Youn Cha Kyung, Kim Jun, Jo Eu-Ri, Oh Jeonghyun, Do Nam Yong, Cho Sung Il
Department of Otolaryngology-Head and Neck Surgery, Chosun University School of Medicine, Gwangju 61453, Korea.
Division of Natural Medical Science, Chosun University School of Medicine, Gwangju 61452, Korea.
Int J Mol Sci. 2016 Nov 18;17(11):1931. doi: 10.3390/ijms17111931.
One of the major adverse effects of cisplatin chemotherapy is hearing loss. Cisplatin-induced ototoxicity hampers treatment because it often necessitates dose reduction, which decreases cisplatin efficacy. This study was performed to investigate the effect of Tempol on cisplatin-induced ototoxicity in an auditory cell line, House Ear Institute-Organ of Corti 1 (HEI-OC1). Cultured HEI-OC1 cells were exposed to 30 μM cisplatin for 24 h with or without a 2 h pre-treatment with Tempol. Cell viability was determined using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and apoptotic cells were identified using terminal deoxynucleotidyl transferase dUTP nick end labeling of nuclei (TUNEL) assay and flow cytometry. The effects of Tempol on cisplatin-induced cleaved poly(ADP-ribose) polymerase, cleaved caspase, and mitochondrial inducible nitric oxide synthase expression were evaluated using western blot analysis. Levels of intracellular reactive oxygen species (ROS) were measured to assess the effects of Tempol on cisplatin-induced ROS accumulation. Mitochondria were evaluated by confocal microscopy, and the mitochondrial membrane potential was measured to investigate whether Tempol protected against cisplatin-induced mitochondrial dysfunction. Cisplatin treatment decreased cell viability, and increased apoptotic features and markers, ROS accumulation, and mitochondrial dysfunction. Tempol pre-treatment before cisplatin exposure significantly inhibited all these cisplatin-induced effects. These results demonstrate that Tempol inhibits cisplatin-induced cytotoxicity in HEI-OC1, and could play a preventive role against cisplatin-induced ototoxicity.
顺铂化疗的主要不良反应之一是听力丧失。顺铂诱导的耳毒性会妨碍治疗，因为它常常需要减少剂量，这会降低顺铂的疗效。本研究旨在调查Tempol对听觉细胞系——House Ear Institute-Organ of Corti 1（HEI-OC1）中顺铂诱导的耳毒性的影响。将培养的HEI-OC1细胞暴露于30 μM顺铂中24小时，顺铂暴露前2小时分别给予或不给予Tempol预处理。使用3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐（MTT）法测定细胞活力，使用细胞核末端脱氧核苷酸转移酶dUTP缺口末端标记法（TUNEL）和流式细胞术鉴定凋亡细胞。使用蛋白质免疫印迹分析评估Tempol对顺铂诱导的裂解聚（ADP-核糖）聚合酶、裂解半胱天冬酶和线粒体诱导型一氧化氮合酶表达的影响。测量细胞内活性氧（ROS）水平，以评估Tempol对顺铂诱导的ROS积累的影响。通过共聚焦显微镜评估线粒体，并测量线粒体膜电位，以研究Tempol是否能预防顺铂诱导的线粒体功能障碍。顺铂处理降低了细胞活力，增加了凋亡特征和标志物、ROS积累以及线粒体功能障碍。在顺铂暴露前进行Tempol预处理可显著抑制所有这些顺铂诱导的效应。这些结果表明，Tempol可抑制顺铂诱导的HEI-OC1细胞毒性，并可能对顺铂诱导的耳毒性起到预防作用。
