Role of reactive oxygen species in gallic acid-induced apoptosis

We tallier demonstrated that gallic acid (3,4,5-trihydrozybenzoic acid) ind uced apoptosis in promyelocytic leukemia HL-60RG cells, which was inhibited by catalase and intracellular Ca2+ chelator, In this study, we further stu died 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 stimul ation with gallic acid by using 5,6-carboxy-2',7'-dichlorofluorescin diacet ate (DCFH-DA). Further studies that used various antioxidants and ROS scave ngers showed that the intracellular peroxide level was well correlated with the potency to induce apoptosis and that the increased intracellular perox ides after gallic acid treatment seemed likely to result from the influx of H2O2 derived from superoxide which were generated extracellularly. In addi tion, gallic acid, HX/XO, and H2O2-induced apoptosis was completely inhibit ed by pretreatment with intracellular Ca2+ chelator 1,2-bis(2-aminophenoxye thane)-N,N,N,N'-tetraacetic acid tetrakis (acetoxymethyl ester) ! (BAPTA-AM ), but increase of intracellular peroxide levels by gallic acid were suppre ssed only slightly, It is suggested that intracellular ROS induced by galli c acid plays an important role in eliciting an eat ly signal in apoptosis, Especially, H2O2 which is derived from superoxide anion generated extracell ularly mag. increase intracellular Ca2+ levels or cooperate with intracellu lar Ca2+, thus resulting in apoptosis induction.