Department of Translational Genomics, Center of Integrated Oncology Cologne-Bonn, University Hospital of Cologne, 50931 Cologne, Germany.
CECAD Cluster of Excellence, University of Cologne, 50931 Cologne, Germany.
Cells. 2020 Oct 9;9(10):2259. doi: 10.3390/cells9102259.
Ferroptosis is a form of regulated necrosis characterized by a chain-reaction of detrimental membrane lipid peroxidation following collapse of glutathione peroxidase 4 (Gpx4) activity. This lipid peroxidation is catalyzed by labile ferric iron. Therefore, iron import mediated via transferrin receptors and both, enzymatic and non-enzymatic iron-dependent radical formation are crucial prerequisites for the execution of ferroptosis. Intriguingly, the dynamin inhibitor dynasore, which has been shown to block transferrin receptor endocytosis, can protect from ischemia/reperfusion injury as well as neuronal cell death following spinal cord injury. Yet, it is unknown how dynasore exerts these cell death-protective effects. Using small interfering RNA suppression, lipid reactive oxygen species (ROS), iron tracers and bona fide inducers of ferroptosis, we find that dynasore treatment in lung adenocarcinoma and neuronal cell lines strongly protects these from ferroptosis. Surprisingly, while the dynasore targets dynamin 1 and 2 promote extracellular iron uptake, their silencing was not sufficient to block ferroptosis suggesting that this route of extracellular iron uptake is dispensable for acute induction of ferroptosis and dynasore must have an additional off-target activity mediating full ferroptosis protection. Instead, in intact cells, dynasore inhibited mitochondrial respiration and thereby mitochondrial ROS production which can feed into detrimental lipid peroxidation and ferroptotic cell death in the presence of labile iron. In addition, in cell free systems, dynasore showed radical scavenger properties and acted as a broadly active antioxidant which is superior to N-acetylcysteine (NAC) in blocking ferroptosis. Thus, dynasore can function as a highly active inhibitor of ROS-driven types of cell death via combined modulation of the iron pool and inhibition of general ROS by simultaneously blocking two routes required for ROS and lipid-ROS driven cell death, respectively. These data have important implications for the interpretation of studies observing tissue-protective effects of this dynamin inhibitor as well as raise awareness that off-target ROS scavenging activities of small molecules used to interrogate the ferroptosis pathway should be taken into consideration.
铁死亡是一种受调控的细胞坏死形式，其特征是谷胱甘肽过氧化物酶 4 (Gpx4) 活性崩溃后，有害的膜脂质过氧化作用发生连锁反应。这种脂质过氧化作用由不稳定的三价铁催化。因此，铁通过转铁蛋白受体的导入以及酶促和非酶促铁依赖性自由基形成都是铁死亡执行的关键前提条件。有趣的是，已显示可阻断转铁蛋白受体内吞作用的动力蛋白抑制剂 dynasore 可防止缺血/再灌注损伤以及脊髓损伤后的神经元细胞死亡。然而，尚不清楚 dynasore 如何发挥这些细胞死亡保护作用。通过使用小干扰 RNA 抑制、脂质活性氧 (ROS)、铁示踪剂和真正的铁死亡诱导剂，我们发现 dynasore 处理肺腺癌细胞系和神经元细胞系可强烈保护其免受铁死亡。令人惊讶的是，尽管 dynasore 的靶点动力蛋白 1 和 2 促进细胞外铁摄取，但它们的沉默不足以阻断铁死亡，这表明该细胞外铁摄取途径对于急性诱导铁死亡是可有可无的，dynasore 必须具有另外的非靶标活性来介导完全的铁死亡保护。相反，在完整细胞中，dynasore 抑制线粒体呼吸，从而抑制线粒体 ROS 的产生，这可以促进在不稳定铁存在下有害的脂质过氧化和铁死亡细胞死亡。此外，在无细胞系统中，dynasore 表现出自由基清除特性，并作为一种广泛有效的抗氧化剂，其阻断铁死亡的能力优于 N-乙酰半胱氨酸 (NAC)。因此，dynasore 可以通过同时阻断 ROS 和脂质 ROS 驱动的细胞死亡分别所需的两种途径来调节铁池和抑制一般 ROS，从而作为一种高度有效的 ROS 驱动型细胞死亡抑制剂发挥作用。这些数据对解释观察到该动力蛋白抑制剂具有组织保护作用的研究具有重要意义，并引起人们注意，应考虑到用于研究铁死亡途径的小分子的非靶标 ROS 清除活性。
