Proteomic analysis of the balance between survival and cell death responses in cisplatin-mediated ototoxicity

Abstract

Cisplatin, a widely used anticancer drug, preferentially damages outer hair cells (OHCs) of the inner ear. In this study, an antibody microarray was used to identify early changes in protein expression in the rat cochlea induced by cisplatin. Only small changes in hearing thresholds (4-34 dB elevation) were detected two days after cisplatin treatment (12 mg/kg). OHC function, measured by otoacoustic emissions, was slightly depressed (10 dB), and little or no receptor cell loss was observed. However, cisplatin induced large changes in the expression of 19 proteins involved in apoptosis, cell survival, or progression through the cell cycle. Fifteen of the proteins are novel to the study of the inner ear. Immunoblotting confirmed increases in the levels of the pro-survival activating transcription factor 2 (ATF2), of pro-apoptotic serine-threonine protein kinase, receptor interacting protein, and a 70/75 kDa nitrotyrosine bearing doublet of unknown function. Anti-nitrotyrosine antibodies localized these oxidatively damaged proteins to the stereocilia of OHCs, the Golgi-centrosome region of Hensen's cells, nuclei of outer pillar cells, and tunnel crossing fibers innervating OHCs. The results of this proteomic analysis reflect the commencement of ototoxic and cell survival responses before the observation of a significant functional or anatomical loss.

Figure 1

Hearing threshold after cisplatin treatment. The ABR recorded before and 48 h after cisplatin treatment shows threshold shifts ranging from 10−34 dB in (A) Wistar and 4−12 dB in (B) Sprague—Dawley rats at 2.5, 5, 10, 20, 40 kHz and clicks. Measurements were done for n = 6 rats for the pretreatment group and n = 3 for the cisplatin treated group. Results are expressed as mean ± standard deviation (SD).

Figure 2

Cisplatin-induced DPOAE changes in Wistar rats. DPOAEs showed a decrease of approximately 10 dB at 8 and 16 kHz in both (A) Wistar and (B) Sprague—Dawley rats 48 h after cisplatin treatment. The pre-NF and post-cis-NF traces indicate the noise floor in DPOAE recordings before and after treatment with cisplatin. The measurements were done in n = 6 rats for the pretreatment group and n = 3 for the cisplatin treated group. The results are expressed as mean ± SD.

Figure 3

Hair cell loss 48 h after cisplatin treatment. Cochleograms indicated that 12 mg/kg cisplatin induced minimal or no hair cell lossat48hpost-treatmentinboth(A)Wistarand(B)Sprague—Dawley rats. The cochleograms from the right ear are given though the left ears also indicated a similar pattern (data not shown).

Figure 4

Antibody microarray of cisplatin-induced changes in cochlea. For array # 1, control proteins from Sprague—Dawley rats were labeled with Cy3 and proteins from cisplatin-treated rats with Cy5. For array # 2, the labeling dyes were swapped (controls labeled with Cy5 and cisplatin-treated with Cy3). Green spots indicate Cy5-labeled proteins outnumber Cy3-labeled proteins. Red spots indicate Cy-3 labeled proteins dominate. Similar results were obtained for samples from Wistar rats (figure not shown).

Figure 5

Cochlear protein expression profile after cisplatin treatment. (A) Wistar and (B) Sprague—Dawley: plots of number of proteins (ordinate) versus fold changes in protein expression. Values greater than 1 indicate an increase in protein level in cisplatin treated group versus control. Values less than 1 reflect a decrease in protein expression. The pattern of distribution in terms of the number of proteins that increased or decreased after cisplatin treatment was similar in both strains. Cut-off lines at fold changes of 0.6 and 1.5 indicate the selection criteria for further protein analysis.

Figure 6

Validation of protein expression by immunoblotting; 60 μg of protein from Sprague—Dawley cochleae were loaded in each lane for control or cisplatin-treated rats. Immunoblotting results indicate cisplatin-dependent increases in the expression of (A) ATF2, (B) RIP, and (C) nitrotyrosine, whereas (D) shows the expression of actin. Numbers give apparent molecular weight in kDa.

Figure 7

Cisplatin-induced localization of nitrotyrosine in cochlea. Images, obtained using confocal microscopy, indicate the presence of nitrotyrosine in stereocilia (S), Hensen's cells (H), outer pillar cell nuclei (P), and tunnel crossing fibers (F). Sprague—Dawley sensory epithelium was labeled with anti-nitrotyrosine, 48 h after cisplatin treatment. Red staining indicates nitrotyrosine, green indicates f-actin, and blue indicates nuclei. The confocal sections are at the level of (A) the stereocilia, (B) outer hair cell nuclei, and (C) Deiters' and pillar cell nuclei. The negative control (D), labeled with a different primary, rules out nonspecific labeling of the stereocilia by the secondary antibody.