ENDOGENOUS MODULATION OF COCHLEA INJURY

描述(申请人提供)：顺铂是一种有效的化疗药物，广泛用于治疗各种恶性肿瘤患者。严重的副作用包括肾毒性、神经毒性和耳毒性，限制了可以使用的剂量。虽然在限制肾毒性方面取得了进展，但耳毒性仍在损害癌症幸存者的生活质量。本申请中概述的实验试图继续确定顺铂耳毒性的机制，以便找到合理的治疗方法，使疗效最大化和毒性最小化。建议的研究将利用从永生鼠耳蜗发展而来的毛细胞系，并结合活体动物实验。这些研究将系统地描述耳蜗中的一种关键酶-NADPH氧化的作用。它产生超氧化物和其他自由基，可以激活凋亡途径中的下游效应器，导致毛细胞死亡和听力丧失。来自当前支持期的数据表明，龙猫耳蜗中存在NADPH氧化，并且这种酶被声音创伤显著激活。用OC-K3细胞系的毛细胞进行的实验表明，这种酶存在于这些细胞中，并在顺铂暴露下被强烈激活。这项拟议的研究将阐明顺铂诱导NADPH氧化激活的方式，导致细胞凋亡的下游信号，以及内源性和外源性化合物保护耳蜗组织的相互作用。我们还将探索在顺铂暴露后，耳蜗中另外两种可能产生自由基的酶的作用：诱导型一氧化氮合酶和黄嘌呤氧化。这些研究将针对四个特定目标：1A)检测耳蜗组织中NADPH氧化的特点；1B)阐明顺铂激活耳蜗NADPH氧化的机制；2)检测腺苷A1受体(A1AR)激活对耳蜗腺苷A1受体(A1AR)激活的细胞保护作用；3)检测天然产物银杏叶(EGb 761)及其成分、萜类和藜芦醇的标准化提取物对顺铂所致耳毒性的保护机制；4)用P53抑制剂Pifitherin阐明顺铂激活P53在导致细胞死亡中的作用。实验将首先在毛细胞系中进行，然后通过使用局部和全身给药顺铂和保护剂的体内实验来证实。毛细胞将通过免疫细胞化学和蛋白质印迹检测iNOS和蛋白激酶C的自由基产生酶，检测NADPH氧化的亚基的RT-PCR，以及检测黄烯氧化物活性的荧光法。核因子-kappaB的检测将使用电泳迁移率改变分析，组织切片上A1 AR、t-Bid、Bax、Bcl2、细胞色素C、caspase-3和Annexin V的免疫细胞化学染色，并使用毛细胞系的流式细胞术。这些实验结果将为顺铂耳毒性的机制提供新的见解，并为化学预防提供新的方法。

DESCRIPTION (provided by applicant): Cisplatin is a potent chemotherapeutic agent widely used to treat patients with a variety of malignant neoplasms. Severe side effects including nephrotoxicity, neurotoxicity and ototoxicity limit doses that can be used. Although progress has been made to limit nephrotoxicity, ototoxicity continues to compromise the quality of life of cancer survivors. Experiments outlined in this application seek to continue to define the mechanisms of cisplatin ototoxicity in order to find rational therapeutic approaches to maximizing efficacy and minimizing toxicity. Studies proposed will utilize hair cell lines developed from the Immortomouse cochlea in combination with live animal experiments. These investigations will systematically characterize the role of a key enzyme in the cochlea, NADPH oxidize. It generates super oxide and other free radicals that can activate downstream effectors in the apoptotic pathway, leading to hair cell death and hearing loss. Data from the current period of support demonstrate the presence of NADPH oxidizes in the chinchilla cochlea, and that this enzyme is dramatically activated by acoustic trauma. Experiments with hair cells of the OC-k3 cell line show that the enzyme is present in these cells and is strongly activated by cisplatin exposure. The proposed research will elucidate the modes of cisplatin-induced activation of NADPH oxidize, downstream signaling which lead to apoptosis and the interaction of endogenous and exogenous compounds that protect the cochlea. We will also explore the roles of two other enzymes that could generate free radicals in the cochlea following cisplatin exposure: inducible nitric oxide synthase and xanthine oxidize. These investigations will address four specific aims: 1A) to examine the characteristics of NADPH oxidize in cochlear tissues; 1B) to elucidate the mechanisms of activation of cochlear NADPH oxidize by cisplatin; 2) to examine the cytoprotective effect of adenosine A1receptor (A1AR) activation in the cochlea; 3) to examine the mechanisms of protection against cisplatin-induced ototoxicity by the standardized extract of the natural product, Gingko biloba (Egb 761) and its components, the terrenes and falconoid; 4) to elucidate the role of p53 activation by cisplatin in causing hair cell death using the p53 inhibitor pifithrin. Experiments will be carried out in hair cell lines initially, and then confirmed by in vivo experiments using local and systemic administration of cisplatin and protective agents. Hair cells will be tested for free radical generating enzymes using immunocytochemistry and Western blotting for INOS and protein kinas C, RT-PCR for the subunits of NADPH oxidize, and Lucien assay for xanthenes oxides activity. Assays for NF-kappaB will be performed using the electrophoretic mobility shift assay, lmmunocytochemical staining for A1 AR, t-BID, Bax, Bcl-2, cytochrome C, caspase- 3, and Annexin V on tissue sections and using flow cytometry with the hair cell lines. The results of these experiments should provide novel insights into the mechanisms of cisplatin-induced ototoxicity and new methods for chemoprevention.