Role of reactive oxygen species in gallic acid-induced apoptosis.
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.
Inoue
M
M
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan. inoue@phar.nagoya-cu.ac.jp
Sakaguchi
N
N
Isuzugawa
K
K
Tani
H
H
Ogihara
Y
Y
D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000502:physiology; D053938:DNA Fragmentation; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes; D005707:Gallic Acid / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D017382:Reactive Oxygen Species / Q000502:physiology*
D000975:Antioxidants; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D005707:Gallic Acid; D002118:Calcium