Bao Lei, Chen Shu-Jen, Conrad Kathleen, Keefer Kerry, Abraham Thomas, Lee John P, Wang JuFang, Zhang Xue-Qian, Hirschler-Laszkiewicz Iwona, Wang Hong-Gang, Dovat Sinisa, Gans Brian, Madesh Muniswamy, Cheung Joseph Y, Miller Barbara A
Departments of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033.
Neural and Behavioral Sciences and Microscopy Imaging Facility.
J Biol Chem. 2016 Nov 18;291(47):24449-24464. doi: 10.1074/jbc.M116.747147. Epub 2016 Sep 30.
Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in modulating cell survival following oxidant injury and is highly expressed in many cancers including neuroblastoma. Here, in xenografts generated from neuroblastoma cells in which TRPM2 was depleted with CRISPR/Cas9 technology and in in vitro experiments, tumor growth was significantly inhibited and doxorubicin sensitivity increased. The hypoxia-inducible transcription factor 1/2α (HIF-1/2α) signaling cascade including proteins involved in oxidant stress, glycolysis, and mitochondrial function was suppressed by TRPM2 depletion. TRPM2-depleted SH-SY5Y neuroblastoma cells demonstrated reduced oxygen consumption and ATP production after doxorubicin, confirming impaired cellular bioenergetics. In cells in which TRPM2 was depleted, mitochondrial superoxide production was significantly increased, particularly following doxorubicin. Ectopic expression of superoxide dismutase 2 (SOD2) reduced ROS and preserved viability of TRPM2-depleted cells, however, failed to restore ATP levels. Mitochondrial reactive oxygen species (ROS) were also significantly increased in cells in which TRPM2 function was inhibited by TRPM2-S, and pretreatment of these cells with the antioxidant MitoTEMPO significantly reduced ROS levels in response to doxorubicin and protected cell viability. Expression of the TRPM2 pore mutant E960D, in which calcium entry through TRPM2 is abolished, also resulted in significantly increased mitochondrial ROS following doxorubicin treatment, showing the critical role of TRPM2-mediated calcium entry. These findings demonstrate the important function of TRPM2 in modulation of cell survival through mitochondrial ROS, and the potential of targeted inhibition of TRPM2 as a therapeutic approach to reduce cellular bioenergetics, tumor growth, and enhance susceptibility to chemotherapeutic agents.
瞬时受体电位香草酸亚家族成员2（TRPM2）离子通道在调节氧化损伤后的细胞存活中具有重要作用，并且在包括神经母细胞瘤在内的许多癌症中高表达。在此，在通过CRISPR/Cas9技术使TRPM2缺失的神经母细胞瘤细胞产生的异种移植瘤以及体外实验中，肿瘤生长受到显著抑制，阿霉素敏感性增加。包括参与氧化应激、糖酵解和线粒体功能的蛋白质在内的缺氧诱导转录因子1/2α（HIF-1/2α）信号级联被TRPM2缺失所抑制。TRPM2缺失的SH-SY5Y神经母细胞瘤细胞在阿霉素处理后显示出氧消耗和ATP生成减少，证实细胞生物能量学受损。在TRPM2缺失的细胞中，线粒体超氧化物生成显著增加，尤其是在阿霉素处理后。超氧化物歧化酶2（SOD2）的异位表达降低了活性氧（ROS）水平并维持了TRPM2缺失细胞的活力，然而，未能恢复ATP水平。在TRPM2功能被TRPM2-S抑制的细胞中，线粒体活性氧也显著增加，用抗氧化剂MitoTEMPO对这些细胞进行预处理可显著降低对阿霉素反应的ROS水平并保护细胞活力。TRPM2孔突变体E960D（通过TRPM2的钙内流被消除）的表达在阿霉素处理后也导致线粒体ROS显著增加，表明TRPM2介导的钙内流的关键作用。这些发现证明了TRPM2在通过线粒体ROS调节细胞存活中的重要功能，以及靶向抑制TRPM2作为一种治疗方法来降低细胞生物能量学、肿瘤生长并增强对化疗药物敏感性的潜力。
