Institute for Innovative Cancer Research, College of Medicine, University of Ulsan, Asan Medical Center, Seoul, 138-736, Korea.
Biometals. 2010 Dec;23(6):997-1013. doi: 10.1007/s10534-010-9346-9. Epub 2010 Jun 4.
Treatment of MCF-7 cells with tamoxifen induced vacuole formation and cell death. Levels of the autophagy marker, microtubule-associated protein light chain 3 (LC3)-II also increased, and GFP-LC3 accumulated in and around vacuoles in MCF-7 cells exposed to tamoxifen, indicating that autophagy is involved in tamoxifen-induced changes. Live-cell confocal microscopy with FluoZin-3 staining and transmission electron microscopy with autometallographic staining revealed that labile zinc(II) ion (Zn(2+)) accumulated in most acidic LC3(+) autophagic vacuoles (AVs). Chelation of Zn(2+) with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) blocked the increase in phospho-Erk and LC3-II levels, and attenuated AV formation and cell death. Conversely, the addition of ZnCl(2) markedly potentiated tamoxifen-induced extracellular signal-regulated kinase (Erk) activation, autophagy and cell death, indicating that Zn(2+) has an important role in these events. Tamoxifen-induced death was accompanied by increased oxidative stress and lysosomal membrane permeabilization (LMP) represented as release of lysosomal cathepsins into cytosol. Treatment with the antioxidant N-acetyl-L-cysteine (NAC) blunted the increase in Zn(2+) levels and reduced LC3-II conversion, cathepsin D release and cell death induced by tamoxifen. And cathepsin inhibitors attenuated cell death, indicating that LMP contributes to tamoxifen-induced cell death. Moreover, TPEN blocked tamoxifen-induced cathepsin D release and increase in oxidative stress. The present results indicate that Zn(2+) contributes to tamoxifen-induced autophagic cell death via increase in oxidative stress and induction of LMP.
用他莫昔芬处理 MCF-7 细胞会诱导空泡形成和细胞死亡。自噬标记物微管相关蛋白轻链 3（LC3）-II 的水平也增加了，并且 GFP-LC3 在暴露于他莫昔芬的 MCF-7 细胞中的空泡内外积累，表明自噬参与了他莫昔芬诱导的变化。使用 FluoZin-3 染色的活细胞共焦显微镜和使用自动金属染色的透射电子显微镜显示，不稳定的锌(II)离子（Zn(2+)）在大多数酸性 LC3(+)自噬空泡（AVs）中积累。用 N,N,N',N'-四（2-吡啶甲基）乙二胺（TPEN）螯合 Zn(2+)可阻断磷酸化 Erk 和 LC3-II 水平的增加，并减弱 AV 形成和细胞死亡。相反，添加 ZnCl(2)显著增强了他莫昔芬诱导的细胞外信号调节激酶（Erk）激活、自噬和细胞死亡，表明 Zn(2+)在这些事件中起重要作用。他莫昔芬诱导的死亡伴随着氧化应激的增加和溶酶体膜通透性（LMP）的增加，表现为溶酶体组织蛋白酶释放到细胞质中。抗氧化剂 N-乙酰-L-半胱氨酸（NAC）的处理减弱了 Zn(2+)水平的增加，并减少了他莫昔芬诱导的 LC3-II 转化、组织蛋白酶 D 释放和细胞死亡。组织蛋白酶抑制剂减轻了细胞死亡，表明 LMP 导致他莫昔芬诱导的细胞死亡。此外，TPEN 阻断了他莫昔芬诱导的组织蛋白酶 D 释放和氧化应激增加。这些结果表明，Zn(2+)通过增加氧化应激和诱导 LMP 促进他莫昔芬诱导的自噬细胞死亡。
