Koumura Tomoko, Nakamura Chika, Nakagawa Yasuhito
Kitasato University, School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
Free Radic Res. 2005 Oct;39(10):1083-9. doi: 10.1080/10715760500264654.
Eicosapentaenoic acid (EPA) was previously shown to induce caspase-independent apoptosis in rat basophilic leukemia cells (RBL2H3 cells) by translocation of apoptosis-inducing factor (AIF) [Free Radic Res (2005) 39, 225-235]. Here, we attempted to investigate the mechanism of EPA-induced apoptosis. A rapid and sustained increase in calcium was observed in mitochondria at 2 h after the addition of EPA prior to apoptosis. Coincidently, hydroperoxide was generated in the mitochondria after exposure to EPA. Production of mitochondrial hydroperoxide was significantly reduced by ruthenium red, an inhibitor of mitochondrial calcium uniporter, and BAPTA-AM, a cytoplasmic calcium chelator, indicating that generation of hydroperoxide is triggered by an accumulation of calcium in the mitochondria. The production of mitochondrial hydroperoxide was markedly attenuated by overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the mitochondria. Apoptosis was therefore, significantly prevented through inhibition of mitochondrial hydroperoxide generation with mitochondrial PHGPx, ruthenium red or BAPTA-AM. However, accumulation of calcium in the mitochondria was not prevented by mitochondrial PHGPx although apoptosis was blocked, indicating that elevated calcium does not directly induce apoptosis. Taken together, our results show that calcium-dependent hydroperoxide accumulation in the mitochondria is critical in EPA-induced apoptosis.
二十碳五烯酸（EPA）先前已被证明可通过凋亡诱导因子（AIF）的易位，在大鼠嗜碱性白血病细胞（RBL2H3细胞）中诱导非半胱天冬酶依赖性凋亡[《自由基研究》（2005年）第39卷，第225 - 235页]。在此，我们试图研究EPA诱导凋亡的机制。在添加EPA后2小时，在凋亡发生之前，观察到线粒体中钙迅速且持续增加。巧合的是，暴露于EPA后线粒体中产生了过氧化氢。线粒体钙单向转运体抑制剂钌红和细胞质钙螯合剂BAPTA - AM可显著降低线粒体过氧化氢的产生，表明过氧化氢的产生是由线粒体中钙的积累触发的。线粒体中磷脂过氧化氢谷胱甘肽过氧化物酶（PHGPx）的过表达可显著减弱线粒体过氧化氢的产生。因此，通过用线粒体PHGPx、钌红或BAPTA - AM抑制线粒体过氧化氢的产生，可显著预防凋亡。然而，尽管凋亡被阻断，但线粒体PHGPx并不能阻止线粒体中钙的积累，这表明钙升高并不直接诱导凋亡。综上所述，我们的结果表明，线粒体中钙依赖性过氧化氢的积累在EPA诱导的凋亡中起关键作用。
