Sun Wen, Wu Xiaxia, Gao Hongwei, Yu Jie, Zhao Wenwen, Lu Jin-Jian, Wang Jinhua, Du Guanhua, Chen Xiuping
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China.
The State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Beijing Key Laboratory of Drug Target Research and Drug Screen, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China.
Free Radic Biol Med. 2017 Jul;108:433-444. doi: 10.1016/j.freeradbiomed.2017.04.010. Epub 2017 Apr 14.
Necroptosis is a form of programmed necrosis mediated by signaling complexes with receptor-interacting protein 1 (RIP1) and RIP3 kinases as the main mediators. However, the underlying execution pathways of this phenomenon have yet to be elucidated in detail. In this study, a RIP1/RIP3 complex was formed in 2-methoxy-6-acetyl-7-methyljuglone (MAM)-treated HCT116 and HT29 colon cancer cells. With this formation, mitochondrial reactive oxygen species (ROS) levels increased, mitochondrial depolarization occurred, and ATP concentrations decreased. This process was identified as necroptosis. This finding was confirmed by experiments showing that MAM-induced cell death was attenuated by the pharmacological or genetic blockage of necroptosis signaling, including RIP1 inhibitor necrostatin-1s (Nec-1s) and siRNA-mediated gene silencing of RIP1 and RIP3, but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM54). Transmission electron microscopy (TEM) analysis further revealed the ultrastructural features of MAM-induced necroptosis. MAM-induced RIP1/RIP3 complex triggered necroptosis through cytosolic calcium (Ca) accumulation and sustained c-Jun N-terminal kinase (JNK) activation. Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate necroptotic features, including mitochondrial ROS elevation, mitochondrial depolarization, and ATP depletion. 2-thenoyltrifluoroacetone (TTFA), which is a mitochondrial complex II inhibitor, was found to effectively reverse both MAM induced mitochondrial ROS generation and cell death, indicating the complex II was the ROS-producing site. The essential role of mitochondrial ROS was confirmed by the protective effect of overexpression of manganese superoxide dismutase (MnSOD). MAM-induced necroptosis was independent of TNFα, p53, MLKL, and lysosomal membrane permeabilization. In summary, our study demonstrated that RIP1/RIP3 complex-triggered cytosolic calcium accumulation is a critical mediator in MAM-induced necroptosis through sustained JNK activation and mitochondrial ROS production. Our study also provided new insights into the molecular regulation of necroptosis in human colon cancer cells.
坏死性凋亡是一种程序性坏死形式，由以受体相互作用蛋白1（RIP1）和RIP3激酶作为主要介质的信号复合物介导。然而，这一现象的潜在执行途径尚未得到详细阐明。在本研究中，在2-甲氧基-6-乙酰基-7-甲基胡桃醌（MAM）处理的HCT116和HT29结肠癌细胞中形成了RIP1/RIP3复合物。随着这种复合物的形成，线粒体活性氧（ROS）水平升高，线粒体去极化发生，并且ATP浓度降低。这一过程被确定为坏死性凋亡。通过实验证实了这一发现，这些实验表明，坏死性凋亡信号的药理学或基因阻断可减轻MAM诱导的细胞死亡，包括RIP1抑制剂坏死抑素-1s（Nec-1s）以及RIP1和RIP3的siRNA介导的基因沉默，但不受半胱天冬酶抑制剂z-vad-fmk或坏死抑制剂2-（1H-吲哚-3-基）-3-戊基氨基马来酰亚胺（IM54）的影响。透射电子显微镜（TEM）分析进一步揭示了MAM诱导的坏死性凋亡的超微结构特征。MAM诱导的RIP1/RIP3复合物通过胞质钙（Ca）积累和持续的c-Jun氨基末端激酶（JNK）激活触发坏死性凋亡。钙螯合剂BAPTA-AM和JNK抑制剂SP600125均可减轻坏死性凋亡特征，包括线粒体ROS升高、线粒体去极化和ATP消耗。发现线粒体复合物II抑制剂2-噻吩甲酰三氟丙酮（TTFA）可有效逆转MAM诱导的线粒体ROS生成和细胞死亡，表明复合物II是ROS产生部位。锰超氧化物歧化酶（MnSOD）过表达的保护作用证实了线粒体ROS的重要作用。MAM诱导的坏死性凋亡独立于TNFα、p53、混合谱系激酶结构域样蛋白（MLKL）和溶酶体膜通透性。总之，我们的研究表明，RIP1/RIP3复合物触发的胞质钙积累是通过持续的JNK激活和线粒体ROS产生在MAM诱导的坏死性凋亡中的关键介质。我们的研究还为人类结肠癌细胞中坏死性凋亡的分子调控提供了新的见解。
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