Caspase Inhibitors, but Not C-Jun NH2-Terminal Kinase Inhibitor Treatment, Prevent Cisplatin-Induced Hearing Loss

[CANCER RESEARCH 64, 9217–9224, December 15, 2004]

Caspase Inhibitors, but not c-Jun NH2-Terminal Kinase Inhibitor Treatment, Prevent Cisplatin-Induced Hearing Loss

Jing Wang,1 Sabine Ladrech,1 Remy Pujol,1 Philippe Brabet,1 Thomas R. Van De Water,2 and Jean-Luc Puel1 1Institut National de la Sante´ et de la Recherche Medicale–UMR 583 and Universite´ de Montpellier 1, Physiopathologie et the´rapie des de´ficits sensoriels et moteurs, Montpellier, France; and 2University of Miami Ear Institute, Department of Otolaryngology, University of Miami School of Medicine, Miami, Florida

ABSTRACT MATERIALS AND METHODS

Cisplatin (CDDP) is a highly effective chemotherapeutic agent but with Animals. The care and use of animals followed the animal welfare guide- significant ototoxic side effects. Apoptosis is an important mechanism of lines of the Institut National de la Sante´et de la Recherche Medicale and was cochlear hair cell loss following exposure to an ototoxic level of CDDP. approved by the French Ministe`re de l’Agriculture et de la Foreˆt. Adult female, This study examines intracellular pathways involved in hair cell death pigmented guinea pigs (250 to 300 g; Charles River Laboratories, Wilmington, induced by CDDP exposure in vivo to develop effective therapeutic strat- MA) were used in this study. egies to protect the auditory receptor from CDDP-initiated hearing loss. Preparation of Otoprotective Molecules for Perfusion. The specific in- Guinea pigs were treated with systemic administration of CDDP. Cochlear hibitors of caspase-3 (z-DEVD-fmk), caspase-8 (z-IETD-fmk), caspase-9 (z- hair cells from CDDP-treated animals exhibited classic apoptotic alter- LEHD-fmk), and cathepsin (z-FA-fmk; Calbiochem, La Jolla, CA) were 83- ations in their morphology. Several important signaling events that reg- mmol/L, 85-mmol/L, 81-mmol/L, and 72-mmol/L stock solution, respectively, ulate the death of CDDP-injured cochlear hair cells were identified. CDDP in 100% DMSO (Merck, Whitehouse Station, NJ), and D-JNKI-1 was a treatment induced the activation and redistribution of cytosolic Bax and 1-mmol/L stock solution in 0.1 mol/L PBS at pH 7.2. the release of cytochrome c from injured mitochondria. Activation of Before perfusion, stock solutions of the putative otoprotective compounds caspase-9 and caspase-3, but not caspase-8, was detected after treatment were diluted in artificial perilymph to final concentrations of 100 ␮mol/L for with CDDP, and the cleavage of fodrin by activated caspase-3 was ob- caspases and cathepsin inhibitor and 10 ␮mol/L for D-JNKI-1. Before surgery, served within damaged hair cells. Intracochlear perfusions with caspase-3 the osmotic minipump (1 ␮L/h, over 7 days; Alza Corp., Palo Alto, CA) was inhibitor (z-DEVD-fmk) and caspase-9 inhibitor (z-LEHD-fmk) prevent filled under sterile conditions with 200 ␮L of artificial perilymph or artificial hearing loss and loss of sensory cells, but caspase-8 inhibitor (z-IETD- perilymph containing one of the putative otoprotective agents. fmk) and cathepsin B inhibitor (z-FA-fmk) do not. Although the stress- Minipump Implantation and Electrophysiologic Recordings. Experi- activated protein kinase/c-Jun NH2-terminal kinase (JNK) signaling path- ments were designed to record compound action potential (CAP) in awake way is activated in response to CDDP toxicity, intracochlear perfusion of animals from a plug fixed on the head of the animal during minipump D-JNKI-1, a JNK inhibitor, did not protect against CDDP ototoxicity but implantation. Briefly, the animals were anesthetized with an intraperitoneal instead potentiated the ototoxic effects of CDDP. The results of the present injection of a 6% solution of sodium pentobarbital (Sanofi, New York, NY; study show that blocking a critical step in apoptosis may be a useful dose, 0.3 mL/kg). Each bulla was opened under sterile conditions, and a strategy to prevent harmful side effects of CDDP ototoxicity in patients recording electrode was positioned on the round window membrane. A hole having to undergo chemotherapy. 0.25 mm in diameter was gently drilled in the base of the right cochlea for installation of the perfusion catheter. INTRODUCTION CAPs of the auditory nerve were elicited by tone bursts of alternating phase (1-ms increase/decrease; 8-ms duration) and applied to the ear at a rate of 10/s Cisplatin (cis-diamine-dichloroplatinum II; CDDP) is a highly ef- from 0 to 100 dB sound pressure level in 5-dB steps in a free field via a JBL fective and widely used anticancer agent (1). The risk of ototoxic and 075 earphone (JBL, Northridge, CA). Cochlear responses were amplified nephrotoxic side effects commonly hinders the use of higher doses (gain, 2000), averaged (128 samples), and stored on a Pentium PC computer that could maximize its antineoplastic effects (2). CDDP has been operating at 100 MHz (Dell Dimension; Dell, Austin, TX). CAP recordings shown to induce auditory sensory cell apoptosis in vitro (3, 4) and in were measured peak to peak between the negative depression N1 and the vivo (5, 6). Devarajan et al. (7) recently have reported CDDP-induced subsequent positive wave P1. The threshold of the CAP was defined as the apoptosis in an immortalized cochlear cell line. In this model, CDDP intensity in decibel sound pressure level needed to elicit a measurable response (Ն5 ␮V). toxicity was associated with an increase in caspase-8 activity, fol- Terminal Deoxynucleotidyl Transferase-Mediated Nick End Labeling lowed by truncation of Bid, translocation of Bax, release of cyto- Assay. ApopTag in situ apoptosis detection kit (Intergen, Purchase, NY) was chrome c, and activation of caspase-9. This suggests that death re- used. The incorporated digoxigenin nucleotides were immunostained with a ceptor and mitochondrial pathways are involved in CDDP-induced FITC-conjugated antidigoxigenin antibody. The tissues were counterstained apoptosis of hair cells. However, results obtained in vitro may not be with propidium iodide (PI). FITC and PI fluorescent signals were observed directly applicable in vivo. with an MRC 1024 laser scanning confocal microscope (Bio-Rad, Hercules, The present study explores the mechanisms of CDDP-induced CA). ototoxicity in vivo and develops an efficacious strategy for protection Hoechst Staining. Cell nuclei morphology was assessed by Hoechst 33342 against CDDP-induced hearing loss. In addition to death receptor and dye (Sigma Chemical Co., St. Louis, MO). Deparaffinized cochlear sections were stained with a 1:100 dilution of rhodamine-conjugated phalloidin (R-415; mitochondrial pathways, we also examine the c-Jun NH2-terminal kinase (JNK) signaling pathway (8) on CDDP-induced apoptosis in Molecular Probes, Eugene, OR) for 1 hour at room temperature. The sections then were counterstained with Hoechst 33342 (0.002%, w/v) at room temper- cochlear sensory cells. ature for 10 minutes and examined on a Leica DMRB microscope (blocks of filters N2.1 and A4; Leica Microsystems, Inc., Wetzlar, Germany). Received 5/5/04; revised 10/6/04; accepted 10/11/04. Immunocytochemistry. Cytochrome c was detected in cryostat sections Grant support: Association pour la Recherche sur le Cancer (ARC) and Ligue Contre (10 ␮m) with mouse monoclonal anti–cytochrome c antibody (1:200; Pharm- le Cancer (Comite´de l’He´rault). The costs of publication of this article were defrayed in part by the payment of page ingen, San Jose, CA) and FITC-conjugated secondary antibody (1:200 dilu- charges. This article must therefore be hereby marked advertisement in accordance with tion; goat antimouse IgG antibody B13113C; BioSys, Ann Arbor, MI). Bax 18 U.S.C. Section 1734 solely to indicate this fact. was detected with rabbit polyclonal antibody to the NH2-terminus of Bax Requests for reprints: Jean-Luc Puel, INSERM U. 583, 80 rue Augustin Fliche, (1:750; Upstate Biotechnology, Lake Placid, NY) that recognizes only the 34295 Montpellier, France. Phone: 33-499-636-009; Fax: 33-499-636-020; E-mail: [email protected]. activated form of Bax and an Alexa 488–labeled secondary antibody (1:1000 ©2004 American Association for Cancer Research. dilution; goat antirabbit IgG antibody; Molecular Probes). Immunostained 9217

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2004 American Association for Cancer Research. CASPASE INHIBITORS PREVENT CDDP OTOTOXICITY specimens were counterstained with PI. The subcellular distribution of Bax Functional and Morphologic Assessment of Hair Cell Integrity. was confirmed by double labeling with two primary antibodies [i.e., a rabbit In the present study, we used two types of CDDP administration [i.e., polyclonal antibody to the NH2-terminus of Bax and a mouse monoclonal a single high dose of CDDP (10 mg/kg, intraperitoneally) and a antibody to mitochondrial heat shock protein (HSP) 70, the specific mitochon- multiple dose of CDDP (2 mg/kg/d, intraperitoneally, for 5 days)] that drial resident protein of the hsp70 family; clone, JG1; 1:500; Affinity BioRe- correspond to the high therapeutic doses of CDDP used to manage agents, Golden, CO]. Secondary antibodies were Alexa 488–labeled goat human cancers (12). When compared with multiple doses of CDDP, antirabbit antibody and Alexa 568–labeled goat antimouse antibody (1:500; a single high-dose administration resulted in a similar pattern of Molecular Probes). hearing loss that occurred more rapidly (i.e., 6 versus 3 days, respec- Activated caspase-3 and cleavage of fodrin were detected using a Tyramide tively) but remained stable for at least 6 days (Fig. 1A). To reduce the Signal Amplification kit (TSA Fluorescence System kit; NEN Life Science Products, Boston, MA). Cryostat sections were incubated with two primary animal mortality that results from single high-dose administration of antibodies [i.e., a monoclonal antibody against calbindin (1:600 dilution; CDDP, quantification of cell death of hair cells and long-term pro- Sigma) and a rabbit polyclonal antibody against active caspase-3 (1:6000; tection were studied after multiple-dose CDDP injections. Even with BioVision Incorporated, Mountain View, CA)] or a rabbit polyclonal antibody this precaution, 6 animals within the group of 42 animals treated with against cleaved ␣-fodrin (Asp1185). This latter antibody recognizes fodrin multiple doses of CDDP died during the course of treatment. Nine fragments that are cleaved exclusively by caspases (1:400; Cell Signaling, San animals within this multiple-dose group did not show a clear pattern Diego, CA). Secondary antibodies were biotinylated antirabbit antibody (1: of hearing loss. Because of this interindividual variability with respect 300; P.A.R.I.S., Compie`gne, France) and Cy3-conjugated goat antimouse IgG to CDDP-induced hearing loss, we ensured that CDDP ototoxicity had antibody (1:500; Jackson ImmunoResearch Labs, West Grove, PA). Fluores- occurred by recording CAP audiograms in each CDDP animal from cent tags were visualized with a confocal microscope (Bio-Rad). In control the nonperfused contralateral cochlea. Only animals with a hearing specimens without primary antibodies, neither FITC nor Cy3 fluorescent tags loss of Ն30 dB at 20 kHz by day 6 in this contralateral ear were were observed. included in this study (i.e., 27 of 42 animals). In vivo Substrate Detection of Activated Caspases. Caspase activity was In the contralateral ears, 3 days after the beginning of CDDP examined in vivo using the fluorescent substrates fam-LETD-fmk (caspase-8 treatment (day 3), only the highest frequencies tested (i.e., 16 to 26 substrate), fam-LEHD-fmk (caspase-9 substrate), and fam-DEVD-fmk kHz) showed measurable hearing losses. During the continuing course (caspase-3 substrate) obtained from Intergen. Fluorescence substrate (2.5ϫ) was perfused directly into the cochleae of untreated and CDDP-treated ani- of the CDDP treatment (i.e., between days 3 and 5), hearing losses mals. The method of perfusion used has been described previously (9). Briefly, continued to develop, progressing to the middle (i.e., 6 to 16 kHz) and Ͻ after the cochlea was exposed ventrally, a 1-hour perfusion of the scala then the lower ( 6 kHz) frequencies. One day after the end of tympani at 2 ␮L/min was performed through a hole made in the lateral treatment (day 6), significant hearing losses were observed across all cochlear wall and allowed to flow out of the cochlea through a hole in the scala of the frequencies, with higher frequencies being most affected (Fig. vestibuli. After the perfusion of the substrate, wash buffer was perfused for 1 1A). hour. Cochleae then were fixed with a final perfusion of fixative for 45 Comparison of surface preparations of untreated cochleae with minutes. The cochleae were removed and double labeled with rhodamine- cochleae subjected to systemic CDDP treatment (Fig. 1B) revealed conjugated phalloidin. that many of the outer hair cells (OHCs) were missing from the Western Blot Analyses. Tissue proteins were homogenized in sample CDDP-exposed cochleae. Counting of remaining hair cells was per- buffer (10) separated on 10% SDS-PAGE and transferred to nitrocellulose formed on surface preparations of the organ of Corti stained with a membranes. Blots were incubated overnight at 4°C with anti-JNK polyclonal rhodamine-phalloidin conjugate. These counts showed a large loss of antibody or anti–phosphoThr183/Tyr185-JNK polyclonal antibody (1:1000; OHCs from the basal turns of the CDDP-treated, nonperfused con- Cell Signaling), anti–c-Jun polyclonal antibody, or anti–phosphoSer73-Jun tralateral cochleae (n ϭ 4), with a typical gradation of base to apex polyclonal antibody (1:1000; Upstate Biotechnology), followed by incubation pattern of hair cell loss and a typical gradient of OHC loss that with peroxidase-conjugated secondary antibody (Roche Applied Science, In- dianapolis, IN). Protein-antibody complexes were revealed with the Western progressed from the first to the third row of OHCs (Fig. 1A, inset). A Lightning Chemiluminescence Reagent (PerkinElmer Inc., Wellesley, MA). few areas of inner hair cell (IHC) loss were observed located in the Image scanning of Western blot analyses was used to quantify phosphorylation most basal portion of CDDP-treated cochleae (Fig. 1A, inset). and expression levels of JNK and c-Jun. DNA Fragmentation and Nuclear Morphology. To determine Transmission Electron Microscopy Assessment of Hair Cell Integrity. the nature of cell death, we performed terminal deoxynucleotidyl The transmission electron microscopy procedure has been described previ- transferase-mediated nick end labeling (TUNEL) on untreated (n ϭ 2) ously (11). Briefly, animals were decapitated during deep anesthesia, and their and CDDP-treated (n ϭ 6) cochleae. TUNEL-positive cells were not cochleae were prepared using our standard protocol. Semithin and ultrathin observed in any of the cochleae from untreated control animals (Fig. radial sections of the plastic embedded organ of Corti were cut from the basal 1C). In contrast, the basal part of cochleae from CDDP-treated ani- and middle turns and observed using a Hitachi 7100 electron microscope mals showed TUNEL-positive labeling of a variety of cell types (Hitachi, Tokyo, Japan) at Centre Re´gional d’Imagerie Cellulaire de Montpel- within the organ of Corti, especially OHCs and occasionally IHCs and lier. support cells (Fig. 1C). Ϯ Statistics. All of the quantitative results are expressed as mean SE. All Cell nuclei within sensory cells of cochleae from untreated control of the statistics were calculated using Sigmaplot 2000 for Windows (version animals stained uniformly with Hoechst dye (Fig. 1D). After CDDP 6.1; Microsoft, Redmond, WA). All of the comparisons between means were treatment, characteristic pathologic changes in the nuclear staining performed using Student’s paired two-tail t tests or a nonparametric Wilcoxon rank test. were observed in the basal and middle turns of cochleae (i.e., the appearance of karyopyknosis in the nuclei of some OHCs and IHCs; Fig. 1D). RESULTS Bax Redistribution. We examined the activation and subcellular localization of Bax by using an antibody directed to the NH2-terminus This study examined the mechanisms by which CDDP damage of Bax. Cochleae from untreated control animals (n ϭ 2) exhibited a induces apoptosis of affected hair cells and evaluated the otoprotec- diffuse, pattern of light immunostaining barely above a background tive effects of five protective molecules on preservation of structural level, indicating an absence of Bax activation (Fig. 2A). Two to 4 integrity and function in CDDP-treated guinea pig cochleae. hours after a single high-dose injection of CDDP (n ϭ 3), the pattern 9218

Fig. 1. A, averaged CAP audiograms after single dose and multiple doses of CDDP. On day 3, the single-dose CDDP treatment induces a similar pattern of hearing loss as the multiple dose administration of CDDP (day 6). Inset, A cytocochleogram derived from nonperfused cochleae of day 6 CDDP-treated animals (n ϭ 4) shows extensive loss of OHCs in all three rows in the basal region of the cochlea. B, surface preparations of organs of Corti stained with rhodamine-labeled phalloidin (red). All of the OHCs (O) and IHCs (I) are present in the untreated control cochlea (p ϭ pillar cells). In contrast, only three OHCs (curved white arrows) remain in the area viewed of this CDDP-damaged cochlea. C, TUNEL staining of transverse tissue sections. DNA strand breaks are stained fluorescent green by FITC-labeled dUTP; nuclei are counterstained red with PI; TUNEL-labeled nuclei appear yellow. Note the TUNEL-positive nuclei present in three OHCs and a single IHC (white arrows) and in several supporting cells in CDDP-treated specimen. D, staining of nuclear chromatin with Hoechst 33342 dye. In untreated control cochleae, all of the hair cells have a normal uniform pattern of nuclear chromatin staining. In CDDP-treated cochleae, OHCs show shrunk, pyknotic nuclei (white arrows). In both insets, representative Hoechst- stained nuclei are shown. Scale bars: B, 15 ␮m, C and D, 20 ␮m, insets, 2.5 ␮m.

of Bax immunostaining of the OHCs of the basal and middle cochlear noted at 6 hours after CDDP treatment (Fig. 4B; n ϭ 9). Similarly, a turns changed to a dense, punctate pattern of immunostaining con- slight but not significant increase (P Ͼ 0.05) of caspase-8–positive sistent with the activation and redistribution of Bax from the cytosol cells was noted at 12 hours (Fig. 4A and B; n ϭ 3). In contrast, to the mitochondria of affected sensory cells (Fig. 2A). Double label- high-dose CDDP-exposed cochleae at 12 hours after treatment ing of cells showed that activation of Bax colocalized with mt HSP 70, showed a robust and significant increase in the number of caspase- the specific mitochondrial resident protein of the hsp70 family, in 9–positive OHCs (Fig. 4A and B; P Ͻ 0.001; n ϭ 3), whereas the treated animals but not in control untreated cochleae (Fig. 2C). proportion of caspase-9–positive IHCs remained approximately the Release of Cytochrome c. Subcellular localization of cytochrome same as measured at 6 hours. Finally, a significantly increased per- c was examined by immunostaining. In the cochleae of untreated, centage of caspase-3–positive IHCs and OHCs was evident at 12 control animals (n ϭ 2), immunolabeling with an anti–cytochrome c hours after high-dose CDDP treatment (Fig. 4A and B; P Ͻ 0.01; antibody showed an unequal, punctate distribution of reaction product n ϭ 3). within the cytoplasm (Fig. 3A). This pattern of immunostaining is Caspase-3 activation also was determined by immunolabeling using consistent with mitochondrial localization of cytochrome c. Six hours an antibody specific for activated caspase-3. Activated caspase-3 was after a single, high-dose injection of CDDP (n ϭ 3), the pattern of hair never detected in the cochlear cells from untreated control animals cell and support cell cytoplasm immunostaining for cytochrome c (Fig. 4C; n ϭ 3). By contrast, marked caspase-3 activation was changed from punctate to a diffuse and weak pattern of immuno- observed in some OHCs and a few IHCs of the basal and middle turns staining with reaction product uniformly distributed throughout the (Fig. 4C; n ϭ 3) of CDDP-treated animals at 12 hours after high-dose cytoplasm of the affected cells (Fig. 3B). The pattern of immuno- CDDP. staining in the CDDP specimens was no longer consistent with mi- Cleavage of Fodrin by Caspases. Using an antibody specific for tochondrial localization of cytochrome c in the auditory hair cells and the Mr 150,000 NH2-terminal fragment of cleaved fodrin, we looked the organ of Corti support cells. for the presence of cleaved fodrin in high-dose CDDP-exposed coch- Specific Caspases. Activation of caspases was examined using lear cells. Cleaved fodrin was never detected in the cochlear cells from fluorescent substrate detection tests in untreated control cochleae and untreated control animals (Fig. 4D; n ϭ 2). In contrast, there was cochleae observed at 6 and 12 hours after a single high-dose injection marked immunostaining for cleaved fodrin in the region of the cutic- of CDDP. In untreated control cochleae (n ϭ 6; Fig. 4A), few hair ular plates of some OHCs and a few IHCs located in the basal and cells contained the green fluorescent labeling that indicated cleaved middle cochlear turns of CDDP-treated animals observed between 6 caspase-8, -9, and -3 substrates. A slight but not significant increase and 48 hours after injection of a single high dose of CDDP (Fig. 4D; (P Ͼ 0.05) of caspase-8–, -9–, and -3–positive auditory hair cells was n ϭ 6). 9219

Fig. 2. Confocal images from transverse sections of the basal cochlear turns from organs of Corti labeled with either PI (red, in A and B) and antibody against Bax-NT (green, in A and B) or with antibody against HSP 70 (red, in C) and antibody against Bax-NT (green, in C). A. An untreated, control cochlea shows a diffuse and light pattern of Bax immunostaining of the organ of Corti. In contrast, 4 hours after a single injection of CDDP, an intense, punctate pattern of staining is seen with the Bax-NT antibody in the OHCs (white arrowhead). B. CDDP-treated cochlea perfused with z-DEVD-fmk or with D-JNKI-1 shows nearly the same pattern of Bax-NT immunostaining 4 hours after treatment as the CDDP- treated, unperfused cochlea (A, right). C. Double labeling with Bax and HSP 70 in an untreated control cochlea shows a punctate staining (red) for HSP 70 and a light, diffuse pattern for Bax (green). The same HSP 70 staining is seen in CDDP-treated cochlea. The dense and punctate Bax staining colocalizes with HSP 70 (yellow) in OHCs; tc, tunnel of Corti. Scale bars: 15 ␮minA and B and 10 ␮minC.

JNK Signaling Pathway. To identify involvement of the JNK CDDP treatment induced strong phosphorylation of JNK (p-JNK) first signal pathway in CDDP-induced apoptosis of cochlear hair cells in detected at 3 hours after treatment, which persisted through 48 hours, vivo, activation of JNK was measured by immunolabeling of Western with a peak level of p-JNK protein reached at 6 hours after injection. blot analyses obtained from untreated cochleae (n ϭ 4) and single In contrast, there was no change in the expression levels of total JNK high-dose (10 mg/kg) CDDP-treated cochleae removed at 3, 6, 12, 24, protein observed during the 48-hour course of CDDP treatment (Fig. and 48 hours after CDDP treatment (n ϭ 4 per time point). High-dose 5A). The level of p-JNK in CDDP-treated cochleae compared with

Fig. 3. Confocal images from transverse sections of the basal cochlear turns from organs of Corti labeled with PI (red) and antibody against cytochrome c (green,in A–D). A. An untreated, control cochlea shows an intense, punctate pattern of cytochrome c staining in the IHC and the OHCs. B. At 6 hours after treatment, the CDDP- exposed cochlea has a diffuse, pale pattern of immunostaining for cytochrome c. C and D. CDDP-treated cochlea perfused with z-DEVD-fmk (C) or with D-JNKI-1 (D) shows a similar diffuse pattern of cytochrome c staining 6 hours after treatment as the CDDP-treated, unperfused cochlea (B); tc, tunnel of Corti; bar, 15 ␮m.

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Fig. 4. A, confocal images of surface preparations of the basal turn from organs of Corti showing in vivo detection of activated caspases (green) in control (left) and CDDP-damaged (right) cochleae. Rhodamine-conjugated phalloidin (red) shows actin labeling in the cuticular plates and stereocilia of the hair cells. CDDP treatment causes no activation of caspase-8. By contrast, 12 hours after injection of CDDP, a robust activation of caspase-9 and -3 is seen in many OHCs and in some IHCs. B, quantitative data for caspase-8, -9, or -3 activation in hair cells from CDDP-exposed cochleae. The number of hair cells containing activated caspase-8, -9, or -3 is counted in the 1-mm length of basal turns of CDDP-exposed cochlear ducts for different time points (before and 6 and 12 hours after treatment). The results are expressed as the mean percentage of caspase-positive cells Ϯ SE (bars) for n Ͼ 4 independent ;P Ͻ 0.01 ,ءء) experiments. A significant increase of activated caspase-9 in OHCs and activated caspase-3 in the OHCs and IHCs appears at 12 hours after CDDP treatment P Ͻ 0.001). C, transverse sections of control and CDDP-treated organ of Corti, double labeled with calbindin (red) and with the antibody specific of the activated form of caspase-3 ,ءءء (green). Caspase-3 immunoreactivity (arrowheads) is seen 12 hours after CDDP treatment in the OHCs (O1,O2, and O3). D, transverse sections double labeled with calbindin (red) and the antibody specific of the cleaved form of ␣-fodrin (green). A robust immunostaining for the cleaved form of ␣-fodrin (green) is seen 48 hours after CDDP treatment in the cuticular plates (arrowheads) of the OHCs and the IHC; bars, A ϭ 20 ␮m, C and D ϭ 10 ␮m. untreated cochleae (Fig. 5A) reached a peak at 6 hours after high-dose after the onset of CDDP treatment (Fig. 6B; P Ͻ 0.001; n ϭ 3). CDDP exposure. Similar results were obtained for phospho-c-Jun Quantitative evaluation performed at the end of CDDP treatment (day (p-c-Jun) and expression levels of c-Jun proteins. A single high dose 6) of the z-DEVD-fmk–perfused cochleae revealed that there were of CDDP induced distinct bands of p-c-Jun labeling that increased in only a few OHCs lost from the basal turn and that there was no intensity during the 3- to 48-hour period following CDDP treatment. apparent damage to the middle or apical turns of these z-DEVD-fmk/ No change in the expression level of c-Jun protein was observed CDDP-treated cochleae (Fig. 6C; n ϭ 3). The otoprotective effect of during the course of CDDP treatment (Fig. 5B). The level of p-c-Jun z-DEVD-fmk against CDDP-induced ototoxicity was confirmed by in CDDP-treated cochleae compared with untreated cochleae (Fig. ultrastructural analysis of the organ of Corti (Fig. 6D), in which no 5B) reached a peak of expression at 12 hours after high-dose CDDP. degeneration-related cellular abnormalities were observed in CDDP- Caspase-3 Inhibitor II and Caspase-9 Inhibitor I, but not Ca- treated cochleae (n ϭ 2) protected by z-DEVD-fmk. thepsin B Inhibitor I and Caspase-8 Inhibitor III, Prevent Hear- Because z-DEVD-fmk also might inhibit cathepsins, we performed ing Loss and Loss of Sensory Cells. Substrate detection and immu- additional experiments using the cathepsin inhibitor (z-FA-fmk). No nolabeling results indicate that procaspase-9 and procaspase-3, but not protective effect against CDDP ototoxicity was observed with perfu- procaspase-8, were activated in the cochlear hair cells of CDDP- sion of 100 ␮mol/L z-FA-fmk (Fig. 6A; n ϭ 5), suggesting that the treated animals. To further investigate involvement of caspase-8 in protective effect of z-DEVD-fmk was caused by inhibition of caspase CDDP-induced apoptosis of cochlear cells, we perfused a caspase-8 activation rather than an effect on cathepsins. inhibitor III (z-IETD-fmk) into the scala tympani (n ϭ 5). There was A Peptide Inhibitor of JNK (D-JNKI-1) Potentiated the Oto- no protective effect on CDDP-induced hearing loss in animals treated toxic Action of CDDP. JNK and c-Jun activation increased over time with 100 ␮mol/L of z-IETD-fmk (Fig. 6A). The roles of caspase-9 and following exposure to high-dose CDDP treatment (Fig. 5A and B). caspase-3 were investigated by perfusing the caspase-9 inhibitor I Thus, we blocked the JNK pathway by using D-JNKI-1, a cell- (z-LEHD-fmk) and caspase-3 inhibitor II (z-DEVD-fmk) into the permeable peptide that competitively blocks the ability of JNK to scala tympani of CDDP-treated animals. Either 100 ␮mol/L of z- phosphorylate its targets. Intracochlear perfusion of 10 ␮mol/L D- LEHD-fmk (n ϭ 5; Fig. 6A)or100␮mol/L of z-DEVD-fmk (n ϭ 6; JNKI-1 solution did not rescue the cochlea from CDDP-initiated Fig. 6A) inhibitors prevented most of the CDDP-initiated hearing loss. ototoxic damage (n ϭ 6); rather, it potentiated CDDP-initiated hearing Although 100 ␮mol/L of z-DEVD-fmk prevented CDDP-induced loss between 10 and 26 kHz (Fig. 5C, left). Statistical analysis hearing loss, it prevented neither the subcellular redistribution of Bax revealed that the potentiation of CDDP-induced hearing loss by D- (Fig. 2B; n ϭ 2) nor the mitochondrial release of cytochrome c into JNKI-1 was significant at the frequencies of 20 and 26 kHz (P Ͻ 0.05) the cells’ cytoplasm (Fig. 3C; n ϭ 2). However, it did significantly when compared with CDDP-treated animals. Morphologic evaluation suppress the occurrence of TUNEL-positive hair cells within 48 hours of the organ of Corti indicated that the combination of D-JNKI-1 with 9221

Fig. 5. A and B, representative Western blot analyses (left) using antibodies to p-JNK and JNK (A) and p-c-Jun and c-Jun (B). Histograms (right) represent p-JNK/JNK (A) and p-c-Jun/c-Jun (B) ratios in CDDP-treated cochleae expressed relative to the same ratio in untreated cochleae (i.e., 0-hour specimens). Values are presented as mean Ϯ SD from three separate experiments. C, audiograms from D-JNKI-1–perfused right cochleae and nonperfused left cochleae in the same CDDP-treated animals (left; n ϭ 6). Hearing losses were similar between 2 and 10 kHz but were greater in the D-JNKI-1–perfused cochleae between 10 and 26 kHz (P Ͻ 0.05). Note the extensive loss of OHCs and of a few IHCs in the basal and middle cochlear turns (right; n ϭ 3). D, Transmission electron micrographs of the organ of Corti from the basal turn of a CDDP-treated cochlea perfused with D-JNKI-1. OHCs and the IHC are absent and have been replaced by expansions of their supporting cells, the Deiters’ cell (D1, D2, and D3) and the phalangeal (ph) and border (b) cells. The curved arrow points to an apoptotic residue; ip, inner pillar cell; bar, 5 ␮m.

CDDP treatment caused greater damage than CDDP treatment alone Death Receptor Pathway. One major signaling pathway that leads (Fig. 5C, right; n ϭ 3). The basal turns of the D-JNKI-1–perfused, to the apoptosis of damaged cells is the Fas/APO-1–dependent (cell CDDP-treated cochleae also lost a few IHCs. Perfusion with D- death receptor) pathway. Activated Fas/APO-1 receptors interact JNKI-1 prevented neither the subcellular redistribution of Bax (Fig. causing the activation of receptor-associated procaspase-8, which can 2B; n ϭ 2) nor the release of cytochrome c from the mitochondria activate downstream effector caspases (15). In contrast to the in vitro (Fig. 3D; n ϭ 2) that occurs following CDDP treatment. Ultrastruc- study showing a transient activation of caspase-8 in an immortalized ϭ tural examination showed that D-JNKI-1–perfused cochleae (n 2) cell line (7), the present study did not observe a significant increase in displayed extensive damage and degeneration of the hair cells and caspase-8 activation in CDDP-treated guinea pig cochleae. It is worth more extensive damage to other cochlear-related structures (Fig. 5D). noting that this immortalized auditory cell line represents OHC pre- The most frequent pathology observed within the somas of OHCs was cursors and not adult-like OHCs (7). Thus, the different patterns of large cytoplasmic vacuoles and chromatin condensations within their caspase-8 activation observed in vitro (7) and in our in vivo study nuclei, and a few OHCs showed degenerative changes indicative of might be caused by the differences in these two experimental models. cellular necrosis. Additional functional data reported in our study reveal that local scala tympani perfusion of z-IETD-fmk, a caspase-8 inhibitor, was ineffec- DISCUSSION tive in preventing either CDDP-induced hair cell death or hearing loss. The pattern of hair cell loss correlated with the electrophysiologic This result suggests that in vivo CDDP-induced apoptosis of cochlear determination of a high-frequency hearing loss. CDDP-induced apo- cells is caspase-8 independent. ptosis of OHCs, IHCs, and nonsensory cells of the organ of Corti as Mitochondrial Pathway. Another cell death pathway that partic- shown by TUNEL labeling, Hoechst staining, and ultrastructural ipates in apoptosis of stress-damaged hair cells is the mitochondria analysis correlates well with the findings of recent reports (5, 13, 14). pathway (16). Mitochondria are a target of reactive oxygen species Identification of intracellular cell death signaling pathways activated and a source for the generation of additional reactive oxygen species. in CDDP-stressed hair cells may offer the possibility of developing a In situ-generated reactive oxygen species can cause cytochrome c strategy for otoprotection against CDDP-induced hearing loss. release in primary cultures of cerebellar granule neurons (17). Cyto- 9222

Fig. 6. A, audiograms from perfused cochleae and contralateral, nonperfused cochleae in CDDP-treated animals. Hearing losses are similar between the z-IETD–perfused, z-FA–perfused cochleae, and contralateral, nonperfused cochleae. In contrast, only high-frequency hearing losses (i.e., between 20 and 26 kHz) are seen in the z-DEVD–perfused and z-LEHD ears; lower frequency hearing losses are totally prevented. B, counting of TUNEL-positive hair cells in a 1-mm length of surface preparation of the organ of Corti from control untreated (n ϭ 3) and 2 days after treatment in CDDP-treated (n ϭ 3) cochleae and z-DEVD–perfused, CDDP-treated cochleae (n ϭ 3). Note the significant increase of TUNEL-positive hair cells in the CDDP-exposed cochleae and the minimal number in the z-DEVD-fmk–perfused, -P Ͻ 0.001). C, a cytocochleo ,ءءء) CDDP-treated cochleae gram from z-DEVD-fmk–perfused cochleae (n ϭ 3). The cytocochleogram derived from z-DEVD-fmk–perfused, CDDP-treated cochleae shows only minimal OHC loss and no IHC loss. D, transmission electron micrographs from the basal turn of a z-DEVD-fmk–perfused cochlea from a CDDP- treated animal. The IHC (I) and the three OHCs (O1,O2, and O3) are well preserved, and their stereocilia (arrows) have a normal appearance. D1–3, Deiter’s cells; op, outer pillar cell; ip, inner pillar cell; ph, phalangeal cell; b, border cell; bar, 5 ␮m.

chrome c release into the cytosol is required for the formation of the vated JNK were measured. Our results show that CDDP treatment apoptosome and the resultant activation of procaspase-9. induced a significant increase in activated JNK and that inhibition of Consistent with other studies (3, 7, 18, 19), we show that an this signal pathway by scala tympani perfusion of D-JNKI-1, a cell- upstream initiator caspase (i.e., caspase-9) and a downstream effector permeable peptide that blocks JNK-mediated activation of c-Jun (26), caspase (i.e., caspase-3) were activated in the CDDP-damaged OHCs prevented neither the CDDP-initiated activation and subcellular re- and some IHCs located in the basal turn of the cochlea. The intraco- distribution of Bax nor the mitochondrial release of cytochrome c. chlear perfusion of caspase-3 inhibitor (z-DEVD-fmk) and caspase-9 Contrarily, inhibition of this signal cascade by D-JNKI-1 increased the inhibitor (z-LEHD-fmk) dramatically reduced the ototoxic effects of sensitivity of cochlear hair cells to damage by CDDP. This result CDDP, as evidenced by a lack of DNA fragmentation with almost no suggests that the JNK pathway is not involved in the CDDP-induced apoptotic cell death of hair cells or other cell types within the cochlea hair cell death but may have a role in DNA repair and maintenance of and almost no loss of hearing in the CDDP-treated animals. z-DEVD- CDDP-damaged sensory cells. fmk is a potent, cell-permeable, and irreversible inhibitor of Clinical Implications. As yet, there is no reliable test to predict caspase-3. We cannot exclude that other members of the caspase-3 which of those patients treated with CDDP will develop clinically subfamily are not involved in CDDP ototoxicity because it is known significant ototoxicity. It now is possible to introduce chemopro- that caspase-2 and -7 also are inhibited by z-DEVD-fmk (20). Mandic tectants, antiapoptotic agents, and other compounds across the round et al. (21) reported on the possible involvement of calcium-dependent window membrane of the cochlea in humans (27, 28). If successful, protease calpains in CDDP toxicity in a human melanoma cell line, the application of an otoprotective strategy for patients who begin to suggesting a role for calpains in CDDP ototoxicity. Although exper- show signs of ototoxic side effects should greatly enhance the quality iments yet have to be done in vivo, the lack of a protective effect of of life for patients who have to undergo high-dose CDDP chemo- calpain inhibitors on auditory hair cell and neurons in culture argues therapy. against this hypothesis (4). The present results suggest that CDDP ototoxicity is mediated through a molecular pathway that involves the ACKNOWLEDGMENTS mitochondria and the sequential activation of initiator and effector caspases. We thank Pr. J.-L. Pujol for helpful comments on chemotherapy/oncology JNK Signaling Pathway. The JNK signaling transduction path- and Dr. C. Bonny for the gift of D-JNKI-1. We also thank Dr. R. V. Lloyd Faulconbridge, Dr. J. Ruel, and Dr. M. Lenoir for their helpful discussion way that phosphorylates the NH2-terminal region of c-Jun (22, 23) is involved in the control of apoptosis in certain models of oxidative concerning experiments. stress damage. A protective role for c-Jun NH2-terminal phosphorylation against DNA damage-induced apoptosis is supported by the REFERENCES results of recent studies (24, 25). These studies have shown that the 1. Trimmer EE, Essigmann JM. Cisplatin. Essays Biochem 1999;34:191–11. JNK signal cascade is activated by CDDP-induced DNA damage and 2. Humes HD. Insights into ototoxicity. Analogies to nephrotoxicity. Ann N Y Acad Sci is necessary for the repair of DNA-CDDP adducts and for cell 1999;884:15–8. 3. Liu W, Staecker H, Stupak H, Malgrange B, Lefebvre P, Van De Water TR. Caspase viability. Therefore, to identify the involvement of this signal pathway inhibitors prevent cisplatin-induced apoptosis of auditory sensory cells. Neuroreport in CDDP-induced apoptosis of cochlear cells in vivo, levels of acti- 1998;9:2609–14. 9223

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2004 American Association for Cancer Research. Caspase Inhibitors, but not c-Jun NH2-Terminal Kinase Inhibitor Treatment, Prevent Cisplatin-Induced Hearing Loss

Jing Wang, Sabine Ladrech, Remy Pujol, et al.

Cancer Res 2004;64:9217-9224.

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