Hack Carsten Theo, Buck Theresa, Bagnjuk Konstantin, Eubler Katja, Kunz Lars, Mayr Doris, Mayerhofer Artur
Biomedical Center Munich (BMC), Cell Biology, Anatomy III, Ludwig-Maximilian-University (LMU), D-82152 Planegg, Germany.
Division of Neurobiology, Department Biology II, Ludwig-Maximilian-University (LMU), D-82152 Planegg, Germany.
Antioxidants (Basel). 2019 Oct 29;8(11):518. doi: 10.3390/antiox8110518.
Recent studies showed that KGN cells, derived from a human granulosa cell tumor (GCT), express NADPH oxidase 4 (NOX4), an important source of HO. Transient receptor potential melastatin 2 (TRPM2) channel is a Ca permeable cation channel that can be activated by HO and plays an important role in cellular functions. It is also able to promote susceptibility to cell death. We studied expression and functionality of TRPM2 in KGN cells and examined GCT tissue microarrays (TMAs) to explore in vivo relevance. We employed live cell, calcium and mitochondrial imaging, viability assays, fluorescence activated cell sorting (FACS) analysis, Western blotting and immunohistochemistry. We confirmed that KGN cells produce HO and found that they express functional TRPM2. HO increased intracellular Ca levels and N-(p-Amylcinnamoyl)anthranilic acid (ACA), a TRPM2 inhibitor, blocked this action. HO caused mitochondrial fragmentation and apoptotic cell death, which could be attenuated by a scavenger (Trolox). Immunohistochemistry showed parallel expression of NOX4 and TRPM2 in all 73 tumor samples examined. The results suggest that GCTs can be endowed with a system that may convey susceptibility to cell death. If so, induction of oxidative stress may be beneficial in GCT therapy. Our results also imply a therapeutic potential for TRPM2 as a drug target in GCTs.
最近的研究表明，源自人颗粒细胞瘤（GCT）的KGN细胞表达NADPH氧化酶4（NOX4），这是HO的一个重要来源。瞬时受体电位香草酸亚型2（TRPM2）通道是一种Ca²⁺通透性阳离子通道，可被HO激活，并在细胞功能中起重要作用。它还能够促进细胞对死亡的易感性。我们研究了TRPM2在KGN细胞中的表达和功能，并检测了GCT组织芯片（TMA）以探索其体内相关性。我们采用了活细胞、钙和线粒体成像、活力测定、荧光激活细胞分选（FACS）分析、蛋白质免疫印迹和免疫组织化学。我们证实KGN细胞产生HO，并发现它们表达功能性TRPM2。HO增加细胞内Ca²⁺水平，而TRPM2抑制剂N-（对戊基肉桂酰基）邻氨基苯甲酸（ACA）可阻断这一作用。HO导致线粒体碎片化和凋亡性细胞死亡，而清除剂（生育三烯酚）可减轻这种情况。免疫组织化学显示，在所有检测的73个肿瘤样本中，NOX4和TRPM2平行表达。结果表明，GCT可能具有一种可传递细胞死亡易感性的系统。如果是这样，诱导氧化应激可能对GCT治疗有益。我们的结果还暗示TRPM2作为GCT的药物靶点具有治疗潜力。
