Inoue M, Sakaguchi N, Isuzugawa K, Tani H, Ogihara Y
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan.
Biol Pharm Bull. 2000 Oct;23(10):1153-7. doi: 10.1248/bpb.23.1153.
We earlier demonstrated that gallic acid (3,4,5-trihydroxybenzoic acid) induced apoptosis in promyelocytic leukemia HL-60RG cells, which was inhibited by catalase and intracellular Ca2+ chelator. In this study, we further studied the involvement of reactive oxygen species (ROS) and intracellular Ca2+ in gallic acid-induced apoptosis. The enhancement of intracellular ROS in HL-60RG cells was detected dose-dependently as early as 5 min after stimulation with gallic acid by using 5,6-carboxy-2',7'-dichlorofluorescin diacetate (DCFH-DA). Further studies that used various antioxidants and ROS scavengers showed that the intracellular peroxide level was well correlated with the potency to induce apoptosis and that the increased intracellular peroxides after gallic acid treatment seemed likely to result from the influx of H2O2 derived from superoxide which were generated extracellularly. In addition, gallic acid, HX/XO, and H2O2-induced apoptosis was completely inhibited by pretreatment with intracellular Ca2+ chelator 1,2-bis(2-aminophenoxyethane)-N,N,N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), but increase of intracellular peroxide levels by gallic acid were suppressed only slightly. It is suggested that intracellular ROS induced by gallic acid plays an important role in eliciting an early signal in apoptosis. Especially, H2O, which is derived from superoxide anion generated extracellularly may increase intracellular Ca2+ levels or cooperate with intracellular Ca2+, thus resulting in apoptosis induction.
我们之前证明了没食子酸（3,4,5 - 三羟基苯甲酸）可诱导早幼粒细胞白血病HL - 60RG细胞凋亡，而过氧化氢酶和细胞内钙离子螯合剂可抑制这种凋亡。在本研究中，我们进一步探讨了活性氧（ROS）和细胞内钙离子在没食子酸诱导凋亡中的作用。通过使用5,6 - 羧基 - 2',7' - 二氯荧光素二乙酸酯（DCFH - DA），早在没食子酸刺激后5分钟就检测到HL - 60RG细胞内ROS呈剂量依赖性增强。使用各种抗氧化剂和ROS清除剂的进一步研究表明，细胞内过氧化物水平与诱导凋亡的能力密切相关，没食子酸处理后细胞内过氧化物增加似乎可能是由于细胞外产生的超氧化物衍生的H2O2流入所致。此外，细胞内钙离子螯合剂1,2 - 双（2 - 氨基苯氧基乙烷） - N,N,N' - 四乙酸四（乙酰氧基甲酯）（BAPTA - AM）预处理可完全抑制没食子酸、黄嘌呤/黄嘌呤氧化酶和H2O2诱导的凋亡，但没食子酸引起的细胞内过氧化物水平升高仅略有抑制。提示没食子酸诱导的细胞内ROS在引发凋亡早期信号中起重要作用。特别是，源自细胞外产生的超氧阴离子的H2O可能会增加细胞内钙离子水平或与细胞内钙离子协同作用，从而导致凋亡诱导。
