17-DMAG induces Hsp70 and protects the auditory hair cells from kanamycin ototoxicity in vitro

Neuroscience Letters 588 (2015) 72–77

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Research article

17-DMAG induces Hsp70 and protects the auditory hair cells from kanamycin ototoxicity in vitro Yun Liu, Yang Yu ∗ , Hanqi Chu, Dan Bing, Shaoli Wang, Liangqiang Zhou, Jin Chen, Qingguo Chen, Chunchen Pan, Yanbo Sun, Yonghua Cui Department of Otolaryngology-Head and Neck Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030, PR China

h i g h l i g h t s • 17-DMAG induces Hsp70 over-expression in the cultured organ of Corti. • 17-DMAG attenuates the ototoxity of kanamycin to hair cell in the organ of Corti. • 17-DMAG might become an effective therapeutic drug for auditory hair cells.

a r t i c l e

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Article history: Received 16 October 2014 Received in revised form 28 December 2014 Accepted 30 December 2014 Available online 31 December 2014 Keywords: 17-DMAG Hsp70 Kanamycin sulfate Ototoxicity

a b s t r a c t Heat shock protein 70 (Hsp70) has been known to be able to play a protective role in the cochlea. The aim of this study was to investigate whether geldanamycin hydrosoluble derivative 17(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) has the ability to induce Hsp70 up-regulation to protect hair cells from kanamycin-induced ototoxicity in vitro. The organ of Corti (OC) explants were isolated from mice at postnatal day 3–5. Then, the explants were exposed to kanamycin with or without pre-incubation with 17-DMAG. The expression of Hsp70 was assessed by reverse transcription-quantitative polymerase chain reaction, ELISA, and immunofluorescent staining. The surviving hair cells were examined by phalloidin labeling and were counted. We found that Hsp70 expression in the explants after pre-incubation with 17-DMAG was significantly increased at both mRNA and protein levels. Immunofluorescent staining showed that Hsp70 was mainly located in the auditory hair cells. Compared with kanamycin group, the loss of hair cells was inhibited significantly in 17-DMAG + kanamycin group. Our study demonstrated that 17-DMAG induces Hsp70 in the hair cells, and has a significant protective effect against kanamycin ototoxicity in vitro. 17-DMAG has the possibility to be a safe and effective anti-ototoxic drug. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction 70-KDa heat shock proteins (Hsp70), as ATP-dependent molecular chaperones, are encoded by Hspa1a and Hspa1b. The function of Hsp70 includes assistance of folding and refolding of proteins, transmembrane transporting of secretory proteins, and targeting of proteins for lysosomal degradation [3,17]. The expression of Hsp70 in most unstressed cells is low but its expression can be rapidly increased by a variety of physical and chemical stresses. It has been reported that Hsp70 can play a role in protection from stress stim-

∗ Corresponding author. Tel.: +86 02783663806. E-mail address: lucy [email protected] (Y. Yu). http://dx.doi.org/10.1016/j.neulet.2014.12.060 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.

ulation [1,3,14]. In the inner ear, Hsp70 was found to be able to be up-regulated by hyperthermia [4], ischemia [19], acoustic noise [15], and drug treatments [18,21,24,30]. Geldanamycin is a natural benzoquinone ansamycin antibiotic and is found to have antiparasite and antineoplastic properties, but with liver-harmful defects [6]. Geldanamycin has been reported to induce Hsp70 up-regulation and have a protective effect on cochlea [30]. 17-(Dimethylaminoethylamino)17-demethoxygeldanamycin (17-DMAG, NSC707545) is a geldanamycin analog and is found to significantly increase Hsp70 expression in the treatment of malignant tumors [11,13,22]. 17DMAG has been found to ameliorate motor neuron degeneration [29], attenuate atherosclerosis inflammation [16], and inhibit lymphoma cells proliferation [12]. So far there is no data about

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17-DMAG in the inner ear. In this study, we will test whether 17DMAG could increase Hsp70 expression in the inner ear to play a protective role. Kanamycin is an aminoglycoside antibiotic and can cause toxic side effects in urologic and auditory systems [7,9]. In clinic, kanamycin-induced hearing loss is a common cause for druginduced deafness. Kanamycin mainly targets hair cells in the inner ear [10,25]. The aim of this study was to investigate whether 17DMAG could protect hair cells against ototoxicity of kanamycin in the cochlea.

2. Materials and methods 2.1. Animal preparation and organ of Corti (OC) explant isolation C57BL/6 J mice were purchased from the Model Animal Research Center of Wuhan University (Wuhan, China). The care and experimental treatment of the animals were approved by the Animal Research Committee of Tongji Medical College, Huazhong University of Science and Technology. Newborn mice (P3-P5) were used. The OC explant was isolated as described by Sobkowicz et al., [26]. After the temporal bone was removed from the skull, the OC explant was isolated in cold, sterile, buffered saline glucose solution. For histological examination, the OC explant was cut into 3 segments corresponding to basal, middle and apical turns. Each segment was cultured in a flat surface preparation (Fig. 1). For reverse transcription-quantitative polymerase chain reaction (RT-qPCR), 10 OC explants isolated from 10 mice were incubated free-floating in the medium and collected as one sample for mRNA extraction. For ELISA examination, 4 explants from 4 mice were gathered together as one sample for protein extraction. Totally, each groups had 6–10 samples. The OC explant isolated from another ear in the same mouse was served as selfcontrol. The data-processing was based on the treated group and the self-control group from the same animal. The explants were cul-

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tured at 37 ◦ C in an incubator with 5% CO2 overnight before further treatment.

2.2. 17-DMAG and kanamycin treatment To examine whether 17-DMAG (BioVision Incorporated, Milpitas, USA) is nontoxic to hair cells, the OC segment explants were treated by 17-DMAG at concentration of 0.5 ␮M, 1 ␮M, 2 ␮M, or 5 ␮M for 24 h. To detect the ototoxic effect of kanamycin sulfate (E004000, Sigma–Aldrich Trading Co., Ltd., Shanghai, China) to hair cells, the OC segment explants were treated with kanamycin sulfate at concentration of 0.2 mM, 0.4 mM, or 1.0 mM for 24 h. To examine the protective effect of 17-DMAG on hair cells, the OC segment explants were pre-incubated with 17-DMAG for 5 h before treatment with kanamycin sulfate.

2.3. Reverse transcription-quantitative polymerase chain reaction The free-floating cultured OC explants were washed for removing the culture medium. TRIzol RNA extraction (Invitrogen, Carlsbad, USA) was used and the cDNA was synthesized with ReverTra Ace (Toyobo, Osaka, Japan). cDNA was amplified using SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan) with LightCycler 480II (Roche, Rotkreuz, Switzerland). The sequences of primers were as followed: Hspa1a (Gene ID:193740) and Hspa1b (Gene ID:15511) forward: 5 -TTC GTG GAG GAG TTC AAG AG-3 ; reverse: 5 -GCG TGA TGG ATG TGT AGA AGT-3 . Actb forward: 5 GCG CAA GTA CTC TGT GTG GA-3 ; reverse: 5 -GAA AGG GTG TAA AAC GCA GC-3 . The amplification protocol comprised the following parameters: 30 s at 95 ◦ C, 40 cycles of 5 s at 95 ◦ C, 10 s at 60 ◦ C and 15 s at 72 ◦ C. Each sample was run in triplicate and the readouts were averaged. The relative gene expression was calculated by 2−Ct method, where Ct = Ct (target gene) −Ct (reference gene) and Ct = Ct (treated) −Ct (control).

Fig. 1. The OC explants isolated from P3-P5 mice.(A): The decapitated mouse head. Arrows indicate two cochleae. (B): The isolated cochlea. (C): The isolated cochlear lateral wall (stria vascularis and spiral ligament) (LW) and OC explants (OC). (D): The OC segment explants on the bottom of the dish for culture. One row of inner hair cells (IHC) and three rows of outer hair cells (OHC) are clearly visible.

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2.4. ELISA examination The ELISA kit, Human/Mouse/Rat Total HSP70 DuoSet IC (DYC1663-2, R&D Systems China Co., Ltd., Shanghai, China) was used. Free-floating OC explants were washed by RNAase-free phosphate-buffered saline (PBS) for removing the culture medium and then lysed in RIPA Lysis buffer (P0013, Beyotime Institute of Biotechnology, Shanghai, China) with 1 mM PMSF (ST506, Beyotime Institute of Biotechnology, Shanghai, China). The total protein was measured by Micro BCA Protein Assay Kit (AR1110, Boster, Wuhan, China). The standard curve was made according to the ELISA manufacturer’s instructions. The data were analyzed by CurveExpert 1.4 software.

2.5. Immunofluorescent staining The OC segment explants were fixed in 4% paraformaldehyde at room temperature for 30 min and then permeabilized with 0.2% Triton X-100 in PBS for 30 min. For hair cell labeling, the epithelia were incubated in tetramethyl rhodamine isothiocyanate (TRITC) -conjugated phalloidin (P1951, Sigma–Aldrich, Saint Louis, USA) (5 ␮g/ml) at room temperature for 30 min. After washout with PBS, the epithelia were mounted with Antifade Mounting Medium (P0126, Beyotime Institute of Biotechnology, Shanghai, China) and examined under a fluorescence microscope (Leica, DM2500, Wetzlar, Germany). The hair cells were counted over a longitudinal distance of 100 ␮m in three separated microscopic fields for each segment. Cells were considered missing when there was a gap in

the normal arrays. A mean value was calculated for each specimen and at least 6 explants were used for each group. For Hsp70 immunofluorescent staining, after fixation and permeabilization, the explants were washed 3 times and incubated in blocking solution (0.8% goat serum, 0.4 Triton and 2% bovine serum albumin in PBS) at room temperature for 3 h and then incubated overnight at 4 ◦ C with anti-Hsp70 antibody (C92F3A-5, Enzo Life Sciences, Inc., Switzerland) (1:50 in blocking solution). After rinse with PBS, the explants were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Boster, Wuhan, China) for 3 h at room temperature. After complete washout, the explants were mounted and observed under a fluorescence microscope (Leica, DM2500, Wetzlar, Germany) or a laser scanning confocal microscope (Olympus Fluoview 500 IX 71, Tokyo, Japan). The images were captured at the same laser intensity and exposure time. 2.6. Statistical analyses All data were presented as the mean ± SEM and statistically analyzed with SPSS (13.0; SPSS Inc., Chicago, USA). One-way ANOVA was used. P < 0.05 was considered as statistical significance. 3. Results 3.1. Kanamycin sulfate caused loss of the auditory hair cells To assess the toxic effect of kanamycin sulfate on the cochlear hair cells, three concentrations (0.2 mM, 0.4 mM and 1.0 mM) were

Fig. 2. (A) Ototoxic effect of kanamycin on hair cells. The survival hair cells were decreased as the kanamycin concentration was increased. (B) No visible toxic effect of 17-DMAG on hair cells. The numbers of OHCs and IHCs had no significant changes in culture under 17-DMAG (0.5–5 ␮M) treatment. Bars represent mean ± SEM.

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performed. After culture for 24 h, the surviving hair cells were counted. Slight hair cell loss was observed at treatment with 0.2 mM of kanamycin. The hair cell loss was increased and clear at 0.4 mM kanamycin treatment. At 1.0 mM concentration, the severe hair cell loss was observed. Fig. 2A shows a dose-dependent deleterious effect of kanamycin on hair cells. The damage of outer hair cells (OHCs) was more serious than that of inner hair cells (IHCs). The concentration of 0.4 mM was chosen for the following experiments

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had increase at 2.5 h, reached the maximum level at 5 to 10 h, and then returned to the normal level at 24 h (Fig. 3A). The expression of Hsp70 at the protein level was also increased. However, different from the changes at the transcription level, the expression of Hsp70 at the protein level was monotonically increased during 24 h testing period (Fig. 3A). Since the expression of Hsp70 at the protein level was already doubled at 5 h, we chose this time point for further experiments. 3.4. The location of Hsp70 induced by 17-DMAG

3.2. The effect of 17-DMAG on hair cells in vitro To assess whether 17-DMAG has toxic effect of on hair cells, four concentrations of 17-DMAG (0.5 ␮M, 1 ␮M, 2 ␮M, and 5 ␮M) were performed. After culture with 17-DMAG for 24 h, the surviving hair cells were counted. Fig. 2B shows that there was no apparent hair cell loss observed after the application of 17-DMAG in any tested concentration. We chose the concentration of 2 ␮M for further experiments.

We also used immunofluorescent staining to examine 17DMAG-induced increase in Hsp70 expression in the cochlea. We found that Hsp70 labeling was intense and had apparent increase in the hair cells, while in the control group without 17-DMAG application, only very weak labeling for Hsp70 is visible (Fig. 3B). In the 17-DMAG application group, the Hsp70 labeling in other cochlear structures was also very weak.

3.3. Increase in Hsp70 expression by 17-DMAG

3.5. Protection of hair cells from kanamycin treatment by 17-DMAG

To determine whether 17-DMAG induced Hsp70 overexpression, RT-qPCR and ELISA detection were used to assess Hsp70 expression at mRNA and protein levels, respectively. To investigate the time course of Hsp70 expression, the explants were treated with 2 ␮M 17-DMAG for 2.5, 5, 10, or 24 h. Compared with the expression in the control group, the mRNA expression of Hsp70

To assess the protective effect, 2 ␮M 17-DMAG was added to the culture medium at 5 h before application of 0.4 mM kanamycin sulfate. Then, the culture was continuous for 24 h under both 17DMAG and kanamycin treatment (Group D + K). In the kanamycin treated group, 17-DMAG was replaced by PBS of the same volume, and then the explants were cultured with kanamycin for

Fig. 3. Increase in Hsp70 expression by 17-DMAG. (A) Time course of Hsp70 expression induced by 17-DMAG. RT-qPCR examination shows that the mRNA is up-regulated almost 6 times after treatment of 2 ␮M 17-DMAG for 5–10 h and then reduces to normal. However, the expression at the protein level examined by ELISA analysis is monotonically increased about 7 times after 24 h of 17-DMAG treatment. (B) Immunofluorescent staining for Hsp70 in the OC segment explants. Hsp70 is labeled by FITCconjugated antibody (green) and the hair cells are labeled by TRITC-conjugated phalloidin (red). The merged images show that the Hsp70 expression in hair cells is abundantly increased. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Protection of hair cells by 17-DMAG. (A) Immunofluorescent images of hair cells of control group (Group C), kanamycin treatment group (Group K, treated with 0.4 mM kanamycin for 24 h), and kanamycin with pre-incubation of 17-DMAG group (Group D + K: treated with 2 ␮M 17-DMAG for 5 h prior to 0.4 mM kanamycin exposure). Hair cells are labeled by phalloidin (red). The stereocilia arranges as an inverted ‘V’ in normal condition. At the presence of ototoxic drug, the stereocilia arranges in disorder. Cells are considered missing when there is a gap in the normal arrays. (B) Number of surviving hair cells in 100 ␮m length of the OC segment explants in three groups. Both numbers of OHCs and IHCs in group D + K are significantly greater than those in group K. Data points represent mean ± SEM. (*P < 0.05,**P < 0.001, group D + K vs. group K).

24 h (Group K). Compared with severe hair cell loss observed in kanamycin treatment group (Group K), mild loss was visible in 17-DMAG pre-incubation group (Group D + K) (Fig. 4). The difference was statistically different (*P < 0.05, **P < 0.001, Group D + K vs. Group K) (Fig. 4B). 4. Discussion Aminoglycosides as antibacterial drugs have been used for decades. However, their ototoxic side effects restrict their widespread use. The intracellular mechanism of aminoglycosides ototoxicity is related to the increased formation of reactive oxygen species (ROS) and the dysfunction of mitochondria, both of which lead to cell apoptosis [7,10,25]. Fig. 2A shows a dose-dependent deleterious effect of kanamycin on hair cells. In clinic, the risk factors for ototoxicity include duration, frequency, renal function, age, and genetic susceptibility besides dosage [9]. Searching for the solution to ototoxicity of aminoglycosides is of current clinical significance. In this study, we have found that a geldanamycin analogue 17DMAG can protect hair cells from kanamycin treatment (Fig. 4). This is consistent with a previous report that geldanamycin protects hair cells from gentamicin treatment [30]. Geldanamycin and 17-DMAG are considered to be Hsp90 inhibitor via binding to the N-terminal domain of Hsp90 and disassociating Hsp90-HSF1 com-

plex, resulting in activation of transcription factor heat shock factor 1 (HSF1) [20]. Hsp70, one of the transcriptional targets of HSF1, has been reported to inhibit the apoptotic pathways by blocking JNK1 activity. Hsp70 has been proved to prevent mitochondrial membrane permeabilization, reduce the release of cytochrome-C, and bind to Apaf-1 to prevent the formation of apoptosome [14]. By use of Hsp70 deficient (Hspa1a (−/−) and Hspa1b (−/−)) and transgenic Hsp70-over expression mice, it has been found that Hsp70 can protect utricle hair cells against aminoglycoside-induced death both in vitro and in vivo experiments [27,28]. It has been proved that both geldanamycin and 17-DMAG could induce Hsp70 upregulation in auditory hair cells in vitro ([30] and Fig. 3). The time course of Hsp70 increased by 17-DMAG is similar to that by geldanamycin stimulation. Moreover, the over-expression of Hsp70 protein persists even longer (more than 24 h) ([30] and Fig. 3). Furthermore, 17-DMAG shows a better protective effect compared with geldanamycin, especially on IHCs ([30] and Fig. 3). These data indicate that Hsp70 may be a key point of protective effect of 17DMAG against kanamycin challenge. Besides Hsp70, other heat shock proteins (Hsps) also have otoprotective effects. It has been reported that heat shock could produce Hsp90 and Hsp27 as well as Hsp70 and inhibit ototoxic drug induced hair cell death [2]. Francis, et al. found that celastrol inhibited ototoxicity via Hsp32 [5]. Hsp90, Hsp27 and Hsp40 have been reported to be up-regulated by 17-DMAG in other cell lines

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and tissues [8,29]. Whether other Hsps were induced by 17-DMAG in the inner ear and involved in protection against ototoxicity, which has not been investigated yet, needs to be further studied in future. In addition to the pharmaceutical approach, heat shock [1], and sound therapy [23] were reported to induce Hsp70 and protect the inner ear too. Hsp70 become a popular potential therapeutic strategy of hair cell injury. We speculate that Hsp70, induced by 17-DMAG, may play an important protective role on hair cells. The further research is needed to reveal the intracellular mechanism underlying the protective effect of Hsp70 against ototoxicity. In summary, we confirmed that 17-DMAG induces Hsp70 overexpression and attenuates the ototoxicity of kanamycin in auditory hair cells of the cultured organ of Corti. Hsp70 may become an important target to the auditory protection. 17-DMAG has the potential to become a safe and effective therapeutic drug for auditory hair cells impairment due to ototoxicity. Preventative protection against ototoxicity may lead to more widespread application of aminoglycosides. Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements This work was supported by the National Natural Science Foundation of China (NSFC) (Grant No. 81070787; Grant No.81271078; Grant No. 81300827). References [1] T.G. Baker, S. Roy, C.S. Brandon, I.K. Kramarenko, S.P. Francis, M. Taleb, K.M. Marshall, R. Schwendener, F.S. Lee, L.L. Cunningham, Heat shock protein-mediated protection against cisplatin-induced hair cell death, J. Assoc. Res. Otolaryngol. (2014) [Epub ahead of print]. [2] L.L. Cunningham, C.S. Brandon, Heat shock inhibits both aminoglycoside- and cisplatin-induced sensory hair cell death, J. Assoc. Res. Otolaryngol. 7 (2006) 299–307. [3] M. Daugaard, M. Rohde, M. Jaattela, The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions, FEBS Lett. 581 (2007) 3702–3710. [4] C.J. Dechesne, H.N. Kim, T.S. Nowak, R.J. Wenthold Jr., Expression of heat shock protein HSP72 in the guinea pig and rat cochlea after hyperthermia: immunochemical and in situ hybridization analysis, Hearing Res. 59 (1992) 195–204. [5] S.P. Francis, I.I. Kramarenko, C.S. Brandon, F.S. Lee, T.G. Baker, L.L. Cunningham, Celastrol inhibits aminoglycoside-induced ototoxicity via heat shock protein 32, Cell Death Dis. 2 (2011) e195. [6] R. Garcia-Carbonero, A. Carnero, L. Paz-Ares, Inhibition of HSP90 molecular chaperones: moving into the clinic, Lancet Oncol. 14 (2013) e358–369. [7] O.W. Guthrie, Aminoglycoside induced ototoxicity, Toxicology 249 (2008) 91–96. [8] E.M. Harrison, E. Sharpe, C.O. Bellamy, S.J. McNally, L. Devey, O.J. Garden, J.A. Ross, S.J. Wigmore, Heat shock protein 90-binding agents protect renal cells from oxidative stress and reduce kidney ischemia-reperfusion injury, Am. J. Physiol. Renal Physiol. 295 (2008) F397–F405. [9] J.L. Houghton, K.D. Green, W. Chen, S. Garneau-Tsodikova, The future of aminoglycosides: the end or renaissance? Chembiochem: Euro. J. Chem. Biol. 11 (2010) 880–902. [10] M.E. Huth, A.J. Ricci, A.G. Cheng, Mechanisms of aminoglycoside ototoxicity and targets of hair cell protection, Int. J. Otolaryngol. 2011 (2011) 937861.

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