Ghahremani Fatemeh, Sabbaghzadeh Reihaneh, Ebrahimi Safieh, Javid Hosein, Ghahremani Javad, Hashemy Seyed Isaac
Department of Biology, Faculty of Science, Hakim Sabzevari University, Sabzevar, Iran.
Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Iran J Basic Med Sci. 2021 Apr;24(4):499-505. doi: 10.22038/ijbms.2021.52902.11945.
Glioblastoma multiforme (GBM), a highly aggressive Grade IV brain tumor, is a significant public health issue due to its poor prognosis and incurability. Neuropeptide substance P (SP) plays a critical role in GBM tumor growth and development via activation of neurokinin-1receptor (NK1R). Moreover, SP is a pro-oxidant factor contributing to oxidative stress in various cell types. However, the link between SP and oxidative stress in cancer cells is not fully investigated. Here, we aimed to identify the effects of SP and NK1R antagonist, aprepitant, on the redox status of GBM cells.
Resazurin assay was employed to determine the effect of aprepitant on viability of U87 glioblastoma cells. 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) assay was employed to measure the levels of intracellular reactive oxygen species (ROS). A quantitative real-time polymerase chain reaction was applied to measure the expression of proteins of the thioredoxin system. Commercial kits (ZellBio GmbH) were also used to measure the enzymatic activity of these proteins.
We found that SP increased ROS level in U87 GBM cells, and aprepitant significantly reduced this effect. Furthermore, we found that SP could also affect the thioredoxin system, a central antioxidant enzyme defense system. SP reduced both expression and enzymatic activity of the thioredoxin system's proteins, Trx and thioredoxin reductase (TrxR) and these effects were significantly reduced by aprepitant.
Our results indicated that SP activation of NK1R represented a link between oxidative stress and GBM and highlighted the need for further validations in future studies.
多形性胶质母细胞瘤（GBM）是一种极具侵袭性的IV级脑肿瘤，因其预后差且无法治愈，成为一个重大的公共卫生问题。神经肽P物质（SP）通过激活神经激肽-1受体（NK1R）在GBM肿瘤的生长和发展中起关键作用。此外，SP是一种促氧化因子，可导致多种细胞类型发生氧化应激。然而，SP与癌细胞氧化应激之间的联系尚未得到充分研究。在此，我们旨在确定SP和NK1R拮抗剂阿瑞匹坦对GBM细胞氧化还原状态的影响。
采用刃天青试验来确定阿瑞匹坦对U87胶质母细胞瘤细胞活力的影响。采用2',7'-二氯二氢荧光素二乙酸酯（H2DCFDA）试验来测量细胞内活性氧（ROS）水平。应用定量实时聚合酶链反应来测量硫氧还蛋白系统蛋白质的表达。还使用商业试剂盒（ZellBio GmbH）来测量这些蛋白质的酶活性。
我们发现SP增加了U87 GBM细胞中的ROS水平，而阿瑞匹坦显著降低了这种作用。此外，我们发现SP还可影响硫氧还蛋白系统，这是一种核心抗氧化酶防御系统。SP降低了硫氧还蛋白系统蛋白质Trx和硫氧还蛋白还原酶（TrxR）的表达及酶活性，而阿瑞匹坦显著降低了这些作用。
我们的结果表明，SP对NK1R的激活代表了氧化应激与GBM之间的联系，并强调了在未来研究中进一步验证的必要性。
