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Role of autophagy in cisplatin-induced ototoxicity
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PEDOT-7715; No. of Pages 6 International Journal of Pediatric Otorhinolaryngology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Role of autophagy in cisplatin-induced ototoxicity Cha Kyung Youn a,b, Jun Kim a, Jun-Hee Park a, Nam Yong Do a, Sung Il Cho a,* a b
Department of Otolaryngology—Head and Neck Surgery, Chosun University School of Medicine, Gwangju, South Korea Division of Natural Medical Science, Chosun University School of Medicine, Gwangju, South Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 February 2015 Received in revised form 4 August 2015 Accepted 5 August 2015 Available online xxx
Objective: Hearing loss is a major side effect of cisplatin chemotherapy. Although cell death in cisplatininduced ototoxicity is primarily caused by apoptosis, the exact mechanism behind the ototoxic effects of cisplatin is not fully understood. Autophagy is generally known as a pro-survival mechanism that protects cells under starvation or stress conditions. However, recent research has reported that autophagy plays a functional role in cell death also. This study aimed to investigate the role of autophagy in cisplatin-induced ototoxicity in an auditory cell line. Methods: Cultured HEI-OC1 cells were exposed to 30 mM cisplatin for 48 h, and cell viability was tested using MTT assays. To evaluate whether autophagy serves to cell death after cisplatin exposure, western blotting and immunofluorescence staining for LC3-II were performed. Markers of two autophagy-related pathways, mTOR and class III PI3K, were also investigated. Results: The formation of the autophagic protein LC3-II in response to 30 mM cisplatin increased with time. The early upregulation of autophagy exerted cytoprotective activity via the class III PI3K pathway. But later increase in autophagy induced cell death by suppressing the mTOR pathway. Conclusion: Our results prove that autophagy could induce cell death during cisplatin-induced ototoxicity, and modulating the autophagic pathway might be another strategy against cisplatin-induced ototoxicity. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Autophagy Cisplatin Ototoxicity Hearing loss Cell death
1. Introduction Cisplatin is a highly effective chemotherapeutic agent for the treatment of human solid tumors such as ovarian, testicular, cervical, head and neck, lung, and bladder cancers. However, the ototoxicity induced by cisplatin is an important obstacle in its utility and therapeutic profile [1]. Although the exact mechanism behind the ototoxic effects induced by cisplatin is not fully understood, the increased production of reactive oxygen species, such as superoxide anions, is believed to play a major role in it. This increased oxygen species production results in calcium influx within hair cells, which leads to apoptosis [2,3]. Autophagy is a cellular process involved in some forms of cell death, but not apoptosis [4]. Autophagy primarily has cytoprotective functions via the normal turnover of long-lived proteins and organelles [5], which maintains cells in a healthy state. However, the excessive activation of autophagy might be harmful and which results in cell death under stress conditions [6]. Autophagy might exert harmful effects, including cell death during cisplatin-mediated
* Corresponding author. Tel.: +82 62 220 3207; fax: +82 62 225 2702. E-mail address: [email protected] (S.I. Cho).
cytotoxicity. Although knowledge of autophagy has increased, its role in cisplatin-induced ototoxicity has not been revealed. This study aimed to investigate the role of autophagy in cisplatin-induced ototoxicity in an auditory cell line. 2. Materials and methods 2.1. Cell culture The House Ear Institute—Organ of Corti 1 (HEI-OC1) cell line was established from the postnatal organ of Corti of a transgenic Immortomouse. It is extremely sensitive to ototoxic drugs, and has molecular markers that are characteristic of organ of Corti cells [7]. HEI-OC1 cells were maintained in high-glucose Dulbecco’s modified Eagle medium (DMEM; Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Lonza, Walkersville, MD, USA) at 33 8C in a humidified incubator with 5% CO2. 2.2. MTT assay Cell viability was determined using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays. HEI-OC1 cells were treated with 30 mM of cisplatin and then these were seeded
http://dx.doi.org/10.1016/j.ijporl.2015.08.012 0165-5876/ß 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: C.K. Youn, et al., Role of autophagy in cisplatin-induced ototoxicity, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.08.012
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at a density of 3 104 cells per well in a 24-well plate and incubated in DMEM containing 10% FBS at 33 8C with 5% CO2. For the MTT assay, 5 mg/mL of MTT solution (Sigma, St Louis, MO, USA) was added to 0.5 mL of cell suspension, and the plates were further incubated for 4 h at 33 8C with 5% CO2. The formazan crystals were centrifuged and the pellets were dissolved by adding 500 mL/well of DMSO. Then the absorption was measured at 570 nm using a spectrophotometer (BioTek, VT, USA). To investigate whether hydroxychloroquine was able to prevent cell death induced by cisplatin, cell viability was determined using MTT assay. Hydroxychloroquine was purchased from Sigma (Saint Louis, MO, USA). 2.3. Apoptosis analysis using flow cytometry Floating cells and trypsin-detached cells after cisplatin exposure were collected and washed twice with ice-cold phosphatebuffered saline (PBS), and then fixed in 70% cold ethanol for 30 min at 4 8C. They were then again washed twice with PBS, resuspended in PI solution (50 mg/mL PI, 50 mg/mL RNase A, and 0.05% Triton X100 in PBS) for 15 min. The DNA content of these cells was analyzed using PI-fluorescent-activated cell sorting (PI-FACS). At least 10,000 events were analyzed, and the percentage of cells in the sub-G0/G1 population, which was considered to be the apoptotic population, was calculated. 2.4. Western blotting The proteins of surviving cells and dead cells after cisplatin exposure were investigated by western blotting. The cells were washed with PBS and lysed at 0 8C for 30 min in lysis buffer (20 mM HEPES pH 7.4, 2 mM EGTA, 50 mM glycerol phosphate, 1% Triton X100, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, 10 mg/mL aprotinin, 1 mM Na3VO4, and 5 mM NaF). The protein content of the samples was measured using a Bio-Rad dye binding microassay (Bio-Rad, Hercules, CA, USA), and the samples were heated at 98 8C for 5 min in Laemmli sample buffer and then subjected to SDS–PAGE. After electrophoresis, the proteins were transferred to nitrocellulose membranes. The membranes were blocked for 2 h in 5% skimmed milk in TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween-20) at room temperature, and then incubated overnight at 4 8C with the following primary antibodies at appropriate dilutions: b-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), LC3 (NanoTools, Teningen, Germany), P-S6 (Cell Signaling Technology), and P-Beclin-1 (Santa Cruz Biotechnology). Unbound antibodies were removed by washing four times with TBST for 15 min. The membranes were then incubated with the appropriate secondary antibodies (1:4000; Santa Cruz Biotechnology) in blocking buffer for 2 h, and washed again. The protein bands were detected using the WEST-ZOL plus western blot detection system (iNtRON Biotechnology, Sungnam, South Korea), and signals were acquired using an image analyzer (Kodak Image Station 4000MM, Kodak, NY, USA).
(1:200) diluted in PBS containing 1% BSA. Cells were then washed twice, and at least 10,000 events were analyzed using flow cytometry. The FL1 channel was used to assess the fluorescence intensity. The fluorescence intensity of unchallenged cells was set to a geometric mean of 101 relative fluorescence units by adjusting the photodiode gain. 2.6. Immunofluorescence staining Autophagosomes of surviving cells after cisplatin exposure were examined using LC3 immunofluorescence staining. Cells growing on slides were washed in PBS and fixed in methanol for 10 min. Slides were then washed in PBS and incubated in PBS containing 1% BSA for 2 h. The attached cells were incubated overnight with anti-LC3B antibodies (1:100 dilution, Cell Signaling Technology) diluted in PBS containing 0.1% BSA. After washing in PBS, the cells were incubated for 2 h with Alexa Fluor 594 chicken anti-rabbit IgG (1:200) diluted in PBS containing 1% BSA. The cells were then washed and imaged using a Zeiss confocal microscope. 2.7. Statistical analysis The results were analyzed statistically using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Student’s t-tests were used for pairs of data, and one-sample t-tests were used for comparison of means. A p value < 0.05 was considered to be statistically significant. 3. Results 3.1. Viability of HEI-OC1 cells after cisplatin injury To investigate cisplatin-induced cell death, the viability of HEIOC1 cell cultures was tested using MTT assays. Fig. 1 shows the viability of HEI-OC1 cells. When the cells were exposed to 30 mM cisplatin, the initiation of cell death was seen after 16 h of cisplatin treatment. The cell viability at 16 h, 24 h, 32 h, and 48 h was 78.9 1.6%, 57.1 5.4%, 35.4 2.5%, and 7.7 5.3%, respectively. The death rate of HEI-OC1 cells increased with time after cisplatin treatment (Fig. 1). 3.2. The ratio of apoptosis after cisplatin injury To investigate the apoptotic cell death induced by cisplatin injury, the cellular DNA content was analyzed. Cells with sub-G0/G1
2.5. Detecting LC3 using flow cytometry To investigate total LC3-II of surviving cells and dead cells after cisplatin exposure, flow cytometry was performed. Floating cells and trypsin-detached cells were collected and washed twice with ice-cold PBS. They were then fixed in methanol for 10 min, washed twice with 1% bovine serum albumin (BSA), and incubated in 1% BSA for 2 h. These cells were incubated overnight with anti-LC3B antibodies (1:100 dilution, Cell Signaling Technology) diluted in PBS containing 0.1% BSA. After washing in 1% BSA, the cells were incubated for 2 h with Alexa Fluor 488 chicken anti-rabbit IgG
Fig. 1. Cell viability was determined using MTT assays. Cell death increased with time after exposure to 30 mM cisplatin. The initiation of cell death was seen after 16 h of cisplatin treatment.
Please cite this article in press as: C.K. Youn, et al., Role of autophagy in cisplatin-induced ototoxicity, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.08.012
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Fig. 2. Cisplatin-induced apoptosis in HEI-OC1 cells. PI fluorescence intensity was measured using flow cytometry. The ratio of cells in the sub-G0/G1 peak (M1 fraction), which were classified as apoptotic cells, increased with time after exposure to 30 mM cisplatin.
DNA content were quantified and classified as apoptotic cells. The proportion of cells in the sub-G0/G1 peak at 16 h, 24 h, 32 h, and 48 h was 1.1 0.3%, 2.8 0.4%, 7.0 0.7%, and 17.5 1.8%, respectively (Fig. 2). The percent of apoptotic cells increased after the exposure to 30 mM cisplatin. The percent of apoptotic to dying cells at 16 h, 24 h, 32 h, and 48 h was 5.2%, 6.5%, 10.8%, and 18.9%, respectively. These results indicate that another mode of cell death in addition to apoptosis could be involved in cisplatin-induced cytotoxicity.
cytometry. After exposure to 30 mM cisplatin, the expression LC3II at 4 h, 8 h, 16 h, 24 h, 32 h, and 48 h was 101.1 0.5%, 110.0 1.5%, 148.6 2.0%, 165.4 2.6%, 165.8 1.5%, and 167.0 7.4%, respectively (Fig. 4). These results indicate that autophagy is a mode of cell death involved in cisplatin-induced cytotoxicity.
3.3. Autophagy accelerates cell death after cisplatin injury To investigate whether autophagy is involved in cisplatininduced cytotoxicity, a reliable marker of autophagy was examined, specifically the conversion of LC3-I to LC3-II [8,9]. This conversion began 16 h after exposure to 30 mM cisplatin, and increased gradually with time. Caspase-3, a marker of apoptosis, also increased with time. To investigate the activation of the mTOR pathway during autophagy P-S6, which is a downstream effector of mTOR, was examined. The expression of P-S6 was induced early (in the first 8 h) in response to cisplatin exposure, and then decreased. To investigate the activation of class III phosphatidylinositol 3-kinase (PI3K) pathway, P-Beclin-1 levels were examined. The expression of P-Beclin-1 was detected early (during the first 4 h) after treatment with cisplatin (Fig. 3). Total LC3-II expression of surviving cells and dead cells was also determined using flow
Fig. 3. Formation of the autophagic protein LC3-II increased with time in response to 30 mM cisplatin. Cleaved caspase-3 increased with time in response to cisplatin. P-S6, a downstream effector of mTOR, increased early (the first 8 h) after cisplatin exposure, and then decreased. P-Beclin1 expression was detected in the first 4 h after cisplatin exposure.
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Fig. 4. LC3-II expression of whole cultured cells including surviving cells and dead cells was determined using flow cytometry (A). The expression increased with time after exposure to 30 mM cisplatin (B, * p < 0.01 using one-sample t-tests).
3.4. Autophagy plays pro-survival and pro-death roles One of the steps in autophagy is autophagosome formation in the cell [10]. The appearance of autophagosomes of surviving cells was examined using LC3 immunofluorescence staining. LC3 puncta formation of surviving cells in response to 30 mM cisplatin exposure increased early (during the first 8 h), and LC3 puncta formation of surviving cells at later time points had lower number of LC3 puncta (Fig. 5). Less activation of autophagy at later time points led to survival of cells. The viability of HEI-OC1 cells did not decrease until 8 h after cisplatin treatment, and then decreased gradually with time (Fig. 1). A comparison of the expression of LC3 puncta and cell viability suggested that autophagy has a prosurvival role in the early phase of cisplatin treatment, and a prodeath role in the late phase. 3.5. Protective effects of hydroxychloroquine on cisplatin-induced ototoxicity Hydroxychloroquine has the ability to block acidification in lysosomes and inhibits the process of autophagy. When the HEIOC1 cells were exposed to 30 mM cisplatin for 24 h, the viability was 63.53 3.5%. The viability of the cells was not affected by 20 mM hydroxychloroquine and it was 105.0 9.7%. After pre-treatment with 20 mM hydroxychloroquine for 2 h, the cells were exposed to 30 mM cisplatin for 24 h. The viability of these cells was 81.16 5.5% (Fig. 6). The pre-treatment group with hydroxychloroquine showed increase in cell viability compared with cisplatin-alone treated group. 4. Discussion Autophagy proceeds with the formation of an autophagosome followed by the fusion of the autophagosome with the lysosome;
the contents are then digested by lysosomal proteases and other hydrolytic enzymes [11]. The formation of autophagosomes is followed by the conversion of the cytosolic isoform of LC3-I protein into LC3-II, which is crucial for the fusion of the autophagosome with the lysosome [12]. Therefore, LC3 is a specific marker for autophagy and the conversion of the unlipidated form of LC3-I into the lipidated form LC3-II is generally used to estimate autophagic activity [13,14]. Autophagy maintains the function for cellular homeostasis and adaptation to environmental stresses, including starvation and hypoxia [15]. The autophagy in response to starvation is generally reported as a cell survival mechanism [16]. However, extensive autophagy or the inappropriate activation of autophagy can result in cell death by eliminating cells. Kroemer and Levine [6] reported that autophagic cell death occurs in the absence of chromatin condensation, but is accompanied by vacuolization of the cytoplasm. Autophagic cell death differs from apoptosis in that it is caspase-independent and therefore occurs in the presence of caspase inhibitors [17]. In the present study, we showed that cell death increases gradually in response to cisplatin. The ratio of apoptotic cells was part of the total cell death. These results suggest that another mode of cell death is involved in cisplatin-induced cytotoxicity. The current data revealed that cell death did not begin until 8 h after cisplatin exposure; thereafter, cell death increased with time. LC3 puncta formation of surviving cells showed an increase in autophagosome formation during the initial 8 h after cisplatin exposure. This suggests that the autophagy occurs during the initial 8 h has a cytoprotective role. The surviving cells at later time points when cell death increased had lower number of LC3 puncta. The result can explain that less activation of autophagy leads to survival of cells. Total LC3-II of surviving cells and dead cells increased with time after cisplatin exposure and as cell viability decreased. Taken together, this suggests that the
Please cite this article in press as: C.K. Youn, et al., Role of autophagy in cisplatin-induced ototoxicity, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.08.012
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Fig. 5. The appearance of autophagosome-associated LC3 vesicles of surviving cells in response to 30 mM cisplatin. The expression of LC3 puncta in living cells is shown (A). LC3 puncta per cell increased during the first 8 h of cisplatin exposure, and the surviving cells at later time points had lower number of LC3 puncta (A and B, * p < 0.01 using Student’s t-tests).
autophagy occurs during the late period of cisplatin cytotoxicity has a role in cell death. One known modulator of autophagy is suppression via mTOR pathway. The upregulation of mTOR deactivates the catalytic activity of Unc-51-like kinase 1 (ULK1), resulting in formation of the ULK1-ATG13-FIP200 complex and subsequent activation of the
Fig. 6. Cells were pretreated with 20 mM hydroxychloroquine (HCQ) for 2 h prior to 24 h cisplatin (CP) exposure. The group (HCQ + CP) showed increase of cell viability compared with cisplatin alone treated group (* p < 0.05 using Student’s t-test).
autophagic cascades [18]. Therefore, activated mTOR suppresses autophagy and inhibited mTOR promotes autophagy [19,20]. The other known modulator of autophagy is the class III phosphatidylinositol 3-kinase (PI3K) complex, which activates Beclin-1 (Atg6) and results in a barkor-Vps15-Vps34-Ambra1 complex and the initiation of autophagosome formation [21,22]. Therefore, P-Beclin1 promotes autophagy through the class III PI3K complex [23,24]. In the present study, the expression of P-S6, which is a downstream effector of mTOR, was induced early (in the first 8 h) in response to cisplatin exposure, and then decreased. The expression of P-Beclin1 increased early (within 4 h) in response to cisplatin exposure. This suggests that early autophagy during cisplatin-induced cytotoxicity results from activation of the class III PI3K pathway, not from decrease of suppression via mTOR pathway, whereas late autophagy during cisplatin-induced cytotoxicity results from decrease of suppression via mTOR pathway. Autophagy is an essential process during early inner ear development because it clears neuroepithelial dying cells and provides the migration of otic neuronal precursors [25]. However, two studies have reported that autophagy causes cell death in the inner ear. Menardo et al. [26] reported that autophagy induces cell death in an age-related hearing loss model. They explained that the early upregulation of autophagy might remove the damaged mitochondria and aberrant proteins induced by oxidative stress. The excessive accumulation of giant non-functional mitochondria and protein aggregates might trigger autophagic
Please cite this article in press as: C.K. Youn, et al., Role of autophagy in cisplatin-induced ototoxicity, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.08.012
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stress, which in turn could induce cell death. Taylor et al. [27] reported ultrastructural changes characteristic of autophagy in hair cells using electron microscopy after aminoglycoside and loop diuretic exposures; therefore, they suggested that autophagy might serve as cell death pathway in aminoglycoside-induced ototoxicity. Hydroxychloroquine is a lysotropic chloroquine derivative that inhibits acidification of the lysosome and blocks the final process of autophagy [28]. In the present study, pre-treatment of hydroxychloroquine protected the auditory cells from cisplatin cytotoxicity. The result also suggests that autophagy has a pro-death role in cisplatin-induced ototoxicity. The present study provides the first evidence for the occurrence of autophagy and its role in cisplatin-induced ototoxicity. In conclusion, autophagy plays a pro-death role in cisplatin-induced ototoxicity via mTOR pathway, and modulating the autophagic pathway might be another strategy against cisplatin-induced ototoxicity. Conflict of interest statement None of the authors has a conflict of interest to declare. Acknowledgement This study was supported by a research fund from Chosun University, 2014. References [1] L.P. Rybak, C.A. Whitworth, D. Mukherjea, V. Ramkumar, Mechanisms of cisplatininduced ototoxicity and prevention, Hear. Res. 226 (2007) 157–167. [2] N. Dehne, J. Lautermann, F. Petrat, U. Rauen, H. de Groot, Cisplatin ototoxicity: involvement of iron and enhanced formation of superoxide anion radicals, Toxicol. Appl. Pharmacol. 174 (2001) 27–34. [3] M.S. Gonc¸alves, A.F. Silveira, A.R. Teixeira, M.A. Hyppolito, Mechanisms of cisplatin ototoxicity: theoretical review, J. Laryngol. Otol. 127 (2013) 536–541. [4] V. Nikoletopoulou, M. Markaki, K. Palikaras, N. Tavernarakis, Crosstalk between apoptosis, necrosis and autophagy, Biochim. Biophys. Acta 1833 (2013) 3448–3459. [5] N. Mizushima, B. Levine, Autophagy in mammalian development and differentiation, Nat. Cell Biol. 12 (2010) 823–830. [6] G. Kroemer, B. Levine, Autophagic cell death: the story of a misnomer, Nat. Rev. Mol. Cell Biol. 9 (2008) 1004–1010.
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Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Role of autophagy in cisplatin-induced ototoxicity Cha Kyung Youn a,b, Jun Kim a, Jun-Hee Park a, Nam Yong Do a, Sung Il Cho a,* a b
Department of Otolaryngology—Head and Neck Surgery, Chosun University School of Medicine, Gwangju, South Korea Division of Natural Medical Science, Chosun University School of Medicine, Gwangju, South Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 February 2015 Received in revised form 4 August 2015 Accepted 5 August 2015 Available online xxx
Objective: Hearing loss is a major side effect of cisplatin chemotherapy. Although cell death in cisplatininduced ototoxicity is primarily caused by apoptosis, the exact mechanism behind the ototoxic effects of cisplatin is not fully understood. Autophagy is generally known as a pro-survival mechanism that protects cells under starvation or stress conditions. However, recent research has reported that autophagy plays a functional role in cell death also. This study aimed to investigate the role of autophagy in cisplatin-induced ototoxicity in an auditory cell line. Methods: Cultured HEI-OC1 cells were exposed to 30 mM cisplatin for 48 h, and cell viability was tested using MTT assays. To evaluate whether autophagy serves to cell death after cisplatin exposure, western blotting and immunofluorescence staining for LC3-II were performed. Markers of two autophagy-related pathways, mTOR and class III PI3K, were also investigated. Results: The formation of the autophagic protein LC3-II in response to 30 mM cisplatin increased with time. The early upregulation of autophagy exerted cytoprotective activity via the class III PI3K pathway. But later increase in autophagy induced cell death by suppressing the mTOR pathway. Conclusion: Our results prove that autophagy could induce cell death during cisplatin-induced ototoxicity, and modulating the autophagic pathway might be another strategy against cisplatin-induced ototoxicity. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Autophagy Cisplatin Ototoxicity Hearing loss Cell death
1. Introduction Cisplatin is a highly effective chemotherapeutic agent for the treatment of human solid tumors such as ovarian, testicular, cervical, head and neck, lung, and bladder cancers. However, the ototoxicity induced by cisplatin is an important obstacle in its utility and therapeutic profile [1]. Although the exact mechanism behind the ototoxic effects induced by cisplatin is not fully understood, the increased production of reactive oxygen species, such as superoxide anions, is believed to play a major role in it. This increased oxygen species production results in calcium influx within hair cells, which leads to apoptosis [2,3]. Autophagy is a cellular process involved in some forms of cell death, but not apoptosis [4]. Autophagy primarily has cytoprotective functions via the normal turnover of long-lived proteins and organelles [5], which maintains cells in a healthy state. However, the excessive activation of autophagy might be harmful and which results in cell death under stress conditions [6]. Autophagy might exert harmful effects, including cell death during cisplatin-mediated
* Corresponding author. Tel.: +82 62 220 3207; fax: +82 62 225 2702. E-mail address: [email protected] (S.I. Cho).
cytotoxicity. Although knowledge of autophagy has increased, its role in cisplatin-induced ototoxicity has not been revealed. This study aimed to investigate the role of autophagy in cisplatin-induced ototoxicity in an auditory cell line. 2. Materials and methods 2.1. Cell culture The House Ear Institute—Organ of Corti 1 (HEI-OC1) cell line was established from the postnatal organ of Corti of a transgenic Immortomouse. It is extremely sensitive to ototoxic drugs, and has molecular markers that are characteristic of organ of Corti cells [7]. HEI-OC1 cells were maintained in high-glucose Dulbecco’s modified Eagle medium (DMEM; Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Lonza, Walkersville, MD, USA) at 33 8C in a humidified incubator with 5% CO2. 2.2. MTT assay Cell viability was determined using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays. HEI-OC1 cells were treated with 30 mM of cisplatin and then these were seeded
http://dx.doi.org/10.1016/j.ijporl.2015.08.012 0165-5876/ß 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: C.K. Youn, et al., Role of autophagy in cisplatin-induced ototoxicity, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.08.012
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at a density of 3 104 cells per well in a 24-well plate and incubated in DMEM containing 10% FBS at 33 8C with 5% CO2. For the MTT assay, 5 mg/mL of MTT solution (Sigma, St Louis, MO, USA) was added to 0.5 mL of cell suspension, and the plates were further incubated for 4 h at 33 8C with 5% CO2. The formazan crystals were centrifuged and the pellets were dissolved by adding 500 mL/well of DMSO. Then the absorption was measured at 570 nm using a spectrophotometer (BioTek, VT, USA). To investigate whether hydroxychloroquine was able to prevent cell death induced by cisplatin, cell viability was determined using MTT assay. Hydroxychloroquine was purchased from Sigma (Saint Louis, MO, USA). 2.3. Apoptosis analysis using flow cytometry Floating cells and trypsin-detached cells after cisplatin exposure were collected and washed twice with ice-cold phosphatebuffered saline (PBS), and then fixed in 70% cold ethanol for 30 min at 4 8C. They were then again washed twice with PBS, resuspended in PI solution (50 mg/mL PI, 50 mg/mL RNase A, and 0.05% Triton X100 in PBS) for 15 min. The DNA content of these cells was analyzed using PI-fluorescent-activated cell sorting (PI-FACS). At least 10,000 events were analyzed, and the percentage of cells in the sub-G0/G1 population, which was considered to be the apoptotic population, was calculated. 2.4. Western blotting The proteins of surviving cells and dead cells after cisplatin exposure were investigated by western blotting. The cells were washed with PBS and lysed at 0 8C for 30 min in lysis buffer (20 mM HEPES pH 7.4, 2 mM EGTA, 50 mM glycerol phosphate, 1% Triton X100, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, 10 mg/mL aprotinin, 1 mM Na3VO4, and 5 mM NaF). The protein content of the samples was measured using a Bio-Rad dye binding microassay (Bio-Rad, Hercules, CA, USA), and the samples were heated at 98 8C for 5 min in Laemmli sample buffer and then subjected to SDS–PAGE. After electrophoresis, the proteins were transferred to nitrocellulose membranes. The membranes were blocked for 2 h in 5% skimmed milk in TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween-20) at room temperature, and then incubated overnight at 4 8C with the following primary antibodies at appropriate dilutions: b-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), LC3 (NanoTools, Teningen, Germany), P-S6 (Cell Signaling Technology), and P-Beclin-1 (Santa Cruz Biotechnology). Unbound antibodies were removed by washing four times with TBST for 15 min. The membranes were then incubated with the appropriate secondary antibodies (1:4000; Santa Cruz Biotechnology) in blocking buffer for 2 h, and washed again. The protein bands were detected using the WEST-ZOL plus western blot detection system (iNtRON Biotechnology, Sungnam, South Korea), and signals were acquired using an image analyzer (Kodak Image Station 4000MM, Kodak, NY, USA).
(1:200) diluted in PBS containing 1% BSA. Cells were then washed twice, and at least 10,000 events were analyzed using flow cytometry. The FL1 channel was used to assess the fluorescence intensity. The fluorescence intensity of unchallenged cells was set to a geometric mean of 101 relative fluorescence units by adjusting the photodiode gain. 2.6. Immunofluorescence staining Autophagosomes of surviving cells after cisplatin exposure were examined using LC3 immunofluorescence staining. Cells growing on slides were washed in PBS and fixed in methanol for 10 min. Slides were then washed in PBS and incubated in PBS containing 1% BSA for 2 h. The attached cells were incubated overnight with anti-LC3B antibodies (1:100 dilution, Cell Signaling Technology) diluted in PBS containing 0.1% BSA. After washing in PBS, the cells were incubated for 2 h with Alexa Fluor 594 chicken anti-rabbit IgG (1:200) diluted in PBS containing 1% BSA. The cells were then washed and imaged using a Zeiss confocal microscope. 2.7. Statistical analysis The results were analyzed statistically using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Student’s t-tests were used for pairs of data, and one-sample t-tests were used for comparison of means. A p value < 0.05 was considered to be statistically significant. 3. Results 3.1. Viability of HEI-OC1 cells after cisplatin injury To investigate cisplatin-induced cell death, the viability of HEIOC1 cell cultures was tested using MTT assays. Fig. 1 shows the viability of HEI-OC1 cells. When the cells were exposed to 30 mM cisplatin, the initiation of cell death was seen after 16 h of cisplatin treatment. The cell viability at 16 h, 24 h, 32 h, and 48 h was 78.9 1.6%, 57.1 5.4%, 35.4 2.5%, and 7.7 5.3%, respectively. The death rate of HEI-OC1 cells increased with time after cisplatin treatment (Fig. 1). 3.2. The ratio of apoptosis after cisplatin injury To investigate the apoptotic cell death induced by cisplatin injury, the cellular DNA content was analyzed. Cells with sub-G0/G1
2.5. Detecting LC3 using flow cytometry To investigate total LC3-II of surviving cells and dead cells after cisplatin exposure, flow cytometry was performed. Floating cells and trypsin-detached cells were collected and washed twice with ice-cold PBS. They were then fixed in methanol for 10 min, washed twice with 1% bovine serum albumin (BSA), and incubated in 1% BSA for 2 h. These cells were incubated overnight with anti-LC3B antibodies (1:100 dilution, Cell Signaling Technology) diluted in PBS containing 0.1% BSA. After washing in 1% BSA, the cells were incubated for 2 h with Alexa Fluor 488 chicken anti-rabbit IgG
Fig. 1. Cell viability was determined using MTT assays. Cell death increased with time after exposure to 30 mM cisplatin. The initiation of cell death was seen after 16 h of cisplatin treatment.
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Fig. 2. Cisplatin-induced apoptosis in HEI-OC1 cells. PI fluorescence intensity was measured using flow cytometry. The ratio of cells in the sub-G0/G1 peak (M1 fraction), which were classified as apoptotic cells, increased with time after exposure to 30 mM cisplatin.
DNA content were quantified and classified as apoptotic cells. The proportion of cells in the sub-G0/G1 peak at 16 h, 24 h, 32 h, and 48 h was 1.1 0.3%, 2.8 0.4%, 7.0 0.7%, and 17.5 1.8%, respectively (Fig. 2). The percent of apoptotic cells increased after the exposure to 30 mM cisplatin. The percent of apoptotic to dying cells at 16 h, 24 h, 32 h, and 48 h was 5.2%, 6.5%, 10.8%, and 18.9%, respectively. These results indicate that another mode of cell death in addition to apoptosis could be involved in cisplatin-induced cytotoxicity.
cytometry. After exposure to 30 mM cisplatin, the expression LC3II at 4 h, 8 h, 16 h, 24 h, 32 h, and 48 h was 101.1 0.5%, 110.0 1.5%, 148.6 2.0%, 165.4 2.6%, 165.8 1.5%, and 167.0 7.4%, respectively (Fig. 4). These results indicate that autophagy is a mode of cell death involved in cisplatin-induced cytotoxicity.
3.3. Autophagy accelerates cell death after cisplatin injury To investigate whether autophagy is involved in cisplatininduced cytotoxicity, a reliable marker of autophagy was examined, specifically the conversion of LC3-I to LC3-II [8,9]. This conversion began 16 h after exposure to 30 mM cisplatin, and increased gradually with time. Caspase-3, a marker of apoptosis, also increased with time. To investigate the activation of the mTOR pathway during autophagy P-S6, which is a downstream effector of mTOR, was examined. The expression of P-S6 was induced early (in the first 8 h) in response to cisplatin exposure, and then decreased. To investigate the activation of class III phosphatidylinositol 3-kinase (PI3K) pathway, P-Beclin-1 levels were examined. The expression of P-Beclin-1 was detected early (during the first 4 h) after treatment with cisplatin (Fig. 3). Total LC3-II expression of surviving cells and dead cells was also determined using flow
Fig. 3. Formation of the autophagic protein LC3-II increased with time in response to 30 mM cisplatin. Cleaved caspase-3 increased with time in response to cisplatin. P-S6, a downstream effector of mTOR, increased early (the first 8 h) after cisplatin exposure, and then decreased. P-Beclin1 expression was detected in the first 4 h after cisplatin exposure.
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Fig. 4. LC3-II expression of whole cultured cells including surviving cells and dead cells was determined using flow cytometry (A). The expression increased with time after exposure to 30 mM cisplatin (B, * p < 0.01 using one-sample t-tests).
3.4. Autophagy plays pro-survival and pro-death roles One of the steps in autophagy is autophagosome formation in the cell [10]. The appearance of autophagosomes of surviving cells was examined using LC3 immunofluorescence staining. LC3 puncta formation of surviving cells in response to 30 mM cisplatin exposure increased early (during the first 8 h), and LC3 puncta formation of surviving cells at later time points had lower number of LC3 puncta (Fig. 5). Less activation of autophagy at later time points led to survival of cells. The viability of HEI-OC1 cells did not decrease until 8 h after cisplatin treatment, and then decreased gradually with time (Fig. 1). A comparison of the expression of LC3 puncta and cell viability suggested that autophagy has a prosurvival role in the early phase of cisplatin treatment, and a prodeath role in the late phase. 3.5. Protective effects of hydroxychloroquine on cisplatin-induced ototoxicity Hydroxychloroquine has the ability to block acidification in lysosomes and inhibits the process of autophagy. When the HEIOC1 cells were exposed to 30 mM cisplatin for 24 h, the viability was 63.53 3.5%. The viability of the cells was not affected by 20 mM hydroxychloroquine and it was 105.0 9.7%. After pre-treatment with 20 mM hydroxychloroquine for 2 h, the cells were exposed to 30 mM cisplatin for 24 h. The viability of these cells was 81.16 5.5% (Fig. 6). The pre-treatment group with hydroxychloroquine showed increase in cell viability compared with cisplatin-alone treated group. 4. Discussion Autophagy proceeds with the formation of an autophagosome followed by the fusion of the autophagosome with the lysosome;
the contents are then digested by lysosomal proteases and other hydrolytic enzymes [11]. The formation of autophagosomes is followed by the conversion of the cytosolic isoform of LC3-I protein into LC3-II, which is crucial for the fusion of the autophagosome with the lysosome [12]. Therefore, LC3 is a specific marker for autophagy and the conversion of the unlipidated form of LC3-I into the lipidated form LC3-II is generally used to estimate autophagic activity [13,14]. Autophagy maintains the function for cellular homeostasis and adaptation to environmental stresses, including starvation and hypoxia [15]. The autophagy in response to starvation is generally reported as a cell survival mechanism [16]. However, extensive autophagy or the inappropriate activation of autophagy can result in cell death by eliminating cells. Kroemer and Levine [6] reported that autophagic cell death occurs in the absence of chromatin condensation, but is accompanied by vacuolization of the cytoplasm. Autophagic cell death differs from apoptosis in that it is caspase-independent and therefore occurs in the presence of caspase inhibitors [17]. In the present study, we showed that cell death increases gradually in response to cisplatin. The ratio of apoptotic cells was part of the total cell death. These results suggest that another mode of cell death is involved in cisplatin-induced cytotoxicity. The current data revealed that cell death did not begin until 8 h after cisplatin exposure; thereafter, cell death increased with time. LC3 puncta formation of surviving cells showed an increase in autophagosome formation during the initial 8 h after cisplatin exposure. This suggests that the autophagy occurs during the initial 8 h has a cytoprotective role. The surviving cells at later time points when cell death increased had lower number of LC3 puncta. The result can explain that less activation of autophagy leads to survival of cells. Total LC3-II of surviving cells and dead cells increased with time after cisplatin exposure and as cell viability decreased. Taken together, this suggests that the
Please cite this article in press as: C.K. Youn, et al., Role of autophagy in cisplatin-induced ototoxicity, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.08.012
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Fig. 5. The appearance of autophagosome-associated LC3 vesicles of surviving cells in response to 30 mM cisplatin. The expression of LC3 puncta in living cells is shown (A). LC3 puncta per cell increased during the first 8 h of cisplatin exposure, and the surviving cells at later time points had lower number of LC3 puncta (A and B, * p < 0.01 using Student’s t-tests).
autophagy occurs during the late period of cisplatin cytotoxicity has a role in cell death. One known modulator of autophagy is suppression via mTOR pathway. The upregulation of mTOR deactivates the catalytic activity of Unc-51-like kinase 1 (ULK1), resulting in formation of the ULK1-ATG13-FIP200 complex and subsequent activation of the
Fig. 6. Cells were pretreated with 20 mM hydroxychloroquine (HCQ) for 2 h prior to 24 h cisplatin (CP) exposure. The group (HCQ + CP) showed increase of cell viability compared with cisplatin alone treated group (* p < 0.05 using Student’s t-test).
autophagic cascades [18]. Therefore, activated mTOR suppresses autophagy and inhibited mTOR promotes autophagy [19,20]. The other known modulator of autophagy is the class III phosphatidylinositol 3-kinase (PI3K) complex, which activates Beclin-1 (Atg6) and results in a barkor-Vps15-Vps34-Ambra1 complex and the initiation of autophagosome formation [21,22]. Therefore, P-Beclin1 promotes autophagy through the class III PI3K complex [23,24]. In the present study, the expression of P-S6, which is a downstream effector of mTOR, was induced early (in the first 8 h) in response to cisplatin exposure, and then decreased. The expression of P-Beclin1 increased early (within 4 h) in response to cisplatin exposure. This suggests that early autophagy during cisplatin-induced cytotoxicity results from activation of the class III PI3K pathway, not from decrease of suppression via mTOR pathway, whereas late autophagy during cisplatin-induced cytotoxicity results from decrease of suppression via mTOR pathway. Autophagy is an essential process during early inner ear development because it clears neuroepithelial dying cells and provides the migration of otic neuronal precursors [25]. However, two studies have reported that autophagy causes cell death in the inner ear. Menardo et al. [26] reported that autophagy induces cell death in an age-related hearing loss model. They explained that the early upregulation of autophagy might remove the damaged mitochondria and aberrant proteins induced by oxidative stress. The excessive accumulation of giant non-functional mitochondria and protein aggregates might trigger autophagic
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stress, which in turn could induce cell death. Taylor et al. [27] reported ultrastructural changes characteristic of autophagy in hair cells using electron microscopy after aminoglycoside and loop diuretic exposures; therefore, they suggested that autophagy might serve as cell death pathway in aminoglycoside-induced ototoxicity. Hydroxychloroquine is a lysotropic chloroquine derivative that inhibits acidification of the lysosome and blocks the final process of autophagy [28]. In the present study, pre-treatment of hydroxychloroquine protected the auditory cells from cisplatin cytotoxicity. The result also suggests that autophagy has a pro-death role in cisplatin-induced ototoxicity. The present study provides the first evidence for the occurrence of autophagy and its role in cisplatin-induced ototoxicity. In conclusion, autophagy plays a pro-death role in cisplatin-induced ototoxicity via mTOR pathway, and modulating the autophagic pathway might be another strategy against cisplatin-induced ototoxicity. Conflict of interest statement None of the authors has a conflict of interest to declare. Acknowledgement This study was supported by a research fund from Chosun University, 2014. References [1] L.P. Rybak, C.A. Whitworth, D. Mukherjea, V. Ramkumar, Mechanisms of cisplatininduced ototoxicity and prevention, Hear. Res. 226 (2007) 157–167. [2] N. Dehne, J. Lautermann, F. Petrat, U. Rauen, H. de Groot, Cisplatin ototoxicity: involvement of iron and enhanced formation of superoxide anion radicals, Toxicol. Appl. Pharmacol. 174 (2001) 27–34. [3] M.S. Gonc¸alves, A.F. Silveira, A.R. Teixeira, M.A. Hyppolito, Mechanisms of cisplatin ototoxicity: theoretical review, J. Laryngol. Otol. 127 (2013) 536–541. [4] V. Nikoletopoulou, M. Markaki, K. Palikaras, N. Tavernarakis, Crosstalk between apoptosis, necrosis and autophagy, Biochim. Biophys. Acta 1833 (2013) 3448–3459. [5] N. Mizushima, B. Levine, Autophagy in mammalian development and differentiation, Nat. Cell Biol. 12 (2010) 823–830. [6] G. Kroemer, B. Levine, Autophagic cell death: the story of a misnomer, Nat. Rev. Mol. Cell Biol. 9 (2008) 1004–1010.
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