Nakazato Tomonori, Sagawa Morihiko, Yamato Kenji, Xian Mingji, Yamamoto Takehiro, Suematsu Makoto, Ikeda Yasuo, Kizaki Masahiro
Division of Hematology, Department of Internal Medicine, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.
Clin Cancer Res. 2007 Sep 15;13(18 Pt 1):5436-45. doi: 10.1158/1078-0432.CCR-07-0481.
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).
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.
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.
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.
我们之前报道过，活性氧（ROS）是多酚（-）-表没食子儿茶素-3-没食子酸酯（EGCG）诱导髓系白血病细胞凋亡的关键介质。本研究旨在进一步探讨EGCG诱导的ROS介导的凋亡机制及其与血红素酶髓过氧化物酶（MPO）的关系。
我们建立了稳定转染表达野生型和突变型MPO的K562细胞。然后，检测对EGCG和其他ROS诱导剂的敏感性，并进一步研究MPO阳性白血病细胞中ROS诱导凋亡的详细分子机制。
EGCG迅速诱导MPO阳性白血病细胞凋亡。用MPO特异性抑制剂4-氨基苯甲酸酰肼和血红素生物合成抑制剂琥珀酰丙酮预孵育髓系白血病细胞，可抑制细胞内MPO活性、ROS产生，并在添加EGCG后诱导凋亡。MPO的过表达使对EGCG耐药的K562细胞对EGCG诱导的凋亡敏感。相反，表达无酶活性MPO突变体的K562细胞对EGCG无反应，表明MPO对确定对EGCG诱导的氧化应激的敏感性很重要。次氯酸清除剂和羟基自由基（·OH）清除剂抑制EGCG诱导的髓系白血病细胞凋亡。用EGCG刺激后，负载氨基苯基荧光素和羟基苯基荧光素髓系白血病细胞的荧光强度均显著增加，表明EGCG在髓系白血病细胞中产生了高毒性的ROS。
这些结果表明，通过过氧化氢/MPO/卤化物系统产生的高毒性ROS如·OH可诱导凋亡，并且ROS可能是EGCG诱导MPO阳性白血病细胞凋亡的直接介质。
