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.
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