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Protective effects of 1,2,3-triazole derivative KPR-A020 against cisplatin-induced ototoxicity in murine cochlear cultures
International Journal of Pediatric Otorhinolaryngology 96 (2017) 59e64
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International Journal of Pediatric Otorhinolaryngology journal homepage: http://www.ijporlonline.com/
Protective effects of 1,2,3-triazole derivative KPR-A020 against cisplatin-induced ototoxicity in murine cochlear cultures Ye-Ri Kim a, b, Da Jung Jung c, Se-Kyung Oh d, Taeho Lee e, In-Kyu Lee f, g, Kyu-Yup Lee, MD, PhD c, *, Un-Kyung Kim, PhD a, b, ** a
Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea Department of Otorhinolaryngology-Head and Neck Surgery, Kyungpook National University Hospital, Kyungpook National University School of Medicine, Daegu, Republic of Korea d Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Republic of Korea e Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University, Daegu, Republic of Korea f Department of Internal Medicine, Kyungpook National University Hospital, Kyungpook National University School of Medicine, Daegu, Republic of Korea g Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University Hospital, Daegu, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2016 Received in revised form 24 February 2017 Accepted 25 February 2017 Available online 28 February 2017
Cisplatin (cis-diaminedichloridoplatinum(II), cis-[PtCl2(NH3)2]) is an effective chemotherapeutic agent in the treatment of several types of malignant solid tumors but its clinical use is associated with ototoxicity. Several studies have investigated the effect of antioxidants on cisplatin-induced ototoxicity in mice. The triazole KPR-A020 has been shown to play a protective role against mitochondrial dysfunction by reducing the production of mitochondrial reactive oxygen species (ROS). The effect of KPR-A020 on cisplatin-induced ototoxicity was examined using cultures of cochlear explants. Healthy mice were randomly divided into 4 groups: control, treated with cisplatin alone (CP), treated with cisplatin and KPR-A020 (CP þ KPR-A020), and treated with KPR-A020 alone (KPR-A020). The cochlear explants were harvested for histological and immunohistochemical examinations. Biochemical analyses of the explants revealed that pre-treatment with KPR-A020 prevented an increase in mitochondrial ROS levels. Moreover, the CP þ KPR-A020 group showed better hair cell survival than the CP group. Immunohistochemical examinations of cochlear explants stained with anti-caspase-3 revealed greater immunopositivity in the CP group. The CP þ KPR-A020 group showed significantly less immunopositivity than the CP group (P < 0.05). Thus, it appears that KPR-A020 protects hair cells in the organ of Corti from cisplatin-induced toxicity by decreasing the production of mitochondrial ROS. The results of this study suggest that KPRA020 can be used as an antioxidant and antiapoptotic agent to prevent hearing loss caused by cisplatin induced-oxidative stress. © 2017 Elsevier B.V. All rights reserved.
Keywords: Triazole KPR-A020 Antioxidants Cochlear explants Cisplatin Ototoxicity
1. Introduction Cisplatin (cis-diaminedichloridoplatinum(II), cis-[PtCl2(NH3)2]) is a highly effective chemotherapeutic agent that is widely used to treat a variety of soft tissue neoplasms, including those associated
* Corresponding author. ** Corresponding author. Department of Biology, College of Natural Sciences Kyungpook National University, Daegu 41566, Republic of Korea. E-mail addresses: [email protected] (Y.-R. Kim), [email protected] (D.J. Jung), [email protected] (S.-K. Oh), [email protected] (T. Lee), [email protected]. kr (I.-K. Lee), [email protected] (K.-Y. Lee), [email protected] (U.-K. Kim). http://dx.doi.org/10.1016/j.ijporl.2017.02.028 0165-5876/© 2017 Elsevier B.V. All rights reserved.
with ovarian, testicular, cervical, lung, bladder, and head and neck cancer. Once in the cytoplasm of a cell, it is well established that cisplatin undergoes a conversion to its aquated derivative, cis[PtCl(NH3)2(OH2)]þ, driven by the approximately 13-fold lower concentration of chloride in the cytoplasm relative to the extracellular fluid [1e3]. This aquated derivative is recognized to be responsible for the anti-cancer activity of the drug, because of its ability to react with DNA, eventually forming a bifunctional adduct, which inhibits DNA replication and initiates apoptosis [4]. However, serious side effects include nephrotoxicity, neurotoxicity, and ototoxicity. In particular, the ototoxicity limits the dose that can be administered. Some audiometric studies have reported
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elevated hearing thresholds in 75e100% of patients treated with cisplatin [5]. This is particularly problematic in children receiving cisplatin. Risk factors that increase the risk for ototoxicity from cisplatin in children include younger age, larger cumulative doses, pre-existing hearing loss, renal disease [6,7], and irradiation of the brain or skull base [8]. Both in vitro and laboratory animal studies show that cisplatin interacts with cochlear tissues such as the outer hair cells of the organ of Corti, the stria vascularis, the spiral ligament, and the spiral ganglionic cells, generating a robust reactive oxygen species (ROS) response [9e13]. ROS that are physiologically produced in mitochondria are involved in signaling pathways that mediate adaptive responses to stress. It also helps to regulate cellular growth and differentiation [14]. However, the overproduction of ROS can be accumulated in a cell which results in altering enzymatic and ion channel activities, and even inducing apoptosis [15]. It has been reported that certain triazole-based compounds, including KPR-A020, inhibit RANKL-induced osteoclast differentiation and bone resorption [16]. Triazoles are heterocyclic compounds comprising three nitrogen and two carbon atoms. Triazoles can bind to various biological molecules, including enzymes and receptors. Consequently, triazole-based compounds have been shown to have diverse biological activities, including antiinflammatory, anti-tumor, anti-tubercular, and anti-fungal activities [17,18]. In addition, many triazole derivatives have low toxicity, high bioavailability, and few side effects [19,20]. Therefore, researchers have attempted to use triazole derivatives as therapeutic agents against infections, cardiac diseases, and seizures [21]. However, there have been few reports describing the direct antioxidant effect of triazole derivatives on oxidant-induced ototoxicity. Different compounds containing 1,2,3-triazoles have been reported to have interesting antiproliferative activities [22e26]. This recently led us to investigate the synthesis of different terpenes coupled to triazole rings using click-chemistry [27e29] and to assess their antiproliferative activities. The antifungal activity of triazoles is well known; fluconazole, itraconazole, voriconazole, and posaconazole are the most used antifungal agents in the clinic [20]. In this study, we investigated the use of KPR-A020, a triazole derivative, as a protective agent against cisplatin-induced ototoxicity by evaluating its effect on mitochondrial ROS production and apoptosis in murine cochlear cultures. 2. Materials and methods 2.1. Culture of murine cochlear explants Primary cochlear explants were prepared from ‘postnatal day 3’ Institute for Cancer Research (ICR) mice, purchased from Hyochang Science (Daegu, Republic of Korea). Culturing of mouse cochlear explants was performed as described previously [30]. Dissected organs of Corti were attached to four-well culture dishes and subsequently incubated in the culture medium under a humidified atmosphere of 5% CO2 at 37 C, which contained high-glucose Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Hyclone) and ampicillin (10 mg/mL; Life Technologies, Carlsbad, CA, USA). The cultured organs of Corti were divided into four groups for KPR-A020 treatment: control (CT, n ¼ 4), cisplatin alone (CP, n ¼ 4), treatment with KPR-A020 prior to cisplatin (CP þ KPR-A020, n ¼ 4), and KPR-A020 alone (KPR-A020, n ¼ 4). After a 16 h incubation, organs of Corti were treated with KPR-A020 (1 mM in DMSO; Sigma, St. Louis, MO, USA) and diluted in culture medium for 1 h under a humidified atmosphere of 5% CO2 at 37 C. After the 1 h incubation, cisplatin (20 mM; Ildong Pharmaceutical Co., Daegu, Republic of Korea) was
added to the CP group and CP þ KPR-A020 group. All animal experiments were conducted in accordance with the guidelines of the ‘Institutional Animal Care and Use Committee’ of Kyungpook National University. These experiments were approved by the ‘Committee on the Ethics of Animal Experiments’ of Kyungpook National University. 2.2. Histological evaluation To investigate the protective effects of KPR-A020 against cisplatin-induced ototoxicity in the organ of Corti, we examined the morphology of inner hair cells (IHCs) and outer hair cells (OHCs) within the organs of Corti. At the end of a 30 h or 48 h incubation period, all cochlear explants were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (PFA, pH 7.4) in PBS for 15 min, and permeabilized with 0.1% Triton X-100 in PBS (PBSTx) for 30 min at room temperature (RT) [9]. Permeabilized samples were blocked with 5% normal goat serum diluted in PBS-Tx for 1 h at RT and then stained with either Alexa Fluor 488- or 555conjugated phalloidin (1:1000; Invitrogen-Molecular Probes, Eugene, OR, USA) in PBS-Tx for 3 h at RT. The specimens were rinsed three times with PBS and mounted on glass slides using Fluoromount (Sigma, St. Louis, MO, USA). Images were captured using Zeiss Axio Imager A2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). 2.3. Determination of mitochondrial ROS levels MitoSOX-red (Invitrogen, Carlsbad, CA, USA) is a novel fluorogenic indicator of superoxide molecules generated specifically from mitochondria. At the end of the 30 h incubation period, all cochlear explants were washed with PBS and stained for 10 min with MitoSOX-red (5 mM) under a humidified atmosphere of 5% CO2 at 37 C. After washing with PBS, the specimens were visualized using a Zeiss Axio Imager A2 fluorescence microscope. 2.4. Immunohistochemical analysis and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay To assess apoptotic cell death in the organs of Corti, we conducted immunohistochemistry using antibodies directed against active caspase-3. We also evaluated DNA fragmentation using the TUNEL assay. At the end of the 48 h incubation period, all cochlear explants were fixed, permeabilized, and blocked as previously described. Following blocking, the specimens were stained with antibodies directed against active caspase-3 (1:1000; Cell Signaling Technology, Beverly, MA, USA) and diluted in the blocking solution at 4 C overnight. Samples were then washed three times with PBS and incubated with Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:1000; Invitrogen, La Jolla, CA, USA) and diluted in the blocking solution for 1 h at RT. Following this, Factin was labeled for 3 h at RT with Alexa Fluor 555-conjugated phalloidin in PBS-Tx and mounted on glass slides using Fluoromount. DNA fragmentation was evaluated as a marker of apoptotic cell death, using the TUNEL assay according to the manufacturer's protocol (Promega, Madison, WI, USA). The cochlear explants were fixed with 4% PFA in PBS for 15 min at RT and then washed with PBS. They were then permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate in distilled water for 10 min at 4 C, followed by staining with TUNEL working solution in the dark for 30 min at 37 C. F-actin was labeled with Alexa Fluor 555-conjugated phalloidin stain in PBS-Tx for 3 h at RT. The specimens were mounted on glass slides using Fluoromount and visualized using a Zeiss Axio Imager A2 fluorescence microscope.
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3. Results and discussion To study the protective effects of KPR-A020, ex vivo cochlear explants in mice were pre-treated with 5 mM of KPR-A020 before the addition of cisplatin. As shown in Fig. 1A, cisplatin treatment led to extensive degeneration of both IHCs and OHCs. Of note, this cisplatin-induced ototoxicity was more severe in basal, rather than apical and middle, turns of the cochlea. In contrast, stereocilia in the CP þ KPR-A020 group were found in intact arrangements in both IHCs and OHCs, similar to those of the CT group. In addition,
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the CP þ KPR-A020 group was found to lose significantly less hair cells compared to the CP group (n ¼ 4 for each group, Fig. 1B). Furthermore, no deleterious effects were observed when the cochlear tissues were treated with KPR-A020 only. These results demonstrate that KPR-A020 prevents cisplatin-induced cochlear hair cell loss. Cisplatin is known to promote the generation of ROS and free radicals in mitochondria of a cell. Therefore, we used MitoSOX-red to investigate whether KPR-A020 could attenuate the disruption of mitochondria arising from cisplatin-induced mitochondrial ROS
Fig. 1. Effect of KPR-A020 on cisplatin-induced ototoxicity. (A) Immunofluorescence images of organ of Corti explants from the various experimental groups (control [CT], cisplatin-treated [CP alone], cisplatin- and KPR-A020-treated [CP þ KPR-A020], and KPR-A020-treated [KPR-A020 alone]). Hair cells were stained with phalloidin (green). (B) The yaxis represents the average number of surviving IHCs and OHCs in each of the three (apical, middle, and base) regions within a 200 mm region of the organ of Corti (n ¼ 4). Data are provided as mean ± SD. *P < 0.05 and **P < 0.001 compared with the cisplatin-treated groups. CP: cisplatin, Scale bar: 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Effects of KPR-A020 on the generation of mitochondrial reactive oxygen species [1] in cisplatin-treated cochlear hair cells. (A) Production of mitochondrial ROS was examined using MitoSOX-red staining of the various groups (control [CT], cisplatin-treated [CP alone], cisplatin- and KPR-A020-treated [CP þ KPR-A020], and KPR-A020-treated [KPR-A020 alone]). (B) Statistical analysis of the data indicates that the number of MitoSOX positive hair cells significantly decreased in the CP þ KPR-A020 group compared to the group treated with CP alone. Data are presented as mean ± SD. *P < 0.05 and **P < 0.001 compared with the CP treatment group. CP: cisplatin. Scale bar: 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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production in the inner ear. Cochlear explants exposed to 20 mM cisplatin exhibited considerable red fluorescent intensity, indicating a higher generation of mitochondrial ROS than in the explants in the CT group (Fig. 2A). In contrast, a significant decrease in the fluorescence was observed in the CP þ KPR-A020 group. In addition, the fluorescence was more pronounced at the apex of the tissue, rather than in the base, in the CP þ KPR-A020 group (n ¼ 4 for each group; Fig. 2B). After establishing that KPR-A020 could inhibit mitochondrial disruption by decreasing the generation of mitochondrial ROS, we detected apoptotic hair cells in cochlear explants. In order to confirm the increase and decrease of apoptosis that occurred after the increase of mitochondrial ROS in the cells, the experiment was carried out after 48 h incubation with cisplatin unlike the previous one. When compared with the group of 30 h incubation, the CP group of 48 h incubation was detected that more stereocilia degenerated than group of 30 h incubation (Fig. 3A and C). Typically, the middle turn of the cochlea is shown. As shown in Fig. 3A,
treated cochlea in the CP group showed substantial activation of cleaved caspase-3. However, green fluorescence, which visualizes levels of cleaved caspase-3 activation, was significantly lower in the CP þ KPR-A020 group than in the CP group (n ¼ 4 for each group; Fig. 3A). The TUNEL signal, which labels nicked DNA fragments, was also visibly decreased in the CP þ KPR-A020 group compared to the CP group. The average number of apoptotic hair cells in each group, as determined by cleaved caspase-3 labeling and TUNEL labeling, clearly demonstrates that KPR-A020 prevents cisplatin-induced apoptosis in cochlear explants (n ¼ 4 for each group; Fig. 3B). Triazoles have multiple biological activities, including potent anti-allergic, anti-inflammatory, and antiviral actions, which may result, at least in part, from their antioxidant and free radicalscavenging abilities. One of the major anticancer mechanisms of the 1,2,3-triazole has been reported to have the binding affinity to the nucleus membrane receptors including glycogen synthase kinase-3, GABA receptors and muscarine receptors, and disturb their own functions which make it as the anticancer drug [31e33].
Fig. 3. Effects of cisplatin and KPR-A020 on apoptosis and DNA damage in cochlear hair cells. Apoptotic cells in cochlear explants were determined by active caspase-3 assays (A, B) and TUNEL assays (C, D). Microscopy images of cells labeled for active caspase-3 or TUNEL (green), and cells labeled with phalloidin [17] (A, C). Hair cells positive for green fluorescence were counted every 200 mm along the apex, mid, and base regions of the organ of Corti (B, D). Values are indicated as the mean ± SD. Asterisks indicate statistically significant differences compared to the CP group (n ¼ 4, *P < 0.05 and **P < 0.001). CP: cisplatin. Scale bars: 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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In Cetin et al., the relationship between substituted functional groups to 1,2,4-triazole rings and the antioxidant potential among tested compounds was found to depend mainly on the presence of electron-rich moieties, which hold high resonance stability [34]. Moreover, KPR-A020 contains electron-rich moieties, and this may account for its antioxidant ability, which is responsible for decreasing mitochondrial ROS levels and preventing apoptosis. It has been reported that the uptake of cisplatin into cochlear hair cells occurs via endocytosis and mechanoelectrical transducer channels [35,36]. Cisplatin promotes apoptosis in these cells by increasing ROS production, which leads to downstream cytokine deprivation, permeabilization of mitochondrial membranes, and ultimately DNA damage [9e13]. Cisplatin-induced ototoxicity was more severe in OHCs than IHCs in our study. Similarly, ototoxicity was more severe in basal regions of the cochlea than apical regions. Our results are consistent with those of previous reports, which demonstrated that cisplatin-induced ototoxicity begins at the base of the cochlea and progresses toward the apex [37]. The basal cochlear region is more metabolically active than the apical region, which may partially explain its enhanced sensitivity to oxidative stress [38]. As expected, the CP group displayed significantly decreased hair cell death and a diminished MitoSOX-red signal compared to the CT group. These results clearly support our hypothesis that an increased mitochondrial ROS level can result in apoptotic cell death, and that elimination of mitochondrial ROS may provide a suitable preventative treatment for cisplatin-induced ototoxicity. Cisplatin-treated cochlear explants produce strong MitoSOX-red fluorescence signals; however, these signals were significantly attenuated when the explants had been pre-treated with KPRA020. These data suggest that KPR-A020 successfully diminished mitochondrial ROS levels in cisplatin-treated cochlear explants and that pre-treatment with KPR-A020 can prevent cisplatin-induced cochlear toxicity. In conclusion, this study has demonstrated that treatment of mouse cochlear cultures with cisplatin leads to apoptotic DNA fragmentation and the resulting ototoxicity can be prevented by KPR-A020 by diminishing the levels of mitochondrial ROS. Further study will be need to demonstrate that KOR-A020 is a promising drug for cisplatin-induced ototoxicity without interfering with anticancer effect of cisplatin via in vivo study. Acknowledgements Our research was supported by the Basic Science Research Program, through the National Research Foundation of Korea, funded by the Ministry of Science, ICT and Future Planning (2014R1A1A3049993). This research was supported by a grant of the Korea Health technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (HI16C1501) and by a grant from the KHIDI, funded by the Ministry of Health & Welfare, Republic of Korea (HI14C0384). References [1] B. Rosenberg, Anticancer activity of cis-dichlorodiammineplatinum(II) and some relevant chemistry, Cancer Treat. Rep. 63 (1979) 1433e1438. [2] E. Smith, A.P. Brock, The effect of reduced osmolarity on platinum drug toxicity, Br. J. Cancer 59 (1989) 873e875. [3] T.W. Hambley, E.C. Ling, V.P. Munk, M.S. Davies, Steric control of stereoselective interactions between the platinum(II) complex [PtCl2(1,4diazacycloheptane)] and DNA: comparison with cis-[PtCl2(NH3)2] and [PtCl2(ethane-1,2-diamine)] using DNA binding and molecular modeling studies, J. Biol. Inorg. Chem. 6 (2001) 534e542. [4] K. Barabas, R. Milner, D. Lurie, C. Adin, Cisplatin: a review of toxicities and therapeutic applications, Vet. Comp. Oncol. 6 (2008) 1e18.
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Protective effects of 1,2,3-triazole derivative KPR-A020 against cisplatin-induced ototoxicity in murine cochlear cultures Ye-Ri Kim a, b, Da Jung Jung c, Se-Kyung Oh d, Taeho Lee e, In-Kyu Lee f, g, Kyu-Yup Lee, MD, PhD c, *, Un-Kyung Kim, PhD a, b, ** a
Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea Department of Otorhinolaryngology-Head and Neck Surgery, Kyungpook National University Hospital, Kyungpook National University School of Medicine, Daegu, Republic of Korea d Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Republic of Korea e Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University, Daegu, Republic of Korea f Department of Internal Medicine, Kyungpook National University Hospital, Kyungpook National University School of Medicine, Daegu, Republic of Korea g Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University Hospital, Daegu, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2016 Received in revised form 24 February 2017 Accepted 25 February 2017 Available online 28 February 2017
Cisplatin (cis-diaminedichloridoplatinum(II), cis-[PtCl2(NH3)2]) is an effective chemotherapeutic agent in the treatment of several types of malignant solid tumors but its clinical use is associated with ototoxicity. Several studies have investigated the effect of antioxidants on cisplatin-induced ototoxicity in mice. The triazole KPR-A020 has been shown to play a protective role against mitochondrial dysfunction by reducing the production of mitochondrial reactive oxygen species (ROS). The effect of KPR-A020 on cisplatin-induced ototoxicity was examined using cultures of cochlear explants. Healthy mice were randomly divided into 4 groups: control, treated with cisplatin alone (CP), treated with cisplatin and KPR-A020 (CP þ KPR-A020), and treated with KPR-A020 alone (KPR-A020). The cochlear explants were harvested for histological and immunohistochemical examinations. Biochemical analyses of the explants revealed that pre-treatment with KPR-A020 prevented an increase in mitochondrial ROS levels. Moreover, the CP þ KPR-A020 group showed better hair cell survival than the CP group. Immunohistochemical examinations of cochlear explants stained with anti-caspase-3 revealed greater immunopositivity in the CP group. The CP þ KPR-A020 group showed significantly less immunopositivity than the CP group (P < 0.05). Thus, it appears that KPR-A020 protects hair cells in the organ of Corti from cisplatin-induced toxicity by decreasing the production of mitochondrial ROS. The results of this study suggest that KPRA020 can be used as an antioxidant and antiapoptotic agent to prevent hearing loss caused by cisplatin induced-oxidative stress. © 2017 Elsevier B.V. All rights reserved.
Keywords: Triazole KPR-A020 Antioxidants Cochlear explants Cisplatin Ototoxicity
1. Introduction Cisplatin (cis-diaminedichloridoplatinum(II), cis-[PtCl2(NH3)2]) is a highly effective chemotherapeutic agent that is widely used to treat a variety of soft tissue neoplasms, including those associated
* Corresponding author. ** Corresponding author. Department of Biology, College of Natural Sciences Kyungpook National University, Daegu 41566, Republic of Korea. E-mail addresses: [email protected] (Y.-R. Kim), [email protected] (D.J. Jung), [email protected] (S.-K. Oh), [email protected] (T. Lee), [email protected]. kr (I.-K. Lee), [email protected] (K.-Y. Lee), [email protected] (U.-K. Kim). http://dx.doi.org/10.1016/j.ijporl.2017.02.028 0165-5876/© 2017 Elsevier B.V. All rights reserved.
with ovarian, testicular, cervical, lung, bladder, and head and neck cancer. Once in the cytoplasm of a cell, it is well established that cisplatin undergoes a conversion to its aquated derivative, cis[PtCl(NH3)2(OH2)]þ, driven by the approximately 13-fold lower concentration of chloride in the cytoplasm relative to the extracellular fluid [1e3]. This aquated derivative is recognized to be responsible for the anti-cancer activity of the drug, because of its ability to react with DNA, eventually forming a bifunctional adduct, which inhibits DNA replication and initiates apoptosis [4]. However, serious side effects include nephrotoxicity, neurotoxicity, and ototoxicity. In particular, the ototoxicity limits the dose that can be administered. Some audiometric studies have reported
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elevated hearing thresholds in 75e100% of patients treated with cisplatin [5]. This is particularly problematic in children receiving cisplatin. Risk factors that increase the risk for ototoxicity from cisplatin in children include younger age, larger cumulative doses, pre-existing hearing loss, renal disease [6,7], and irradiation of the brain or skull base [8]. Both in vitro and laboratory animal studies show that cisplatin interacts with cochlear tissues such as the outer hair cells of the organ of Corti, the stria vascularis, the spiral ligament, and the spiral ganglionic cells, generating a robust reactive oxygen species (ROS) response [9e13]. ROS that are physiologically produced in mitochondria are involved in signaling pathways that mediate adaptive responses to stress. It also helps to regulate cellular growth and differentiation [14]. However, the overproduction of ROS can be accumulated in a cell which results in altering enzymatic and ion channel activities, and even inducing apoptosis [15]. It has been reported that certain triazole-based compounds, including KPR-A020, inhibit RANKL-induced osteoclast differentiation and bone resorption [16]. Triazoles are heterocyclic compounds comprising three nitrogen and two carbon atoms. Triazoles can bind to various biological molecules, including enzymes and receptors. Consequently, triazole-based compounds have been shown to have diverse biological activities, including antiinflammatory, anti-tumor, anti-tubercular, and anti-fungal activities [17,18]. In addition, many triazole derivatives have low toxicity, high bioavailability, and few side effects [19,20]. Therefore, researchers have attempted to use triazole derivatives as therapeutic agents against infections, cardiac diseases, and seizures [21]. However, there have been few reports describing the direct antioxidant effect of triazole derivatives on oxidant-induced ototoxicity. Different compounds containing 1,2,3-triazoles have been reported to have interesting antiproliferative activities [22e26]. This recently led us to investigate the synthesis of different terpenes coupled to triazole rings using click-chemistry [27e29] and to assess their antiproliferative activities. The antifungal activity of triazoles is well known; fluconazole, itraconazole, voriconazole, and posaconazole are the most used antifungal agents in the clinic [20]. In this study, we investigated the use of KPR-A020, a triazole derivative, as a protective agent against cisplatin-induced ototoxicity by evaluating its effect on mitochondrial ROS production and apoptosis in murine cochlear cultures. 2. Materials and methods 2.1. Culture of murine cochlear explants Primary cochlear explants were prepared from ‘postnatal day 3’ Institute for Cancer Research (ICR) mice, purchased from Hyochang Science (Daegu, Republic of Korea). Culturing of mouse cochlear explants was performed as described previously [30]. Dissected organs of Corti were attached to four-well culture dishes and subsequently incubated in the culture medium under a humidified atmosphere of 5% CO2 at 37 C, which contained high-glucose Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Hyclone) and ampicillin (10 mg/mL; Life Technologies, Carlsbad, CA, USA). The cultured organs of Corti were divided into four groups for KPR-A020 treatment: control (CT, n ¼ 4), cisplatin alone (CP, n ¼ 4), treatment with KPR-A020 prior to cisplatin (CP þ KPR-A020, n ¼ 4), and KPR-A020 alone (KPR-A020, n ¼ 4). After a 16 h incubation, organs of Corti were treated with KPR-A020 (1 mM in DMSO; Sigma, St. Louis, MO, USA) and diluted in culture medium for 1 h under a humidified atmosphere of 5% CO2 at 37 C. After the 1 h incubation, cisplatin (20 mM; Ildong Pharmaceutical Co., Daegu, Republic of Korea) was
added to the CP group and CP þ KPR-A020 group. All animal experiments were conducted in accordance with the guidelines of the ‘Institutional Animal Care and Use Committee’ of Kyungpook National University. These experiments were approved by the ‘Committee on the Ethics of Animal Experiments’ of Kyungpook National University. 2.2. Histological evaluation To investigate the protective effects of KPR-A020 against cisplatin-induced ototoxicity in the organ of Corti, we examined the morphology of inner hair cells (IHCs) and outer hair cells (OHCs) within the organs of Corti. At the end of a 30 h or 48 h incubation period, all cochlear explants were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (PFA, pH 7.4) in PBS for 15 min, and permeabilized with 0.1% Triton X-100 in PBS (PBSTx) for 30 min at room temperature (RT) [9]. Permeabilized samples were blocked with 5% normal goat serum diluted in PBS-Tx for 1 h at RT and then stained with either Alexa Fluor 488- or 555conjugated phalloidin (1:1000; Invitrogen-Molecular Probes, Eugene, OR, USA) in PBS-Tx for 3 h at RT. The specimens were rinsed three times with PBS and mounted on glass slides using Fluoromount (Sigma, St. Louis, MO, USA). Images were captured using Zeiss Axio Imager A2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). 2.3. Determination of mitochondrial ROS levels MitoSOX-red (Invitrogen, Carlsbad, CA, USA) is a novel fluorogenic indicator of superoxide molecules generated specifically from mitochondria. At the end of the 30 h incubation period, all cochlear explants were washed with PBS and stained for 10 min with MitoSOX-red (5 mM) under a humidified atmosphere of 5% CO2 at 37 C. After washing with PBS, the specimens were visualized using a Zeiss Axio Imager A2 fluorescence microscope. 2.4. Immunohistochemical analysis and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay To assess apoptotic cell death in the organs of Corti, we conducted immunohistochemistry using antibodies directed against active caspase-3. We also evaluated DNA fragmentation using the TUNEL assay. At the end of the 48 h incubation period, all cochlear explants were fixed, permeabilized, and blocked as previously described. Following blocking, the specimens were stained with antibodies directed against active caspase-3 (1:1000; Cell Signaling Technology, Beverly, MA, USA) and diluted in the blocking solution at 4 C overnight. Samples were then washed three times with PBS and incubated with Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:1000; Invitrogen, La Jolla, CA, USA) and diluted in the blocking solution for 1 h at RT. Following this, Factin was labeled for 3 h at RT with Alexa Fluor 555-conjugated phalloidin in PBS-Tx and mounted on glass slides using Fluoromount. DNA fragmentation was evaluated as a marker of apoptotic cell death, using the TUNEL assay according to the manufacturer's protocol (Promega, Madison, WI, USA). The cochlear explants were fixed with 4% PFA in PBS for 15 min at RT and then washed with PBS. They were then permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate in distilled water for 10 min at 4 C, followed by staining with TUNEL working solution in the dark for 30 min at 37 C. F-actin was labeled with Alexa Fluor 555-conjugated phalloidin stain in PBS-Tx for 3 h at RT. The specimens were mounted on glass slides using Fluoromount and visualized using a Zeiss Axio Imager A2 fluorescence microscope.
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3. Results and discussion To study the protective effects of KPR-A020, ex vivo cochlear explants in mice were pre-treated with 5 mM of KPR-A020 before the addition of cisplatin. As shown in Fig. 1A, cisplatin treatment led to extensive degeneration of both IHCs and OHCs. Of note, this cisplatin-induced ototoxicity was more severe in basal, rather than apical and middle, turns of the cochlea. In contrast, stereocilia in the CP þ KPR-A020 group were found in intact arrangements in both IHCs and OHCs, similar to those of the CT group. In addition,
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the CP þ KPR-A020 group was found to lose significantly less hair cells compared to the CP group (n ¼ 4 for each group, Fig. 1B). Furthermore, no deleterious effects were observed when the cochlear tissues were treated with KPR-A020 only. These results demonstrate that KPR-A020 prevents cisplatin-induced cochlear hair cell loss. Cisplatin is known to promote the generation of ROS and free radicals in mitochondria of a cell. Therefore, we used MitoSOX-red to investigate whether KPR-A020 could attenuate the disruption of mitochondria arising from cisplatin-induced mitochondrial ROS
Fig. 1. Effect of KPR-A020 on cisplatin-induced ototoxicity. (A) Immunofluorescence images of organ of Corti explants from the various experimental groups (control [CT], cisplatin-treated [CP alone], cisplatin- and KPR-A020-treated [CP þ KPR-A020], and KPR-A020-treated [KPR-A020 alone]). Hair cells were stained with phalloidin (green). (B) The yaxis represents the average number of surviving IHCs and OHCs in each of the three (apical, middle, and base) regions within a 200 mm region of the organ of Corti (n ¼ 4). Data are provided as mean ± SD. *P < 0.05 and **P < 0.001 compared with the cisplatin-treated groups. CP: cisplatin, Scale bar: 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Effects of KPR-A020 on the generation of mitochondrial reactive oxygen species [1] in cisplatin-treated cochlear hair cells. (A) Production of mitochondrial ROS was examined using MitoSOX-red staining of the various groups (control [CT], cisplatin-treated [CP alone], cisplatin- and KPR-A020-treated [CP þ KPR-A020], and KPR-A020-treated [KPR-A020 alone]). (B) Statistical analysis of the data indicates that the number of MitoSOX positive hair cells significantly decreased in the CP þ KPR-A020 group compared to the group treated with CP alone. Data are presented as mean ± SD. *P < 0.05 and **P < 0.001 compared with the CP treatment group. CP: cisplatin. Scale bar: 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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production in the inner ear. Cochlear explants exposed to 20 mM cisplatin exhibited considerable red fluorescent intensity, indicating a higher generation of mitochondrial ROS than in the explants in the CT group (Fig. 2A). In contrast, a significant decrease in the fluorescence was observed in the CP þ KPR-A020 group. In addition, the fluorescence was more pronounced at the apex of the tissue, rather than in the base, in the CP þ KPR-A020 group (n ¼ 4 for each group; Fig. 2B). After establishing that KPR-A020 could inhibit mitochondrial disruption by decreasing the generation of mitochondrial ROS, we detected apoptotic hair cells in cochlear explants. In order to confirm the increase and decrease of apoptosis that occurred after the increase of mitochondrial ROS in the cells, the experiment was carried out after 48 h incubation with cisplatin unlike the previous one. When compared with the group of 30 h incubation, the CP group of 48 h incubation was detected that more stereocilia degenerated than group of 30 h incubation (Fig. 3A and C). Typically, the middle turn of the cochlea is shown. As shown in Fig. 3A,
treated cochlea in the CP group showed substantial activation of cleaved caspase-3. However, green fluorescence, which visualizes levels of cleaved caspase-3 activation, was significantly lower in the CP þ KPR-A020 group than in the CP group (n ¼ 4 for each group; Fig. 3A). The TUNEL signal, which labels nicked DNA fragments, was also visibly decreased in the CP þ KPR-A020 group compared to the CP group. The average number of apoptotic hair cells in each group, as determined by cleaved caspase-3 labeling and TUNEL labeling, clearly demonstrates that KPR-A020 prevents cisplatin-induced apoptosis in cochlear explants (n ¼ 4 for each group; Fig. 3B). Triazoles have multiple biological activities, including potent anti-allergic, anti-inflammatory, and antiviral actions, which may result, at least in part, from their antioxidant and free radicalscavenging abilities. One of the major anticancer mechanisms of the 1,2,3-triazole has been reported to have the binding affinity to the nucleus membrane receptors including glycogen synthase kinase-3, GABA receptors and muscarine receptors, and disturb their own functions which make it as the anticancer drug [31e33].
Fig. 3. Effects of cisplatin and KPR-A020 on apoptosis and DNA damage in cochlear hair cells. Apoptotic cells in cochlear explants were determined by active caspase-3 assays (A, B) and TUNEL assays (C, D). Microscopy images of cells labeled for active caspase-3 or TUNEL (green), and cells labeled with phalloidin [17] (A, C). Hair cells positive for green fluorescence were counted every 200 mm along the apex, mid, and base regions of the organ of Corti (B, D). Values are indicated as the mean ± SD. Asterisks indicate statistically significant differences compared to the CP group (n ¼ 4, *P < 0.05 and **P < 0.001). CP: cisplatin. Scale bars: 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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In Cetin et al., the relationship between substituted functional groups to 1,2,4-triazole rings and the antioxidant potential among tested compounds was found to depend mainly on the presence of electron-rich moieties, which hold high resonance stability [34]. Moreover, KPR-A020 contains electron-rich moieties, and this may account for its antioxidant ability, which is responsible for decreasing mitochondrial ROS levels and preventing apoptosis. It has been reported that the uptake of cisplatin into cochlear hair cells occurs via endocytosis and mechanoelectrical transducer channels [35,36]. Cisplatin promotes apoptosis in these cells by increasing ROS production, which leads to downstream cytokine deprivation, permeabilization of mitochondrial membranes, and ultimately DNA damage [9e13]. Cisplatin-induced ototoxicity was more severe in OHCs than IHCs in our study. Similarly, ototoxicity was more severe in basal regions of the cochlea than apical regions. Our results are consistent with those of previous reports, which demonstrated that cisplatin-induced ototoxicity begins at the base of the cochlea and progresses toward the apex [37]. The basal cochlear region is more metabolically active than the apical region, which may partially explain its enhanced sensitivity to oxidative stress [38]. As expected, the CP group displayed significantly decreased hair cell death and a diminished MitoSOX-red signal compared to the CT group. These results clearly support our hypothesis that an increased mitochondrial ROS level can result in apoptotic cell death, and that elimination of mitochondrial ROS may provide a suitable preventative treatment for cisplatin-induced ototoxicity. Cisplatin-treated cochlear explants produce strong MitoSOX-red fluorescence signals; however, these signals were significantly attenuated when the explants had been pre-treated with KPRA020. These data suggest that KPR-A020 successfully diminished mitochondrial ROS levels in cisplatin-treated cochlear explants and that pre-treatment with KPR-A020 can prevent cisplatin-induced cochlear toxicity. In conclusion, this study has demonstrated that treatment of mouse cochlear cultures with cisplatin leads to apoptotic DNA fragmentation and the resulting ototoxicity can be prevented by KPR-A020 by diminishing the levels of mitochondrial ROS. Further study will be need to demonstrate that KOR-A020 is a promising drug for cisplatin-induced ototoxicity without interfering with anticancer effect of cisplatin via in vivo study. Acknowledgements Our research was supported by the Basic Science Research Program, through the National Research Foundation of Korea, funded by the Ministry of Science, ICT and Future Planning (2014R1A1A3049993). This research was supported by a grant of the Korea Health technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (HI16C1501) and by a grant from the KHIDI, funded by the Ministry of Health & Welfare, Republic of Korea (HI14C0384). References [1] B. Rosenberg, Anticancer activity of cis-dichlorodiammineplatinum(II) and some relevant chemistry, Cancer Treat. Rep. 63 (1979) 1433e1438. [2] E. Smith, A.P. Brock, The effect of reduced osmolarity on platinum drug toxicity, Br. J. Cancer 59 (1989) 873e875. [3] T.W. Hambley, E.C. Ling, V.P. Munk, M.S. Davies, Steric control of stereoselective interactions between the platinum(II) complex [PtCl2(1,4diazacycloheptane)] and DNA: comparison with cis-[PtCl2(NH3)2] and [PtCl2(ethane-1,2-diamine)] using DNA binding and molecular modeling studies, J. Biol. Inorg. Chem. 6 (2001) 534e542. [4] K. Barabas, R. Milner, D. Lurie, C. Adin, Cisplatin: a review of toxicities and therapeutic applications, Vet. Comp. Oncol. 6 (2008) 1e18.
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