Role of Reactive Oxygen Species in Gallic Acid-Induced Apoptosis

Abstract

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, 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, H₂O₂ which is derived from superoxide anion generated extracellularly may increase intracellular Ca²⁺ levels or cooperate with intracellular Ca²⁺, thus resulting in apoptosis induction.