German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
FASEB J. 2010 Aug;24(8):2938-50. doi: 10.1096/fj.10-155846. Epub 2010 Mar 24.
Oxidative stress and increased release of reactive oxygen species (ROS) are associated with apoptosis induction. Here we report ROS-mediated induction of apoptosis by xanthohumol (XN) from hops. XN at concentrations of 1.6-25 microM induced an immediate and transient increase in superoxide anion radical (O(2)(-)) formation in 3 human cancer cell lines (average+/-SD EC(50) of maximum O(2)(-) induction=3.1+/-0.8 microM), murine macrophages (EC(50)=4.0+/-0.3 microM), and BPH-1 benign prostate hyperplasia cells (EC(50)=4.3+/-0.1 microM), as evidenced by the O(2)(-)-specific indicator dihydroethidium. MitoSOX Red costaining and experiments using isolated mouse liver mitochondria (EC(50)=11.4+/-1.8 microM) confirmed mitochondria as the site of intracellular O(2)(-) formation. Antimycin A served as positive control (EC(50)=12.4+/-0.9 microM). XN-mediated O(2)(-) release was significantly reduced in BPH-1 rho(0) cells harboring nonfunctional mitochondria (EC(50)>25 microM) and by treatment of BPH-1 cells with vitamin C, N-acetylcysteine (NAC), or the superoxide dismutase mimetic MnTMPyP. In addition, we demonstrated a rapid 15% increase in oxidized glutathione and a dose-dependent overall thiol depletion within 6 h (IC(50)=24.3+/-11 microM). Respiratory chain complexes I-III were weakly inhibited by XN in bovine heart submitochondrial particles, but electron flux from complex I and II to complex III was significantly inhibited in BPH-1 cells, with IC(50) values of 28.1 +/- 2.4 and 24.4 +/- 5.2 microM, respectively. Within 15 min, intracellular ATP levels were significantly reduced by XN at 12.5 to 50 microM concentrations (IC(50)=26.7+/-3.7 microM). Concomitantly, XN treatment caused a rapid breakdown of the mitochondrial membrane potential and the release of cytochrome c, leading to apoptosis induction. Pre- or coincubation with 2 mM NAC and 50 microM MnTMPyP at various steps increased XN-mediated IC(50) values for cytotoxicity in BPH-1 cells from 6.7 +/- 0.2 to 12.2 +/- 0.1 and 41.4 +/- 7.6 microM, and it confirmed XN-induced O(2)(-) as an essential trigger for apoptosis induction. In summary, we have identified mitochondria as a novel cellular target of XN action, resulting in increased O(2)(-*) production, disruption of cellular redox balance and mitochondrial integrity, and subsequent apoptosis.
氧化应激和活性氧（ROS）的释放增加与细胞凋亡的诱导有关。在这里，我们报告了黄腐酚（XN）通过hops 诱导的 ROS 介导的细胞凋亡。XN 在 1.6-25 microM 的浓度下，在 3 个人类癌细胞系（最大 O2（-）诱导的平均+/-SD EC50）中诱导超氧阴离子自由基（O2（-））的形成立即和短暂增加（3.1+/-0.8 microM），鼠巨噬细胞（EC50=4.0+/-0.3 microM）和 BPH-1 良性前列腺增生细胞（EC50=4.3+/-0.1 microM），这一点可以通过 O2（-）特异性指示剂二氢乙啶证明。MitoSOX Red 染色和使用分离的鼠肝线粒体的实验（EC50=11.4+/-1.8 microM）证实线粒体是细胞内 O2（-）形成的部位。抗霉素 A 作为阳性对照（EC50=12.4+/-0.9 microM）。XN 介导的 O2（-）释放在含有非功能线粒体的 BPH-1 rho（0）细胞中显著降低（EC50>25 microM），并且通过用维生素 C、N-乙酰半胱氨酸（NAC）或超氧化物歧化酶模拟物 MnTMPyP 处理 BPH-1 细胞。此外，我们还证明了在 6 小时内氧化型谷胱甘肽迅速增加了 15%，并且总巯基耗竭呈剂量依赖性（IC50=24.3+/-11 microM）。XN 在牛心亚线粒体颗粒中对呼吸链复合物 I-III 的抑制作用较弱，但电子从复合物 I 和 II 到复合物 III 的通量在 BPH-1 细胞中受到显著抑制，IC50 值分别为 28.1 +/- 2.4 和 24.4 +/- 5.2 microM。XN 在 12.5 至 50 microM 浓度下可在 15 分钟内显著降低细胞内 ATP 水平（IC50=26.7+/-3.7 microM）。同时，XN 处理导致线粒体膜电位迅速崩溃和细胞色素 c 的释放，从而诱导细胞凋亡。XN 介导的 BPH-1 细胞的细胞毒性的 IC50 值在预先或同时与 2 mM NAC 和 50 microM MnTMPyP 孵育的各个步骤中从 6.7 +/- 0.2 增加到 12.2 +/- 0.1 和 41.4 +/- 7.6 microM，这证实了 XN 诱导的 O2（-）作为诱导细胞凋亡的必要触发因素。总之，我们已经确定线粒体是 XN 作用的新型细胞靶标，导致 O2（-*）产生增加、细胞氧化还原平衡和线粒体完整性破坏以及随后的细胞凋亡。
