosis, we transfected the full length MPO cDNA and empty vector into MPO-negative K562 cells (designated K562/MPO and K562/Cont cells, respectively). In contrast to K562/Cont cells, K562/MPO cells enhanced MPO activity and ROS production, and sensitized EGCG-resistant K562 cells to apoptosis induced by ROS-producing agents. In addition, an enzymatically inactive MPO mutant expressing K562 (K562/H502A) cells could not respond to EGCG, suggesting that MPO is important for determining the sensitivity to EGCG-induced oxidative stress. MPO catalyzes the formation of HOCl, a powerful oxidant formed from chloride ions and H2O2. We next examined the relationship between EGCG-induced apoptosis and the H2O2/MPO/halide system in MPO-positive myeloid leukemic cells. Further examination revealed that both HOCl scavengers (methionine, taurine) and the hydroxyl radical (·OH) scavenger thiourea inhibited EGCG-induced apoptosis in myeloid leukemic cells. To determine which ROS play a key role in oxidative stress-induced apoptosis mediated through MPO, we used the novel fluorescence probes AFP and HPF to detect highly toxic ROS (·OH). It is noteworthy that the fluorescence intensity of both APF- and HPF-loaded myeloid leukemic cells significantly increased upon stimulation with EGCG, suggesting that EGCG generated highly toxic ROS (·OH) in MPO-positive leukemic cells. Interestingly, the combination of EGCG and other ROS-producing agents (As2O3, doxorubicin, and daunorubicin) enhanced apoptosis through an increase in the production of highly toxic ROS in myeloid leukemic cells. Taken together, these observations indicate that highly toxic ROS (·OH) generated via the H2O2/MPO/halide system induce apoptosis, and that such ROS may be the direct mediators of oxidative stress-induced apoptosis in MPO-positive leukemic cells. “MPO-targeted therapy” will lead to new insights that should help in overcoming drug resistance in refractory AML.