Targeting nitrative stress for treatment of cisplatin ototoxicity

摘要 在理解硝化应激如何有效地抑制硝化应激方面存在关键差距 在其他模型中，细胞死亡改变了耳蜗蛋白信号，从而在顺铂诱导的耳毒性中诱导细胞凋亡。 这一缺口的持续存在对40%-80%接受顺铂治疗的癌症来说是一个重要的问题 因使用顺铂而遭受严重和在某些情况下永久性听力损失的患者。直到 潜在的硝化应激机制被描绘为这一新的干预靶点的前景 减轻顺铂的剂量限制副作用可能仍未实现。我们的长期目标是更好地 了解耳蜗硝化应激在获得性听力损失中的功能和机制作用。这个 目的是描述顺铂诱导的硝化应激，特别是硝化的信号通路。 耳蜗LMO4，促进耳毒性，因为顺铂处理硝酸盐和下调LMO4蛋白。 LMO4是一种转录调节因子，控制调节细胞存活和细胞死亡的途径。中环 假设顺铂诱导的硝化应激下调耳蜗LMO4并损害STAT3- 介导抗细胞凋亡信号以促进耳毒性。了解硝化的机理 耳蜗LMO4促进顺铂诱导的耳毒性可能有助于制定策略 防止这种令人衰弱的不利影响。在强有力的初步数据的指导下，这项研究将追求三个具体的 目的：(1)建立顺铂诱导的LMO4硝化与耳毒性之间的因果联系；(2)确定 顺铂诱导的LMO4硝化对JAK/STAT信号的影响；以及(3)确定耳保护效果 药物对硝化的抑制作用。在目标1中，将分析阻断后顺铂诱导的细胞凋亡。 定点突变法硝化LMO4及其抑制蛋白酶体降解的研究 Lactacystin。LMO4蛋白水平和顺铂引起的耳毒性之间的联系将通过以下方式确定 检测LMO4基因敲除和过度表达小鼠的耳蜗细胞凋亡/听力损失。在目标2中，顺铂- 将使用质谱仪分析耳蜗LMO4蛋白质-蛋白质相互作用的诱导变化- 基于蛋白质组学的方法，同时将分析JAK/STAT相关的凋亡和炎症信号 使用靶向基因阵列。在目标3中，SRI110的耳保护效果，一种过氧亚硝酸盐的分解 催化剂，将使用CBA/J小鼠进行评估；SRI110对其抗癌活性的潜在干扰 顺铂将使用SCID小鼠进行分析。这项创新性的研究改变了现状 从氧化应激到硝化应激在顺铂耳毒性中的关键作用。值得注意的是，结果 有望在垂直方向上促进对硝化应激如何调节耳蜗细胞凋亡的理解 顺铂致耳毒性。这些发现将在缓解顺铂方面具有重要的翻译应用- 在硝化应激起关键作用的情况下，可用于诱发听力损失和预防其他耳部疾病。

Abstract A critical gap exists in understanding how nitrative stress, which has been effectively targeted to inhibit cell death in other models, alters cochlear protein signaling to induce apoptosis in cisplatin-induced ototoxicity. Continued existence of this gap represents an important problem for the 40%-80% of cisplatin-treated cancer patients who suffer with significant and in some cases permanent hearing loss as a result of cisplatin use. Until the underlying nitrative stress mechanism is delineated the promise of this new interventional target for mitigating a dose-limiting side-effect of cisplatin likely will remain unrealized. The long-term goal is to better understand the functional as well as mechanistic role of cochlear nitrative stress in acquired hearing loss. The objective is to delineate signaling pathways by which cisplatin-induced nitrative stress, particularly nitration of cochlear LMO4, facilitates ototoxicity, because cisplatin treatment nitrates and downregulates LMO4 protein. LMO4 is a transcriptional regulator that controls pathways regulating cell survival and cell death. The central hypothesis is that cisplatin-induced nitrative stress downregulates cochlear LMO4 and compromises STAT3- mediated anti-apoptotic signaling to facilitate ototoxicity. Understanding the mechanisms whereby nitrated cochlear LMO4 promotes cisplatin-induced ototoxicity is likely to contribute to the development of strategies to prevent this debilitating adverse effect. Guided by strong preliminary data this study will pursue three specific aims: (1) establish the causal link between cisplatin-induced LMO4 nitration and ototoxicity; (2) determine the effects of cisplatin-induced LMO4 nitration on JAK/STAT signaling; and (3) determine the otoprotective efficacy of pharmacological inhibition of nitration. In Aim 1, cisplatin-induced apoptosis will be analyzed after blocking nitration of LMO4 by site-directed mutagenesis and inhibiting proteasomal degradation of nitrated-LMO4 by lactacystin. The link between LMO4 protein levels and cisplatin-induced ototoxicity will be ascertained by testing cochlear apoptosis/hearing loss in LMO4 knockout and overexpressing mice. In Aim 2, cisplatin- induced changes in protein-protein interactions of cochlear LMO4 will be analyzed using a mass spectrometry- based proteomics approach while JAK/STAT related apoptotic and inflammatory signaling will be analyzed using targeted gene arrays. In Aim 3, the otoprotective efficacy of SRI110, a peroxynitrite decomposition catalyst, will be assessed using CBA/J mice; potential interference of SRI110 with anti-cancer activity of cisplatin will be analyzed using SCID mice. This innovative research departs from the status quo by shifting the focus from oxidative stress to the pivotal role of nitrative stress in cisplatin ototoxicity. Significantly, outcomes are expected to vertically advance understanding of how nitrative stress regulates cochlear apoptosis in cisplatin-induced ototoxicity. Findings will have important translational applications in mitigating cisplatin- induced hearing loss and preventing other otopathologies where nitrative stress plays a crucial role.