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Protective role of edaravone against cisplatin-induced ototoxicity in an auditory cell line
Hearing Research xxx (2015) 1e6
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Protective role of edaravone against cisplatin-induced ototoxicity in an auditory cell line Gi Jung Im a, Jiwon Chang b, *, Sehee Lee a, June Choi a, Hak Hyun Jung a, Hyung Min Lee b, Sung Hoon Ryu b, Su Kyoung Park b, Jin Hwan Kim b, Hyung-Jong Kim b a
Department of Otolaryngology-Head and Neck Surgery, Korea University College of Medicine, Anam-Dong 5-Ga 126-1, Sungbuk-Gu, Seoul, South Korea Department of Otolaryngology-Head and Neck Surgery, Kangnam Sacred Heart Hospital, Hallym University College of Medicine, 948-1, Daerim 1-dong, Yeongdeunpo-gu, Seoul, South Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 February 2015 Received in revised form 31 July 2015 Accepted 12 August 2015 Available online xxx
Edaravone is a neuroprotective agent with a potent free radical scavenging and antioxidant actions. In the present study we investigated the influence of edaravone on cisplatin ototoxicity in auditory cells. Cell viability was determined using a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide cell proliferation assay. Oxidative stress and apoptosis were assessed by reactive oxygen species (ROS) measurement, Hoechst 33258 staining, caspase-3 activity assay, and immunoblotting of PARP. Pretreatment with 100 mM of edaravone prior to application of 15 mM of cisplatin increased cell viability after 48 h of incubation in HEI-OC1 cells (from 51.9% to 64. 6% viability) and also, attenuated the cisplatin-induced increase in reactive oxygen species (ROS) (from 2.3 fold to 1.9 fold). Edaravone also decreased the activation of caspase-3 and reduced levels of cleaved poly-ADP-ribose polymerase (PARP). We propose that edaravone protects against cisplatin-induced ototoxicity by preventing apoptosis, and limiting ROS production in HEI-OC1 cells. © 2015 Elsevier B.V. All rights reserved.
Keywords: Edaravone Cisplatin Ototoxicity Cell culture Apoptosis
1. Introduction Cisplatin (cis-diamminedichloroplatinum II) is an important chemotherapeutic agent used in the treatment of solid tumors such as ovarian, cervical, testicular, lung, and head and neck cancers. Cisplatin acts in the tumor cells through mechanisms such as DNA damage and production of reactive oxygen species (ROS), which lead to cell death by apoptosis. Cell death can also occur via necrosis when the cell is exposed to high concentrations of cisplatin (Wang and Lippard, 2005). However, the administration of cisplatin is accompanied by dose-limiting adverse effects such as nephrotoxicity, ototoxicity, neurotoxicity, gastrointestinal tract toxicity and bone marrow toxicity (Saleh and El-Demerdash, 2005). Nephrotoxicity can be managed with saline hydration and administration of diuretics, and some other side effects can be reduced with fractionated doses of medication. However, there is currently no way to cure or prevent ototoxicity.
* Corresponding author. Department of Otolaryngology-Head and Neck Surgery, Kangnam Sacred Heart Hospital, Hallym University College of Medicine, 948-1, Daerim 1-dong, Yeongdeunpo-gu, 150-950 Seoul, South Korea. E-mail address: [email protected] (J. Chang).
Ototoxicity is a medication-induced auditory or vestibular functional loss that results in hearing loss or disequilibrium (Roland and Cohen, 1998). Cisplatin induces bilateral and irreversible hearing loss, and the elevation of hearing threshold has been reported in 75e100% of patients treated with cisplatin (McKeage, 1995). There are several mechanisms by which cisplatin damage the auditory and vestibular system and trigger ototoxicity. The first mechanism is through the covalent binding of cisplatin to guanine bases in DNA forming inter- and intra-strand chain crosslinking which induces the p53 and apoptosis. A second mechanism refers to the generation of free radicals, specifically ROS, which can increase lipid peroxidation, alter enzyme and structural proteins, and cause apoptotic cell death (Casares et al., 2012; Gutteridge and Halliwell, 2010). In organotypic cultures, when activated by cisplatin, the nicotinamide adenine dinucleotide phosphate oxidase 3 isoform (NOX 3) produces superoxide radical (O2$) (B anfi et al., 2004; Rybak, 2007). The superoxide radical may interact with unsaturated fatty acids in the lipid bilayer of the cell membrane to generate aldehyde 4-hydroxynonenal, which is highly toxic and may lead to cell death. Also the increase in aldehyde 4hydroxynonenal concentration is associated with an increased calcium influx to the outer hair cells, leading to apoptosis (Ikeda
http://dx.doi.org/10.1016/j.heares.2015.08.004 0378-5955/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Im, G.J., et al., Protective role of edaravone against cisplatin-induced ototoxicity in an auditory cell line, Hearing Research (2015), http://dx.doi.org/10.1016/j.heares.2015.08.004
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G.J. Im et al. / Hearing Research xxx (2015) 1e6
et al., 1993). The superoxide anion may also inactivate antioxidant enzymes (Pigeolet et al., 1990), and cause the pro-apoptotic Bax protein to release cytochrome c from the damaged mitochondria to activate caspases 9 and 3 (Rybak et al., 2009). Edaravone (MCI-186, 3-methyl-1-phenyl-pyrazolin-5-one) which is clinically used to treat acute cerebral infarction and acute myocardial infarction (Mishina et al., 2005; Tsujita et al., 2004) is a free radical scavenger and can interact with both peroxyl and hydroxyl radicals to form oxidized compounds (Asplund et al., 2009). Edaravone is reported to reduce the amount of oxidative DNA damage and lipid peroxidation in barotraumatic inner ear (Maekawa et al., 2009). Edaravone also suppresses streptomycininduced vestibulotoxicity, and protects the cochlea from acoustic trauma in guinea pigs (Horiike et al., 2003; Takemoto et al., 2004). Furthermore, edaravone protects hair cells of zebrafish against cisplatin and preserved ultrastructure of mitochondria (Hong et al., 2013). While previous experimental studies have investigated the antioxidant activity of edaravone, the effect of edaravone on cisplatin induced ototoxicity has not been evaluated thoroughly. The purpose of the present study is to investigate the mechanism of edaravone on cisplatin ototoxicity in auditory cell line. 2. Methods 2.1. HEI-OC1 cell culture The HEI-OC1 cell line is extremely sensitive to ototoxic drugs, expresses several molecular markers which are characteristic of organ of Corti sensory cells (Kalinec et al., 2003), and therefore the HEI-OC1 cell line can be a useful study model of ototoxic drugs. The cells were maintained in high-glucose Dulbecco's modified eagle's medium (Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco BRL, Grand Island, NY, USA) and 50-U/mL interferon-g (PEPROTECH, USA) without antibiotics at 33 C and 10% CO2 in air. 2.2. MTT assay to identify cell viability The uptake and conversion of 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazdiumbromide (MTT, Sigma, St Louis, MO, USA) to crystals of dark violet formazan depends on cell viability. Thus, HEI-
OC1 cells (2 x 104 cells/well of 48-well plate) were incubated with 15 mM of cisplatin (a concentration which is known to result in 50% of cell viability in our experiments) for 48 h, and the dosedependent or time-dependent effects of cisplatin were measured using an MTT assay. After pre-test of application of various concentrations of edaravone, the concentration of 100 mM edaravone was adopted since higher concentration seemed to influence the growth of HEI-OC1 cell (Fig 1). In order to examine the effects of edaravone on cisplatin ototoxicity in the auditory cell line, the cells were pretreated with edaravone (100 mM) for 24 h, and then were exposed to cisplatin (15 mM, 48 h). These protocols were maintained throughout the experiments. For the MTT assay, 200 mL of the MTT solution (5 mg/mL) was added to cells and the plates were incubated for 3 h at 33 C in a 10% CO2 and 90% air mixture. The resulting insoluble violet formazan crystals were centrifuged and the pellets dissolved in DMSO (500 mL/well). Optical density was measured using a microplate reader at 570 nm (Spectra Max, Molecular Devices, Sunnyvale, CA, USA). 2.3. Intracellular ROS measurement The intracellular ROS level was measured using a fluorescent dye, 20 , 70 -dichlorofluorescein diacetate (DCFH-DA; Calbiochem, Darmstadt, Germany). In the presence of an oxidant, DCFH is converted into highly fluorescent 20 , 70 -dichlorofluorescein (DCF). For ROS assay, HEI-OC1 cells were treated with 15 mM cisplatin for 48 h in the presence or absence of edaravone (100 mM, 24 h pretreatment). Following this incubation, the cells were treated with 50 mM DCFH-DA and were further incubated for 30 min. The samples were measured using a FACScan flow cytometry (BD Biosciences, Heidelberg, Germany) at a 488 nm excitation wavelength and a 530 nm emission wavelength (10,000 cells/sample), and the mean fluorescence intensity was calculated by histogram statistics using BD CellQuest Pro software (BD Biosciences). 2.4. Fluorescence microscopy Cells were grown on the Cell Culture Slides (SPL, Gyeonggido, Korea). Cells were washed twice with serum-free medium without phenol red and incubated with 50 mM DCFH-DA in serum-free medium without phenol red for 30 min at 33 C. After three washings with serum free medium without phenol red, cells were fixed with 3.7% glutaraldehyde for 10 min at room temperature. Cells were incubated with 10 mg/mL Hoechst 33258 (Sigma, St Louis, MO, USA) for 20 min at room temperature in the dark. After washing twice with PBS and mounting, the fluorescence images from multiple fields of view were obtained using an olympus ix71 microscopy with a long-term real-time live cell image system (LAMBDA DG-4, Sutter Int., Novato, CA, USA). 2.5. Measurement of caspase-3 activity
Fig. 1. Effect of edaravone on HEI-OC1 cells HEI-OC1 cells were cultured with various concentrations (0, 50, 100, 150, 200, 250, 300 mM) of edaravone for 72 h. The concentrations more than 150 mM had effect the growth of HEI-OC1 cell (cell viability of 103.5 ± 4.8%). In order to rule out the growth effect of edaravone, we adopted the 100 mM of edarvaone for the further study (cell viability of 101.4 ± 3.0%).
The enzymatic activity of caspase-3 was assayed with a caspase3/CPP32 fluorometric assay kit (Biovision, Milpitas, California, USA) according to the manufacturer's protocol. Auditory cell line lysate was prepared in a lysis buffer on ice for 10 min and centrifuged for 5 min at 14,000 rpm. The protein concentration in each lysate was measured. The catalytic activity of caspase-3 in the cell lysate was measured by proteolytic cleavage of 50 mM DEVD-pNA and fluorometric substrate for 2 h at 37 C. The mixture incubated with no DEVD-pNA substrate was used as a negative control. The plates were read by microplate reader (Spectra Max, Molecular Devices, Sunnyvale, CA, USA) at a 400 nm excitation filter and a 505 nm emission filter.
Please cite this article in press as: Im, G.J., et al., Protective role of edaravone against cisplatin-induced ototoxicity in an auditory cell line, Hearing Research (2015), http://dx.doi.org/10.1016/j.heares.2015.08.004
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2.6. Immunoblotting of PARP Following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): poly-ADP-ribose polymerase (PARP). For the assay, HEI-OC1 cells were cultured and treated with 15 mM cisplatin for 48 h in the presence or absence of edaravone (100 mM, 24 h pretreatment). Cell lysates were used in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA). The proteins (20 mg/sample) were immediately heated for 5 min at 100 C and were subjected to SDS-PAGE on gels. Separated proteins were transferred to nitrocellulose membranes, and western blotting was performed using a gel loading kit and protein transfer system kit (BioRad, Hercules, CA, USA). Membranes were blocked by treatment with 5% skim milk in Tris buffer solution (TBS) and subsequently incubated with the primary polyclonal antibodies at a final dilution of 1: 1000. After three washes in TBS containing 0.1% Tween (TBST), membranes were incubated with peroxidaseconjugated secondary antibodies (final dilution, 1: 2000) in blocking buffer for 1 h and subsequently washed. Detection was performed by chemiluminescence using an ECL solution (Gendepot, Barker TX, USA). Beta-actin was used as a loading control. For the calculation of band density, Image J was used (Imagej.nih.gov/ij/ index.html). 2.7. Statistical analysis All values are represented as mean ± SD. For data analysis, we used the SPSS 20.0 statistical program. For the comparison of multiple groups in the MTT assay, flow cytometry analysis, ROS measurement, and caspase-3 activity, ANOVA was used. A p value of <0.05 was considered statistically significant. For multiple comparisons, Bonferroni correction was done.
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it was significantly higher than the cisplatin alone treated group (p < 0.01). 3.2. Hoechst 33258 staining Apoptosis was evaluated by the appearance of condensed and fragmented nuclei upon Hoechst 33258 staining. As compared with normal and edaravone applied cells (Fig. 3A, B), nuclei of HEI-OC1 cells after exposure to 15 mM cisplatin for 48 h showed condensed and fragmented DNA (Fig. 3C) and decreased numbers due to apoptosis. However, when cells were pretreated with 100 mM edaravone (Fig 3D), nuclei of HEI-OC1 cells had less condensed and fragmented DNA after the exposure to cisplatin. 3.3. Intracellular ROS measurement DCFH was converted into highly fluorescent 20 , 70 -dichlorofluorescein (DCF) when 15 mM cisplatin was applied (Fig 3G), but when the cells were pretreated with 100 mM edaravone, the amount of ROS increase was less (Fig 3H). We measure the intracellular levels of ROS generated by HEIOC1 cell in response to cisplatin and edaravone using the fluorescent probe DCFH-DA. As shown in Fig 4A, HEI-OC1 cells treated with 15 mM cisplatin exhibited markedly increased fluorescence intensity in comparison to the control group. Pretreatment with 100 mM edaravone for 24 h reduced the cisplatin induced generation of ROS. The mean probe intensity in comparison to that of the control is shown in Fig 4B. Treatment with 15 mM cisplatin increased the fluorescence intensity by 2.3 fold, whereas pretreatment with 100 mM edaravone decreased the fluorescence intensity by 1.9 folds (p < 0.01). 3.4. Measurement of caspase-3 activity
3. Results 3.1. MTT assay Cell viability was determined by MTT assay to verify whether edaravone was able to prevent apoptosis induced by cisplatin. When cultured cells were exposed to 15 mM cisplatin and 100 mM edaravone, cell viability was 51.9%, 101.4%, respectively. But when the cells were exposed to 15 mM cisplatin after had been pretreated with 100 mM edaravone for 24 h, cell viability was 64.6% (Fig 2) and
Caspase-3 activity is involved in cisplatin-induced toxicity and related to apoptotic changes in cisplatin ototoxicity. The administration of 15 mM cisplatin increased the activity of caspase-3 (9.96 ± 2.03 fold over the normal control) (Fig 5). The pretreatment of HEI-OC1 cells with 100 mM edaravone significantly decreased caspase-3 activity (7.03 ± 1.92 fold) as compared with cells treated with cisplatin alone (P < 0.01). 3.5. Immunoblotting of PARP The cleavages of PARP facilitate cellular disassembly and serve as a marker of cells undergoing apoptosis. Cisplatin increased cleaved PARP (4.9 ± 0.68 fold over the normal control) (Fig 6). The pretreatment of HEI-OC1 cells with 100 mM edaravone significantly decreased cleaved PARP (1.7 ± 0.82 fold) as compared with cells treated with cisplatin alone (P < 0.01). Thus, this result also supports the existence of an antiapoptotic effect of edaravone. 4. Discussions
Fig. 2. MTT assay When cultured cells were exposed to 15 mM cisplatin and 100 mM edaravone, cell viability was 51.9 (±7.1)% and 101.4 (±3.0)%, respectively. But when the cells were exposed to 15 mM cisplatin after pretreatment with 100 mM edaravone for 24 h, cell viability was 64.6 (±8.5)% which was significantly higher than in the cells treated with cisplatin alone (p < 0.01).
Many therapeutic agents such as metformin (Chang et al., 2014), alpha-lipoic acid (Kim et al., 2014), p-methionine (Campbell et al., 2003), N-acetylcystein (Dickey et al., 2004; Choe et al., 2004) and sodium thiosulfate (Wang et al., 2003) have been introduced to prevent cisplatin induced ototoxicity. Nonetheless, thus far, there is no ideal protective agent in clinical use. Edaravone is a neuroprotective agent with potent free radical scavenging and antioxidant actions without causing any serious side effects (Takumida and Anniko, 2006; Higashi et al., 2006). Edaravone is reported to reduce the amount of oxidative DNA damage (Maekawa et al., 2009) and decrease the amount of ROS
Please cite this article in press as: Im, G.J., et al., Protective role of edaravone against cisplatin-induced ototoxicity in an auditory cell line, Hearing Research (2015), http://dx.doi.org/10.1016/j.heares.2015.08.004
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Fig. 3. Measurement of intracellular ROS production and Hoechst 33258 staining Apoptosis was evaluated based on the appearance of condensed and fragmented nuclei in Hoechst 33258 staining (AeD). As compared to control cells and those treated with edaravone (A, B), cells exposed to 15 mM cisplatin for 48 h displayed nuclei that were condensed and fragmented (C; arrows). Group C also exhibited reduced cell density compared to groups A and B. However, when the cells were pretreated with 100 mM edaravone (D), they were less condensed and less fragmented. DCFH was converted into highly fluorescent 20 , 70 -dichlorofluorescein (DCF) when 15 mM cisplatin was applied (G), but when the cells were pretreated with 100 mM edaravone, the amount of ROS increase was less (H).
Fig. 4. Intracellular ROS measurement (A) HEI-OC1 cells treated with 15 mM cisplatin(3rd row) exhibited markedly increased fluorescence intensity in comparison to the control group(1st row). Pretreatment with 100 mM edaravone for 24 h (4th row) reduced the cisplatin induced generation of ROS. (B) Treatment with 15 mM cisplatin increased the fluorescence intensity by 2.3 (±0.2) fold, whereas pretreatment with 100 mM edaravone decreased the fluorescence intensity by 1.9 (±0.3) folds (p < 0.01).
Please cite this article in press as: Im, G.J., et al., Protective role of edaravone against cisplatin-induced ototoxicity in an auditory cell line, Hearing Research (2015), http://dx.doi.org/10.1016/j.heares.2015.08.004
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apoptosis. In Hoechst stain, the cells pretreated with edaraovone before cisplatin treatment had less condensed and fragmented nuclei. Also, the amount of ROS production was reduced when edaravone was used. Although edaravone is rather a safe agent when administered to the patients (Takumida and Anniko, 2006), its clinical application in cisplatin-induced ototoxicity remains unclear, because the problem as to whether edaravone enhances or suppresses the efficacy of cisplatin in the treatment of cancer cells remains unsettled yet. Also, since our experiment was conducted with an HEI-OC1 cell line, its conditions are not typical condition of cochlear cells as it is cultured under permissive conditions (33.8 C, 10% CO2). Therefore, in order to validate the protective effect of edaravone in cisplatin ototoxicity, these results obtained in vitro should be corroborated by in vivo studies. In conclusion, we found that edaravone protected cisplatin induced apoptotic cascade, reduced ROS production and enhanced cell viability in an auditory cell line. Fig. 5. Measurement of caspase-3 activity The administration of 15 mM cisplatin increased the activity of caspase-3 (9.96 ± 2.03) fold over the normal control. The pretreatment of HEI-OC1 cells with 100 mM edaravone significantly decreased caspase3 activity (7.03 ± 1.92 fold) as compared with cells treated with cisplatin alone (p < 0.01).
Acknowledgement This study was supported by Hallym University Research Fund (HURF-201510). References
and inhibits oxidation. It is assumed that edaravone inhibits lipoxygenase metabolism by trapping hydroxyl radicals, prevents peroxidation, and enhances of prostacyclin production (Takumida and Anniko, 2006; Watanabe et al., 1988). In our study, edaravone protected cisplatin induced cell death in auditory cell line by reducing ROS and preventing a sequential apoptotic cascade. Edaravone significantly decreased caspase-3 activity and reduced the cleaved PARP. Caspase-3 is reported to activate DNA fragmentation factor, which in turn activate endonucleases to cleave nuclear DNA, and ultimately leads to cell death (Lee et al., 2007). In addition, caspase-3 is responsible for the proteolytic cleavage of many key proteins, such as PARP, which is fundamental for cell viability. The cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing
Fig. 6. Immunoblotting of PARP Cisplatin increased cleaved PARP (4.9 ± 0.68 fold over the normal control). The pretreatment of HEI-OC1 cells with 100 mM edaravone significantly decreased cleaved PARP (1.7 ± 0.82 fold) as compared with cells treated with cisplatin alone (p < 0.01) (A, B).
Asplund, M.S., Lidian, A., Linder, B., Takumida, M., Anniko, M., 2009. Protective effect of edaravone against tobramycin-induced ototoxicity. Acta Otolaryngol. 129 (2009), 8e13. nfi, B., Malgrange, B., Knisz, J., Steger, K., Dubois-Dauphin, M., Krause, K.-H., 2004. Ba NOX3, a superoxide-generating NADPH oxidase of the inner ear. J. Biol. Chem. 279, 46065e47232. Campbell, K.C., Meech, R.P., Rybak, L.P., Hughes, L.F., 2003. The effect of D-methionine on cochlear oxidative state with and without cisplatin administration: mechanisms of otoprotection. J. Am. Acad. Audiol. 14 (3), 144e156. n, A., Jorge, E., GarcíaCasares, C., Ramírez-Camacho, R., Trinidad, A., Rolda Berrocal, J.R., 2012. Reactive oxygen species in apoptosis induced by cisplatin: review of physiopathological mechanisms in animal models. Eur. Arch. Otorhinolaryngol. 269 (12), 2455e2459. Chang, J., Jung, H.H., Yang, J.Y., Lee, S., Choi, J., Im, G.J., Chae, S.W., 2014. Protective effect of metformin against cisplatin-induced ototoxicity in an auditory cell line. J. Assoc. Res. Otolaryngol. 15 (2), 149e158. Choe, W.T., Chinosornvatana, N., Chang, W.K., 2004. Prevention of cisplatin ototoxicity using transtympanic N-acetylcysteine and lactate. Otol. Neurotol. 25, 910e915. Dickey, T.D., Mulcoon, L.L., Kraemer, D.F., Neuwelt, E.A., 2004. Protection against cisplatin induced ototoxicity by N-acetyl-cysteine in rat model. Hear Res. 193, 25e30. Gutteridge, J.M., Halliwell, B., 2010. Antioxidants: molecules, medicines and myths. Biochem. Biophys. Res. Commun. 393 (4), 561e564. Higashi, Y., Jitsuiki, D., Chayama, K., Yoshizumi, M., 2006. Edaravone (3-methyl-1phenyl-2-pyrazolin-5-one), a novel free radical scavenger, for treatment of cardiovascular diseases. Recent Pat. Cardiovasc Drug Discov. 1 (1), 85e93. Hong, S.J., Im, G.J., Chang, J., Chae, S.W., Lee, S.H., Kwon, S.Y., Jung, H.H., Chung, A.Y., Park, H.C., Choi, J., 2013. Protective effects of edaravone against cisplatininduced hair cell damage in zebrafish. Int. J. Pediatr. Otorhinolaryngol. 77 (6), 1025e1031. Horiike, O., Shimogori, H., Ikeda, T., Yamashita, H., 2003. Protective effect of edaravone against streptomycin-induced vestibulotoxicity in the guinea pig. Eur. J. Pharmacol. 464, 75e78. Ikeda, K., Sunose, H., Takasaka, T., 1993. Effects of free radicals on the intracellular calcium concentration in the isolated hair cell of the guinea pig cochlea. Acta Otolaryngol. 113, 137e141. Kalinec, G.M., Webster, P., Lim, D.J., Kalinec, F., 2003. A cochlear cell line as an in vitro system for drug ototoxicity screening. Audiol. Neurootol. 8, 177e189. Kim, J., Cho, H.J., Sagong, B., Kim, S.J., Lee, J.T., So, H.S., Lee, I.K., Kim, U.K., Lee, K.Y., Choo, Y.S., 2014. Alpha-lipoic acid protects against cisplatin-induced ototoxicity via the regulation of MAPKs and proinflammatory cytokines. Biochem. Biophys. Res. Commun. 449 (2), 183e189. Lee, M.K., Kang, S.J., Poncz, M., Sibg, K.J., Park, K.S., 2007. Resveratrol protects SHSY5Y neuroblastoma cells from apoptosis induced by dopamine. Exp. Mol. Med. 39 (3), 376e384. Maekawa, H., Matsunobu, T., Tsuda, H., Onozato, K., Masuda, Y., Tanabe, T., Shiotani, A., 2009. Therapeutic effect of edaravone on inner ear barotrauma in the guinea pig. Neurochem. Int. 54, 513e518. McKeage, M.J., 1995. Comparative adverse effect profiles of platinum drugs. Drug Saf. 13, 228e244.
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Mishina, M., Komaba, Y., Kobayashi, S., Tanaka, N., Kominami, S., Fukuchi, T., et al., 2005. Efficacy of edaravone, a free radical scavenger, for the treatment of acute lacunar infarction. Neurol. Med. Chir. (Tokyo) 45, 344e348 discussion 348. Pigeolet, E., Corbisier, P., Houbion, A., Lambert, D., Michiels, C., Raes, M., et al., 1990. Glutathione peroxidase, superoxide dismutase and catalase inactivation by peroxides and oxygen derived free radicals. Mech. Ageing Dev. 50, 283e297. Roland Jr., J.T., Cohen, N.L., 1998. Vestibular and auditory ototoxicity. In: Cummings, C.W., Frederickson, J.M., Harker, L.A., Krause, C.J., Schuller, D.F., Richardson, M.A. (Eds.), Otolaryngology & Head and Neck Surgery, third ed. Mosby, St. Louis. 3186e3197. Rybak, L.P., 2007. Mechanisms of cisplatin ototoxicity and progress in otoprotection. Curr. Opin. Otolaryngol. Head. Neck Surg. 15, 364e369. Rybak, L.P., Mukherjea, D., Jajoo, S., Ramkumar, V., 2009. Cisplatin ototoxicity and protection: clinical and experimental studies. Tohoku J. Exp. Med. 219, 177e186. Saleh, S., El-Demerdash, E., 2005. Protective effects of L-arginine against cisplatininduced renal oxidative stress and toxicity: role nitric oxide. Basic Clin. Pharmacol. Toxicol. 97, 91e97.
Takemoto, T., Sugahara, K., Okuda, T., Shimogori, H., Yamashita, H., 2004. The clinical free radical scavenger, edaravone, protects cochlear hair cells from acoustic trauma. Eur. J. Pharmaology 487, 113e116. Takumida, M., Anniko, M., 2006. Protective effect of edaravone against the ototoxicity of Pseudomonas aeruginosa exotoxin A. Acta Otolaryngol. 126 (1), 15e19. Tsujita, K., Shimomura, H., Kawano, H., Hokamaki, J., Fukuda, M., Yamashita, T., et al., 2004. Effects of edaravone on reperfusion injury in patients with acute myocardial infarction. Am. J. Cardiol. 94, 481e484. Wang, D., Lippard, S.J., 2005. Cellular processing of platinum anticancer drugs. Nat. Rev. Drug Discov. 4, 307e320. Wang, J., Lloyd Faulconbridge, R.V., Fetoni, A., Guitton, M.J., Pujol, R., Puel, J.L., 2003. Local application of sodium thiosulfate prevents cisplatin-induced hearing loss in the guinea pig. Neuropharmacology 45 (3), 380e393. Watanabe, T., Morita, I., Nishi, H., Murota, S., 1988. Preventive effect of MCI-186 on 15-HPETE induced vascular endothelial cell injury in vitro. Prostagl. Leukot. Essent. Fat. Acids 33 (1), 81e87.
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Protective role of edaravone against cisplatin-induced ototoxicity in an auditory cell line Gi Jung Im a, Jiwon Chang b, *, Sehee Lee a, June Choi a, Hak Hyun Jung a, Hyung Min Lee b, Sung Hoon Ryu b, Su Kyoung Park b, Jin Hwan Kim b, Hyung-Jong Kim b a
Department of Otolaryngology-Head and Neck Surgery, Korea University College of Medicine, Anam-Dong 5-Ga 126-1, Sungbuk-Gu, Seoul, South Korea Department of Otolaryngology-Head and Neck Surgery, Kangnam Sacred Heart Hospital, Hallym University College of Medicine, 948-1, Daerim 1-dong, Yeongdeunpo-gu, Seoul, South Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 February 2015 Received in revised form 31 July 2015 Accepted 12 August 2015 Available online xxx
Edaravone is a neuroprotective agent with a potent free radical scavenging and antioxidant actions. In the present study we investigated the influence of edaravone on cisplatin ototoxicity in auditory cells. Cell viability was determined using a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide cell proliferation assay. Oxidative stress and apoptosis were assessed by reactive oxygen species (ROS) measurement, Hoechst 33258 staining, caspase-3 activity assay, and immunoblotting of PARP. Pretreatment with 100 mM of edaravone prior to application of 15 mM of cisplatin increased cell viability after 48 h of incubation in HEI-OC1 cells (from 51.9% to 64. 6% viability) and also, attenuated the cisplatin-induced increase in reactive oxygen species (ROS) (from 2.3 fold to 1.9 fold). Edaravone also decreased the activation of caspase-3 and reduced levels of cleaved poly-ADP-ribose polymerase (PARP). We propose that edaravone protects against cisplatin-induced ototoxicity by preventing apoptosis, and limiting ROS production in HEI-OC1 cells. © 2015 Elsevier B.V. All rights reserved.
Keywords: Edaravone Cisplatin Ototoxicity Cell culture Apoptosis
1. Introduction Cisplatin (cis-diamminedichloroplatinum II) is an important chemotherapeutic agent used in the treatment of solid tumors such as ovarian, cervical, testicular, lung, and head and neck cancers. Cisplatin acts in the tumor cells through mechanisms such as DNA damage and production of reactive oxygen species (ROS), which lead to cell death by apoptosis. Cell death can also occur via necrosis when the cell is exposed to high concentrations of cisplatin (Wang and Lippard, 2005). However, the administration of cisplatin is accompanied by dose-limiting adverse effects such as nephrotoxicity, ototoxicity, neurotoxicity, gastrointestinal tract toxicity and bone marrow toxicity (Saleh and El-Demerdash, 2005). Nephrotoxicity can be managed with saline hydration and administration of diuretics, and some other side effects can be reduced with fractionated doses of medication. However, there is currently no way to cure or prevent ototoxicity.
* Corresponding author. Department of Otolaryngology-Head and Neck Surgery, Kangnam Sacred Heart Hospital, Hallym University College of Medicine, 948-1, Daerim 1-dong, Yeongdeunpo-gu, 150-950 Seoul, South Korea. E-mail address: [email protected] (J. Chang).
Ototoxicity is a medication-induced auditory or vestibular functional loss that results in hearing loss or disequilibrium (Roland and Cohen, 1998). Cisplatin induces bilateral and irreversible hearing loss, and the elevation of hearing threshold has been reported in 75e100% of patients treated with cisplatin (McKeage, 1995). There are several mechanisms by which cisplatin damage the auditory and vestibular system and trigger ototoxicity. The first mechanism is through the covalent binding of cisplatin to guanine bases in DNA forming inter- and intra-strand chain crosslinking which induces the p53 and apoptosis. A second mechanism refers to the generation of free radicals, specifically ROS, which can increase lipid peroxidation, alter enzyme and structural proteins, and cause apoptotic cell death (Casares et al., 2012; Gutteridge and Halliwell, 2010). In organotypic cultures, when activated by cisplatin, the nicotinamide adenine dinucleotide phosphate oxidase 3 isoform (NOX 3) produces superoxide radical (O2$) (B anfi et al., 2004; Rybak, 2007). The superoxide radical may interact with unsaturated fatty acids in the lipid bilayer of the cell membrane to generate aldehyde 4-hydroxynonenal, which is highly toxic and may lead to cell death. Also the increase in aldehyde 4hydroxynonenal concentration is associated with an increased calcium influx to the outer hair cells, leading to apoptosis (Ikeda
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Please cite this article in press as: Im, G.J., et al., Protective role of edaravone against cisplatin-induced ototoxicity in an auditory cell line, Hearing Research (2015), http://dx.doi.org/10.1016/j.heares.2015.08.004
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et al., 1993). The superoxide anion may also inactivate antioxidant enzymes (Pigeolet et al., 1990), and cause the pro-apoptotic Bax protein to release cytochrome c from the damaged mitochondria to activate caspases 9 and 3 (Rybak et al., 2009). Edaravone (MCI-186, 3-methyl-1-phenyl-pyrazolin-5-one) which is clinically used to treat acute cerebral infarction and acute myocardial infarction (Mishina et al., 2005; Tsujita et al., 2004) is a free radical scavenger and can interact with both peroxyl and hydroxyl radicals to form oxidized compounds (Asplund et al., 2009). Edaravone is reported to reduce the amount of oxidative DNA damage and lipid peroxidation in barotraumatic inner ear (Maekawa et al., 2009). Edaravone also suppresses streptomycininduced vestibulotoxicity, and protects the cochlea from acoustic trauma in guinea pigs (Horiike et al., 2003; Takemoto et al., 2004). Furthermore, edaravone protects hair cells of zebrafish against cisplatin and preserved ultrastructure of mitochondria (Hong et al., 2013). While previous experimental studies have investigated the antioxidant activity of edaravone, the effect of edaravone on cisplatin induced ototoxicity has not been evaluated thoroughly. The purpose of the present study is to investigate the mechanism of edaravone on cisplatin ototoxicity in auditory cell line. 2. Methods 2.1. HEI-OC1 cell culture The HEI-OC1 cell line is extremely sensitive to ototoxic drugs, expresses several molecular markers which are characteristic of organ of Corti sensory cells (Kalinec et al., 2003), and therefore the HEI-OC1 cell line can be a useful study model of ototoxic drugs. The cells were maintained in high-glucose Dulbecco's modified eagle's medium (Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco BRL, Grand Island, NY, USA) and 50-U/mL interferon-g (PEPROTECH, USA) without antibiotics at 33 C and 10% CO2 in air. 2.2. MTT assay to identify cell viability The uptake and conversion of 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazdiumbromide (MTT, Sigma, St Louis, MO, USA) to crystals of dark violet formazan depends on cell viability. Thus, HEI-
OC1 cells (2 x 104 cells/well of 48-well plate) were incubated with 15 mM of cisplatin (a concentration which is known to result in 50% of cell viability in our experiments) for 48 h, and the dosedependent or time-dependent effects of cisplatin were measured using an MTT assay. After pre-test of application of various concentrations of edaravone, the concentration of 100 mM edaravone was adopted since higher concentration seemed to influence the growth of HEI-OC1 cell (Fig 1). In order to examine the effects of edaravone on cisplatin ototoxicity in the auditory cell line, the cells were pretreated with edaravone (100 mM) for 24 h, and then were exposed to cisplatin (15 mM, 48 h). These protocols were maintained throughout the experiments. For the MTT assay, 200 mL of the MTT solution (5 mg/mL) was added to cells and the plates were incubated for 3 h at 33 C in a 10% CO2 and 90% air mixture. The resulting insoluble violet formazan crystals were centrifuged and the pellets dissolved in DMSO (500 mL/well). Optical density was measured using a microplate reader at 570 nm (Spectra Max, Molecular Devices, Sunnyvale, CA, USA). 2.3. Intracellular ROS measurement The intracellular ROS level was measured using a fluorescent dye, 20 , 70 -dichlorofluorescein diacetate (DCFH-DA; Calbiochem, Darmstadt, Germany). In the presence of an oxidant, DCFH is converted into highly fluorescent 20 , 70 -dichlorofluorescein (DCF). For ROS assay, HEI-OC1 cells were treated with 15 mM cisplatin for 48 h in the presence or absence of edaravone (100 mM, 24 h pretreatment). Following this incubation, the cells were treated with 50 mM DCFH-DA and were further incubated for 30 min. The samples were measured using a FACScan flow cytometry (BD Biosciences, Heidelberg, Germany) at a 488 nm excitation wavelength and a 530 nm emission wavelength (10,000 cells/sample), and the mean fluorescence intensity was calculated by histogram statistics using BD CellQuest Pro software (BD Biosciences). 2.4. Fluorescence microscopy Cells were grown on the Cell Culture Slides (SPL, Gyeonggido, Korea). Cells were washed twice with serum-free medium without phenol red and incubated with 50 mM DCFH-DA in serum-free medium without phenol red for 30 min at 33 C. After three washings with serum free medium without phenol red, cells were fixed with 3.7% glutaraldehyde for 10 min at room temperature. Cells were incubated with 10 mg/mL Hoechst 33258 (Sigma, St Louis, MO, USA) for 20 min at room temperature in the dark. After washing twice with PBS and mounting, the fluorescence images from multiple fields of view were obtained using an olympus ix71 microscopy with a long-term real-time live cell image system (LAMBDA DG-4, Sutter Int., Novato, CA, USA). 2.5. Measurement of caspase-3 activity
Fig. 1. Effect of edaravone on HEI-OC1 cells HEI-OC1 cells were cultured with various concentrations (0, 50, 100, 150, 200, 250, 300 mM) of edaravone for 72 h. The concentrations more than 150 mM had effect the growth of HEI-OC1 cell (cell viability of 103.5 ± 4.8%). In order to rule out the growth effect of edaravone, we adopted the 100 mM of edarvaone for the further study (cell viability of 101.4 ± 3.0%).
The enzymatic activity of caspase-3 was assayed with a caspase3/CPP32 fluorometric assay kit (Biovision, Milpitas, California, USA) according to the manufacturer's protocol. Auditory cell line lysate was prepared in a lysis buffer on ice for 10 min and centrifuged for 5 min at 14,000 rpm. The protein concentration in each lysate was measured. The catalytic activity of caspase-3 in the cell lysate was measured by proteolytic cleavage of 50 mM DEVD-pNA and fluorometric substrate for 2 h at 37 C. The mixture incubated with no DEVD-pNA substrate was used as a negative control. The plates were read by microplate reader (Spectra Max, Molecular Devices, Sunnyvale, CA, USA) at a 400 nm excitation filter and a 505 nm emission filter.
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2.6. Immunoblotting of PARP Following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): poly-ADP-ribose polymerase (PARP). For the assay, HEI-OC1 cells were cultured and treated with 15 mM cisplatin for 48 h in the presence or absence of edaravone (100 mM, 24 h pretreatment). Cell lysates were used in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA). The proteins (20 mg/sample) were immediately heated for 5 min at 100 C and were subjected to SDS-PAGE on gels. Separated proteins were transferred to nitrocellulose membranes, and western blotting was performed using a gel loading kit and protein transfer system kit (BioRad, Hercules, CA, USA). Membranes were blocked by treatment with 5% skim milk in Tris buffer solution (TBS) and subsequently incubated with the primary polyclonal antibodies at a final dilution of 1: 1000. After three washes in TBS containing 0.1% Tween (TBST), membranes were incubated with peroxidaseconjugated secondary antibodies (final dilution, 1: 2000) in blocking buffer for 1 h and subsequently washed. Detection was performed by chemiluminescence using an ECL solution (Gendepot, Barker TX, USA). Beta-actin was used as a loading control. For the calculation of band density, Image J was used (Imagej.nih.gov/ij/ index.html). 2.7. Statistical analysis All values are represented as mean ± SD. For data analysis, we used the SPSS 20.0 statistical program. For the comparison of multiple groups in the MTT assay, flow cytometry analysis, ROS measurement, and caspase-3 activity, ANOVA was used. A p value of <0.05 was considered statistically significant. For multiple comparisons, Bonferroni correction was done.
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it was significantly higher than the cisplatin alone treated group (p < 0.01). 3.2. Hoechst 33258 staining Apoptosis was evaluated by the appearance of condensed and fragmented nuclei upon Hoechst 33258 staining. As compared with normal and edaravone applied cells (Fig. 3A, B), nuclei of HEI-OC1 cells after exposure to 15 mM cisplatin for 48 h showed condensed and fragmented DNA (Fig. 3C) and decreased numbers due to apoptosis. However, when cells were pretreated with 100 mM edaravone (Fig 3D), nuclei of HEI-OC1 cells had less condensed and fragmented DNA after the exposure to cisplatin. 3.3. Intracellular ROS measurement DCFH was converted into highly fluorescent 20 , 70 -dichlorofluorescein (DCF) when 15 mM cisplatin was applied (Fig 3G), but when the cells were pretreated with 100 mM edaravone, the amount of ROS increase was less (Fig 3H). We measure the intracellular levels of ROS generated by HEIOC1 cell in response to cisplatin and edaravone using the fluorescent probe DCFH-DA. As shown in Fig 4A, HEI-OC1 cells treated with 15 mM cisplatin exhibited markedly increased fluorescence intensity in comparison to the control group. Pretreatment with 100 mM edaravone for 24 h reduced the cisplatin induced generation of ROS. The mean probe intensity in comparison to that of the control is shown in Fig 4B. Treatment with 15 mM cisplatin increased the fluorescence intensity by 2.3 fold, whereas pretreatment with 100 mM edaravone decreased the fluorescence intensity by 1.9 folds (p < 0.01). 3.4. Measurement of caspase-3 activity
3. Results 3.1. MTT assay Cell viability was determined by MTT assay to verify whether edaravone was able to prevent apoptosis induced by cisplatin. When cultured cells were exposed to 15 mM cisplatin and 100 mM edaravone, cell viability was 51.9%, 101.4%, respectively. But when the cells were exposed to 15 mM cisplatin after had been pretreated with 100 mM edaravone for 24 h, cell viability was 64.6% (Fig 2) and
Caspase-3 activity is involved in cisplatin-induced toxicity and related to apoptotic changes in cisplatin ototoxicity. The administration of 15 mM cisplatin increased the activity of caspase-3 (9.96 ± 2.03 fold over the normal control) (Fig 5). The pretreatment of HEI-OC1 cells with 100 mM edaravone significantly decreased caspase-3 activity (7.03 ± 1.92 fold) as compared with cells treated with cisplatin alone (P < 0.01). 3.5. Immunoblotting of PARP The cleavages of PARP facilitate cellular disassembly and serve as a marker of cells undergoing apoptosis. Cisplatin increased cleaved PARP (4.9 ± 0.68 fold over the normal control) (Fig 6). The pretreatment of HEI-OC1 cells with 100 mM edaravone significantly decreased cleaved PARP (1.7 ± 0.82 fold) as compared with cells treated with cisplatin alone (P < 0.01). Thus, this result also supports the existence of an antiapoptotic effect of edaravone. 4. Discussions
Fig. 2. MTT assay When cultured cells were exposed to 15 mM cisplatin and 100 mM edaravone, cell viability was 51.9 (±7.1)% and 101.4 (±3.0)%, respectively. But when the cells were exposed to 15 mM cisplatin after pretreatment with 100 mM edaravone for 24 h, cell viability was 64.6 (±8.5)% which was significantly higher than in the cells treated with cisplatin alone (p < 0.01).
Many therapeutic agents such as metformin (Chang et al., 2014), alpha-lipoic acid (Kim et al., 2014), p-methionine (Campbell et al., 2003), N-acetylcystein (Dickey et al., 2004; Choe et al., 2004) and sodium thiosulfate (Wang et al., 2003) have been introduced to prevent cisplatin induced ototoxicity. Nonetheless, thus far, there is no ideal protective agent in clinical use. Edaravone is a neuroprotective agent with potent free radical scavenging and antioxidant actions without causing any serious side effects (Takumida and Anniko, 2006; Higashi et al., 2006). Edaravone is reported to reduce the amount of oxidative DNA damage (Maekawa et al., 2009) and decrease the amount of ROS
Please cite this article in press as: Im, G.J., et al., Protective role of edaravone against cisplatin-induced ototoxicity in an auditory cell line, Hearing Research (2015), http://dx.doi.org/10.1016/j.heares.2015.08.004
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Fig. 3. Measurement of intracellular ROS production and Hoechst 33258 staining Apoptosis was evaluated based on the appearance of condensed and fragmented nuclei in Hoechst 33258 staining (AeD). As compared to control cells and those treated with edaravone (A, B), cells exposed to 15 mM cisplatin for 48 h displayed nuclei that were condensed and fragmented (C; arrows). Group C also exhibited reduced cell density compared to groups A and B. However, when the cells were pretreated with 100 mM edaravone (D), they were less condensed and less fragmented. DCFH was converted into highly fluorescent 20 , 70 -dichlorofluorescein (DCF) when 15 mM cisplatin was applied (G), but when the cells were pretreated with 100 mM edaravone, the amount of ROS increase was less (H).
Fig. 4. Intracellular ROS measurement (A) HEI-OC1 cells treated with 15 mM cisplatin(3rd row) exhibited markedly increased fluorescence intensity in comparison to the control group(1st row). Pretreatment with 100 mM edaravone for 24 h (4th row) reduced the cisplatin induced generation of ROS. (B) Treatment with 15 mM cisplatin increased the fluorescence intensity by 2.3 (±0.2) fold, whereas pretreatment with 100 mM edaravone decreased the fluorescence intensity by 1.9 (±0.3) folds (p < 0.01).
Please cite this article in press as: Im, G.J., et al., Protective role of edaravone against cisplatin-induced ototoxicity in an auditory cell line, Hearing Research (2015), http://dx.doi.org/10.1016/j.heares.2015.08.004
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apoptosis. In Hoechst stain, the cells pretreated with edaraovone before cisplatin treatment had less condensed and fragmented nuclei. Also, the amount of ROS production was reduced when edaravone was used. Although edaravone is rather a safe agent when administered to the patients (Takumida and Anniko, 2006), its clinical application in cisplatin-induced ototoxicity remains unclear, because the problem as to whether edaravone enhances or suppresses the efficacy of cisplatin in the treatment of cancer cells remains unsettled yet. Also, since our experiment was conducted with an HEI-OC1 cell line, its conditions are not typical condition of cochlear cells as it is cultured under permissive conditions (33.8 C, 10% CO2). Therefore, in order to validate the protective effect of edaravone in cisplatin ototoxicity, these results obtained in vitro should be corroborated by in vivo studies. In conclusion, we found that edaravone protected cisplatin induced apoptotic cascade, reduced ROS production and enhanced cell viability in an auditory cell line. Fig. 5. Measurement of caspase-3 activity The administration of 15 mM cisplatin increased the activity of caspase-3 (9.96 ± 2.03) fold over the normal control. The pretreatment of HEI-OC1 cells with 100 mM edaravone significantly decreased caspase3 activity (7.03 ± 1.92 fold) as compared with cells treated with cisplatin alone (p < 0.01).
Acknowledgement This study was supported by Hallym University Research Fund (HURF-201510). References
and inhibits oxidation. It is assumed that edaravone inhibits lipoxygenase metabolism by trapping hydroxyl radicals, prevents peroxidation, and enhances of prostacyclin production (Takumida and Anniko, 2006; Watanabe et al., 1988). In our study, edaravone protected cisplatin induced cell death in auditory cell line by reducing ROS and preventing a sequential apoptotic cascade. Edaravone significantly decreased caspase-3 activity and reduced the cleaved PARP. Caspase-3 is reported to activate DNA fragmentation factor, which in turn activate endonucleases to cleave nuclear DNA, and ultimately leads to cell death (Lee et al., 2007). In addition, caspase-3 is responsible for the proteolytic cleavage of many key proteins, such as PARP, which is fundamental for cell viability. The cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing
Fig. 6. Immunoblotting of PARP Cisplatin increased cleaved PARP (4.9 ± 0.68 fold over the normal control). The pretreatment of HEI-OC1 cells with 100 mM edaravone significantly decreased cleaved PARP (1.7 ± 0.82 fold) as compared with cells treated with cisplatin alone (p < 0.01) (A, B).
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