International Journal of Pediatric Otorhinolaryngology 78 (2014) 474–478
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Protective effect of silymarin against cisplatin-induced ototoxicity Sung Il Cho *, Ji-Eun Lee, Nam Yong Do Department of Otolayngology-Head and Neck Surgery, 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 17 September 2013 Received in revised form 3 December 2013 Accepted 17 December 2013 Available online 27 December 2013
Objectives: Silymarin is a plant extract with strong antioxidant properties in addition to antiinflammatory and anticarcinogenic actions. The aim of this study was to investigate the potential preventive effect of silymarin on cisplatin ototoxicity in an auditory cell line, HEI-OC1 cells. Methods: Cultured HEI-OC1 cells were exposed to cisplatin (30 mM) with or without pre-treatment with silymarin (50 mM). Cell viability was evaluated using MTT assay. Hoechst 33258 staining was used to identify cells undergoing apoptosis. Western blot analysis was done to evaluate whether silymarin inhibits cisplatin-induced caspase and PARP activation. Cell-cycle analysis was done by flow cytometry to investigate whether silymarin is capable of protecting cisplatin-induced cell cycle arrest. Results: Cell viability significantly increased in cells pretreated with silymarin compared with cells exposed to cisplatin alone. Pre-treatment of silymarin appeared to protect against cisplatin-induced apoptotic features on Hoechst 33258 staining. Cisplatin increased cleaved caspase-3 and PARP on Western blot analysis. However, pre-treatment with silymarin inhibited the expression of cleaved caspase-3 and PARP. Silymarin did attenuate cell cycle arrest and apoptosis in HEI-OC1 cells. Conclusions: Our results demonstrate that silymarin treatment inhibited cisplatin-induced cytotoxicity in the auditory cell line, HEI-OC1. Silymarin may be a potential candidate drug to eliminate cisplatin induced ototoxicity. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Silymarin Cisplatin Ototoxicity Apoptosis
Introduction Cisplatin is one of the most prescribed drugs for the treatment of human solid tumors such as ovarian, testicular, cervical, head and neck, lung, and bladder cancer. However, ototoxicity-induced by cisplatin has been shown to be an important obstacle for its utility and therapeutic profile [1,2]. Though the exact mechanisms of ototoxic effects by cisplatin is not widely revealed, reactive oxygen species (ROS) such as superoxide anion are believed to play a major role. Increased ROS results in depletion of the cochlear antioxidant enzymes [3,4]. This can lead to calcium influx within hair cells, leading to apoptosis [5]. Ototoxicity of cisplatin can be reduced by various antioxidants by counteracting this response [6]. Silymarin is a lipophilic extract from the seeds of the milk thistle (Silybum marianum) and composed of three isomers of flavonolignans (silybin, silydianin, and silychristin), and two flavonoids (tamoxifen and quercetin) [7,8]. Silymarin has been
used in the treatment of liver diseases such as cirrhosis, viral hepatitis due to its hepatoprotective effect. The effect is mediated by scavenging of free radicals, decreasing formation of ROS and inhibition of fatty acid peroxidation. Another mechanism involves anti-apoptotic actions and anti-inflammatory actions [9,10]. Silymarin has no significant adverse reactions in human studies and has been reported to be safe in animal models [11]. In addition, silymarin has been shown to exert anti-neoplastic effects in a variety of cancer models [12,13]. Anti-oxidant and anti-apoptotic properties of silymarin may also have protective role against cisplatin-induced ototoxicity. In this study, we aimed to investigate the effect of silymarin on cisplatin-induced ototoxicity in an auditory cell line and establish potential application for prevention of ototoxicity after cisplatin chemotherapy. Materials and methods Cell culture
* Corresponding author at: Department of Otolaryngology-Head and Neck Surgery, Chosun University Hospital, 365 Pilmun-daero, Dong-gu, Gwangju 501717, South Korea. Tel.: +82 62 220 3207; fax: +82 62 225 2702. E-mail address: [email protected] (S.I. Cho). 0165-5876/$ – see front matter ß 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijporl.2013.12.024
The establishment of House Ear Institute-Organ of Corti 1 (HEIOC1) cell line was derived from postnatal organ of Corti of a transgenic immortomouse. The auditory cell line is extremely sensitive to ototoxic drug and express molecular markers, which
S.I. Cho et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 474–478
are characteristic of organ of Corti cells [14]. HEI-OC1 cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; GIBCO BRL, NY, USA) supplemented with 10% fetal bovine serum (FBS; Lonza Walkersvile, MD, USA) at 33 8C in a humidified incubator with 5% CO2. MTT assay Cell viability was determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. HEI-OC1 cells were seeded at 3 104 cells per well of a 24-well plate and were incubated in DMEM with 10% FBS at 33 8C with 5% CO2. Cells were divided into four groups: control, silymarin, cisplatin, silymarin plus cisplatin. Silymarin was purchased from Sigma (Saint Louis, MO, USA). Control group, silymarin, and cisplatin treated groups were incubated for 48 h. A 30 mM concentration of cisplatin for 48 h was used as adequate concentration to study cisplatininduced cytotoxicity on the HEI-OC1 cells [15]. In order to investigate the effects of silymarin on cisplatin ototoxicity, the cells were pretreated with silymarin (50 mM) for 1 h, and exposed to cisplatin (30 mM) for 48 h. For the MTT assay, 5 mg/ml of MTT solution (Sigma, Saint 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 dissolved by the addition of 500 mL/well of DMSO. Absorption was measured using a spectrophotometer (BioTek, VT, USA) at 570 nm. Hoechst 33258 staining Apoptotic cell death was determined by evaluating the nuclear morphology using Hoechst 33258 staining. Cells were incubated with 10 mg/mL of the Hoechst 33258 (Sigma, Saint Louis, MO, USA) for 30 min. Membrane-permeable Hoechst 33258 was a blue fluorescent dye and stained the cell nucleus. After washing twice with phosphate buffered saline (PBS), the cells were detached by trypsinization and fixed with 4% paraformaldehyde for 10 min at room temperature (RT). The cells were placed over the slides and mounted with glycerol after drying with air. The cells were observed under a fluorescence microscope (DM5000, Leica, Wetzlar, Germany).
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Propidium iodide staining The floating and trypsin-detached HEI-OC 1 cells were collected and washed once with ice-cold PBS, followed by fixing in 70% cold ethanol for 30 min at 4 8C. The cells were then stained in PBS and propidium iodide (50 mg/ml), RNase A (50 mg/ml), and 0.05% Triton X-100 for 45 min at RT. The DNA content of the HEI-OC1 cells was analyzed by fluorescent-activated cell sorting (FACSort, Becton Dickinson). At least 10,000 events were analyzed, and the percentage of cells in sub-G1 population was calculated. Aggregates of cell debris at the origin of histogram were excluded from the sub-G1 cells. Statistical analysis Statistical analysis of the results was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). The Student’s t-test was used for pairs of data. A p < 0.01 was considered statistically significant. Results MTT assay To investigate whether silymarin was able to prevent apoptosis induced by cisplatin, cell viability in HEI-OC1 cultures was determined by MTT assay. HEI-OC1 cells were treated with various concentrations of silymarin and with 30 mM of cisplatin for 48 h, the cell viability of HEI-OC1 cells was maximally protected at 50 mM of silymarin. Silymarin in concentrations over 100 mM had cytotoxic effects on the HEI-OC1 cells. Therefore, a 50 mM concentration of silymarin was used as the optimal experimental concentration for this study (Fig. 1). Fig. 2 shows cell viability of HEI-OC1 cells. When the cells were exposed with 30 mM of cisplatin for 48 h, the cell viability was 23 0.9%. The cell viability of the HEI-OC1 cells was not affected by a 50 mM concentration of silymarin and it was 98 2.8%. After pre-treatment with 50 mM of silymarin for 1 h, the cells were exposed with 30 mM of cisplatin for 48 h. The cell viability was 70 6.8% (Fig. 2). Pretreatment of HEI-OC1 cells with silymarin significantly prevented the loss of cell viability induced by cisplatin treatment.
Western blot analysis 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 X-100, 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 contents were measured using a Bio-Rad dye binding microassay (Bio-rad, Hercules, CA, USA), and heated at 98 8C for 5 min in Laemmli sample buffer and were subjected to SDS-PAGE on gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes. The membranes were blocked for 2 h in 5% skim milk with TBST (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween-20) at RT and then incubated overnight at 4 8C with primary antibodies at appropriate dilutions; actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase 3 and PARP (Cell Signaling Technology, Danvers, MA, USA). Unbound antibody was removed by four washes for 15 min with TBST. The membranes were then incubated with the appropriate secondary antibodies (1:4000; Santa Cruz Biotechnology, Inc.) in blocking buffer for 2 h, and washed again. The protein bands were detected using the Super Signal West Pico chemiluminescence kit (Thermo Scientific, Waltham, MA, USA) and signals were acquired by image analyzer (LAS-3000 Imaging System, FujiFilm, Tokyo, Japan).
Fig. 1. The effect of silymarin on cisplatin-induced cell viability in HEI-OC1 cells. Cell viability using MTT assay was not significantly affected until a 50 mM concentration silymarin was reached. However, silymarin in concentrations over 100 mM had cytotoxic effects. The maximal protective effect against cisplatin cytotoxicity was observed at a concentration of 50 mM of silymarin on the HEI-OC1 cells. * Optimal experimental concentration of silymarin.
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Hoechst 33258 stain Apoptosis was examined by the appearance of nuclei on Hoechst 33258 staining. The nuclei of the normal control cells showed round-shaped with homogenous intensity (Fig. 3A). After HEI-OC1 cells were exposed to 30 mM of cisplatin for 48 h, the reduction of cell viability was shown. Cisplatin induced apoptotic morphology, condensation and fragmentation with heterogenous intensity in the nuclei (Fig. 3B). The cells treated with 50 mM of silymarin for 48 h showed the normal appearance like the nuclei of control cells (Fig. 3C). Pre-treatment of cells with 50 mM silymarin for 1 h prior to 30 mM cisplatin exposure for 48 h reduced the apoptotic features of nuclei (Fig. 3D). Therefore, silymarin appeared to protect against cisplatin-induced apoptotic features. Western blot analysis of caspase-3 and PARP
Fig. 2. MTT assay. HEI-OC1 cells were exposed 30 mM of cisplatin for 48 h. Cisplatin group shows decrease of cell viability. Cells were pretreated with 50 mM silymarin for 1 h prior to 48 h cisplatin exposure. The group (cisplatin + silymarin) shows increase of cell viability compared with cisplatin alone treated group (*p < 0.01 by Student’s t-test).
To evaluate whether silymarin inhibited cisplatin-induced apoptotic cascades, Western blot analysis was done. Cisplatin caused marked increase of the cleavage of caspase-3 and PARP. When cisplatin was pre-treated with silymarin, the expression of cleaved caspase-3 and PARP was reduced (Fig. 4). These results suggest that silymarin is able to protect HEI-OC1 cells from cisplatin-induced apoptosis by inhibiting activation of apoptotic effectors.
Fig. 3. Hoechst 33258 stain. (A) Control cells show round-shaped neclei with homogenous intensity. (B) Cells were treated with cisplatin for 48 h. The loss of HEI-OC1 cell was noted. Arrows indicate apoptotic nuclear profiles of pyknotic and condensed nuclei. (C) Cells treated with silymarin alone show normal features like control. (D) Inhibitory effect of silymarin on apoptotic features in cisplatin-treated HEI-OC1 cells is seen. Silymarin reduces the cisplatin-induced cell death.
S.I. Cho et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 474–478
Fig. 4. Western blotting of caspase-3 and PARP. Cisplatin increased cleaved caspase3 and PARP. Those expression was inhibited by pre-treatment of silymarin prior to cisplatin exposure.
Propidium iodide staining To investigate the effects of silymarin on the cell cycle, the cellular DNA content was analyzed. Cells with sub-G1 DNA content were quantified and classified as apoptotic cells. Control cells and silymarin-treated cells had no significant change in cell cycle phase. The proportion of sub-G1 were 0.21 0.03% and 0.51 0.15% respectively. The ratio of apoptotic cells significantly increased after the exposure of 30 mM cisplatin for 48 h. (p < 0.01) The proportion of sub-G1 was 25.97 2.1%. After 1 h of pretreatment with 50 mM silymarin prior to cisplatin exposure for 48 h, the ratio of apoptotic cells significantly decreased to 6.53 1.12% (p < 0.01). Fig. 5 is representative histograms showing that silymarin inhibits the cisplatin-induced cell cycle arrest. Discussion Cisplatin is a highly effective chemotherapeutic agent commonly used in the treatment of several malignant tumors. However, the clinical use of cisplatin is limited by its side effects
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including ototoxicity [16]. Cisplatin induces monoadducts at nucleophilic sites such as guanine or adenine and can subsequently lead to intrastrand and interstrand crosslinks in the DNA. These lesions can prevent normal cell cycle progression and activate apoptotic cascades via two main pathways: extrinsic (cytoplasmic) and intrinsic (mitochondrial) pathway [17]. Caspase cascades are activated and result in DNA fragmentation and cell apoptosis. When DNA is damaged, the cell cycle is suspended at the G1 phase. Moreover, cisplatin generates free radicals, specifically ROS. The generation of ROS can deplete cochlear tissue of antioxidant protective molecules such as glutathione and antioxidant enzymes and increase lipid peroxidation, and then lead to calcium influx and apoptosis in cells of the cochlea [18,19]. Antioxidants compound can make upstream protection of the cochlea before death pathways and may function as free radical scavengers [6]. Therefore, the development of therapies to prevent cisplatin-induced ototoxicity has focused on administration of antioxidants. Silymarin has been shown to have antioxidative activity and capability to protect normal cells [20]. Moreover, previous studies have reported that silymarin exerts antitumor activities in some cancer cells [21–25]. Silymarin has been revealed to have potent scavenging activity against hydroxyl radicals and consequently protect lipid peroxidation of the cell membrane [26,27]. It has besides antifibrotic, immunomodulating, and antiinflammatory effects [28]. Cell viability was assessed using MTT assay, which is an important way for measuring cell viability by detecting damage of the function of mitochondria, cisplatin led to increased loss of HEIOC1 cells, while the pre-treatment of silymarin prior to cisplatin exposure prevented cell loss, suggesting silymarin may protect against cisplatin-induced damage to auditory cells. The apoptosis was investigated by utilizing Hoechst 33258 staining. The morphological characteristics of apoptosis involve nuclear fragmentation, chromatin condensation, cell shrinkage [29]. In this study, the results of Hoechst 33258 staining showed that cisplatin promoted apoptotic changes like nuclear condensation, fragmentation and cell death, and silymarin effectively protected against cisplatin-induced apoptosis in the HEI-OC1 cells. Two apoptotic pathways converge to produce caspase 3 activation, resulting in cell death via cleaving proteins necessary for cell
Fig. 5. Propidium iodide staining. PI florescent intensity was measured by flow cytometry. The ratio of cells with sub-G1 DNA content (M1 fraction) significantly increases after exposure of 30 mM of cisplatin for 48 h. The proportion of sub-G1 DNA content is markedly decreased in pretreated cells with 50 mM silymarin for 1 h prior to 48 h cisplatin exposure.
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survival, including Bcl-2, inhibitors of deoxyribonucleases and cytoskeletal proteins [30,31]. And, caspase activates cleavage of PARP which is known to detect and repair the DNA strand breaks generated by toxic agents [32]. We demonstrated that cisplatin enhanced caspase-3 and PARP activity, while silymarin attenuated the activation of caspase-3 and cleavage of PARP in HEI-OC1 cells. This results suggest that silymarin serves important functions in anti-apoptosis through inhibition of caspase-dependant pathway. The interaction between cisplatin and DNA leads to cell cycle arrest. Endonuclease action at a late stage in the apoptotic cascade makes small fragments of DNA. When cells are fixed in ethanol and subsequently rehydrated, some of the lower-molecular-weight DNA leaches out and lessen the DNA content. These cell can be analyzed as a sub-G1 peak in a DNA histogram. Therefore, an increase of sub-G1 population indicates the apoptosis of cells [33,34]. In cell-cycle phase distribution, cisplatin significantly increased the number of cells in the sub-G1 phase, which represented the apoptotic cells. Silymarin decreased the number of cells in the sub-G1 phase compared with the cisplatin-treated cells. Silymarin successfully inhibited the cell cycle arrest by cisplatin. In this study, we analyzed the protective effects of silymarin in cultured HEI-OC1 cells and demonstrated that silymarin significantly suppresses cisplatin-induced apoptosis and increases cell viability in the HEI-OC1 cells. Silymarin can play a preventive role against cisplatin-induced ototoxicity by inhibiting apoptotic cascade that leads to cell death, and it may be a potential candidate drug to eliminate cisplatin induced ototoxicity. Further studies may reveal potential clinical usefulness of silymarin as a chemopreventive agent 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 research fund from Chosun University, 2013. References [1] N. Pabla, Z. Dong, Cisplatin nephrotoxicity: mechanisms and renoprotective strategies, Kidney Int. 73 (2008) 994–1007. [2] J.T. Hartmann, H.P. Lipp, Toxicity of platinum compounds, Expert Opin. Pharmacother. 4 (2003) 889–901. [3] N. Dehne, J. Lautemann, 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. [4] E. Pigeolet, P. Corbisier, A. Houbion, D. Lambert, C. Michiels, M. Raes, et al., Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals, Mech. Ageing Dev. 51 (1990) 283–297. [5] W.J. Clerici, D.L. DiMartino, M.R. Prasad, Direct effects of reactive oxygen species on cochlear outer hair cell shape in vitro, Hear. Res. 84 (1995) 30–40. [6] L.P. Rybak, C.A. Whitworth, Ototoxicity: therapeutic opportunities, Drug Discov. Today 10 (2005) 1313–1321. [7] V. Kren, D. Walterova´, Silybin and silymarin – new effects and applications, Biomed. Pap. Med. Fac. Univ. Palacky Olomouc. Czech Repub. 149 (2005) 29–41. [8] L. Abenavoli, R. Capasso, N. Milic, F. Capasso, Milk thistle in liver diseases: past, present, future, Phytother. Res. 24 (2010) 1423–1432.
[9] R. Saller, R. Meier, R. Brignoli, The use of silymarin in the treatment of liver diseases, Drugs 61 (2001) 2035–2063. [10] M. Vaid, S.K. Katiyar, Molecular mechanisms of inhibition of photocarcinogenesis by silymarin, a phytochemical from milk thistle (Silybum marianum L. Gaertn.), Int. J. Oncol. 36 (2010) 1053–1060 (Review). [11] F.S. Hogan, N.K. Krishnegowda, M. Mikhailova, M.S. Kahlenberg, Flavonoid, silibinin, inhibits proliferation and promotes cell-cycle arrest of human colon cancer, J. Surg. Res. 143 (2007) 58–65. [12] C.W. Cheung, N. Gibbons, D.W. Johnson, D.L. Nicol, Silibinin – a promising new treatment for cancer, Anticancer Agents Med. Chem. 10 (2010) 186–195. [13] K. Ramasamy, R. Agarwal, Multitargeted therapy of cancer by silymarin, Cancer Lett. 269 (2008) 352–362. [14] G.M. Kalinec, P. Webster, D.J. Lim, F. Kalinec, A cochlear cell line as an in vitro system for drug ototoxicity screening, Audiol. Neurootol. 8 (2003) 177–189. [15] G.J. Im, J.W. Chang, J. Choi, S.W. Chae, E.J. Ko, H.H. Jung, Protective effect of Korean red ginseng extract on cisplatin ototoxicity in HEI-OC1 auditory cells, Phytother. Res. 24 (2010) 614–621. [16] D. Wang, S.J. Lippard, Cellular processing of platinum anticancer drugs, Nat. Rev. Drug Discov. 4 (2005) 307–320. [17] T. Langer, A. Am Zehnhoff-Dinnesen, S. Radtke, J. Meitert, O. Zolk, Understanding platinum-induced ototoxicity, Trends Pharmacol. Sci. 34 (2013) 458–469. [18] C. Casares, R. Ramı´rez-Camacho, A. Trinidad, A. Rolda´n, E. Jorge, J.R. Garcı´aBerrocal, Reactive oxygen species in apoptosis induced by cisplatin: review of physiopathological mechanisms in animal models, Eur. Arch. Otorhinolaryngol. 269 (2012) 2455–2459. [19] J.E. Lee, T. Nakagawa, T.S. Kim, T. Endo, A. Shiga, F. Iguchi, et al., Role of reactive radicals in degeneration of the auditory system of mice following cisplatin treatment, Acta Oto-Laryngol. 124 (2004) 1131–1135. [20] H.H. Mansour, H.F. Hafez, N.M. Fahmy, Silymarin modulates cisplatin-induced oxidative stress and hepatotoxicity in rats, J. Biochem. Mol. Biol. 39 (2006) 656–661. [21] X. Zi, D.K. Feyes, R. Agarwal, Anticarcinogenic effect of a flavonoid antioxidant, silymarin, in human breast cancer cells MDA-MB 468: induction of G1 arrest through an increase in Cip1/p21 concomitant with a decrease in kinase activity of cyclin-dependent kinases and associated cyclins, Clin. Cancer Res. 4 (1998) 1055–1064. [22] G. Sharma, R.P. Singh, D.C. Chan, R. Agarwal, Silibinin induces growth inhibition and apoptotic cell death in human lung carcinoma cells, Anticancer Res. 23 (2003) 2649–2655. [23] Q. Huang, L.J. Wu, S. Tashiro, S. Onodera, L.H. Li, T. Ikejima, Silymarin augments human cervical cancer HeLa cell apoptosis via P38/JNK MAPK pathways in serumfree medium, J. Asian Nat. Prod. Res. 7 (2005) 701–709. [24] G. Deep, R.P. Singh, C. Agarwal, D.J. Kroll, R. Agarwal, Silymarin and silibinin cause G1 and G2-M cell cycle arrest via distinct circuitries in human prostate cancer PC3 cells: a comparison of flavanone silibinin with flavanolignan mixture silymarin, Oncogene 25 (2006) 1053–1069. [25] X. Zhong, Y. Zhu, Q. Lu, J. Zhang, Z. Ge, S. Zheng, Silymarin causes caspases activation and apoptosis in K562 leukemia cells through inactivation of Akt pathway, Toxicology 227 (2006) 211–216. [26] L. Mira, M. Silva, C.F. Manso, Scavenging of reactive oxygen species by silibinin dihemisuccinate, Biochem. Pharmacol. 48 (1994) 753–759. [27] P. Muriel, T. Garciapin˜a, V. Perez-Alvarez, M. Mourelle, Silymarin protects against paracetamol-induced lipid peroxidation and liver damage, J. Appl. Toxicol. 12 (1992) 439–442. [28] G. Karimi, M. Vahabzadeh, P. Lari, M. Rashedinia, M. Moshiri, Silymarin, a promising pharmacological agent for treatment of diseases, Iran J. Basic Med. Sci. 14 (2011) 308–317. [29] T. Huang, A.G. Cheng, H. Stupak, W. Liu, A. Kim, H. Staecker, et al., Oxidative stressinduced apoptosis of cochlear sensory cells: otoprotective strategies, Int. J. Dev. Neurosci. 18 (2000) 259–270. [30] Y.A. Lazebnik, S.H. Kaufmann, S. Desnoyers, G.G. Poirier, W.C. Earnshaw, Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE, Nature 371 (1994) 346–347. [31] K. Watanabe, S. Inai, K. Jinnouchi, S. Baba, T. Yagi, Expression of caspase-activated deoxyribonuclease (CAD) and caspase 3 (CPP32) in the cochlea of cisplatin (CDDP)-treated guinea pigs, Auris Nasus Larynx 30 (2003) 219–225. [32] P. Decker, S. Muller, Modulating poly(ADP-ribose) polymerase activity: potential for the prevention and therapy of pathogenic situations involving DNA damage and oxidative stress, Curr. Pharm. Biotechnol. 3 (2002) 275–283. [33] M.G. Ormerod, The study of apoptotic cells by flow cytometry, Leukemia 12 (1998) 1013–1025. [34] I. Vermes, C. Haanen, C. Reutelingsperger, Flow cytometry of apoptotic cell death, J. Immunol. Methods 243 (2000) 167–190.
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Protective effect of silymarin against cisplatin-induced ototoxicity Sung Il Cho *, Ji-Eun Lee, Nam Yong Do Department of Otolayngology-Head and Neck Surgery, 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 17 September 2013 Received in revised form 3 December 2013 Accepted 17 December 2013 Available online 27 December 2013
Objectives: Silymarin is a plant extract with strong antioxidant properties in addition to antiinflammatory and anticarcinogenic actions. The aim of this study was to investigate the potential preventive effect of silymarin on cisplatin ototoxicity in an auditory cell line, HEI-OC1 cells. Methods: Cultured HEI-OC1 cells were exposed to cisplatin (30 mM) with or without pre-treatment with silymarin (50 mM). Cell viability was evaluated using MTT assay. Hoechst 33258 staining was used to identify cells undergoing apoptosis. Western blot analysis was done to evaluate whether silymarin inhibits cisplatin-induced caspase and PARP activation. Cell-cycle analysis was done by flow cytometry to investigate whether silymarin is capable of protecting cisplatin-induced cell cycle arrest. Results: Cell viability significantly increased in cells pretreated with silymarin compared with cells exposed to cisplatin alone. Pre-treatment of silymarin appeared to protect against cisplatin-induced apoptotic features on Hoechst 33258 staining. Cisplatin increased cleaved caspase-3 and PARP on Western blot analysis. However, pre-treatment with silymarin inhibited the expression of cleaved caspase-3 and PARP. Silymarin did attenuate cell cycle arrest and apoptosis in HEI-OC1 cells. Conclusions: Our results demonstrate that silymarin treatment inhibited cisplatin-induced cytotoxicity in the auditory cell line, HEI-OC1. Silymarin may be a potential candidate drug to eliminate cisplatin induced ototoxicity. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Silymarin Cisplatin Ototoxicity Apoptosis
Introduction Cisplatin is one of the most prescribed drugs for the treatment of human solid tumors such as ovarian, testicular, cervical, head and neck, lung, and bladder cancer. However, ototoxicity-induced by cisplatin has been shown to be an important obstacle for its utility and therapeutic profile [1,2]. Though the exact mechanisms of ototoxic effects by cisplatin is not widely revealed, reactive oxygen species (ROS) such as superoxide anion are believed to play a major role. Increased ROS results in depletion of the cochlear antioxidant enzymes [3,4]. This can lead to calcium influx within hair cells, leading to apoptosis [5]. Ototoxicity of cisplatin can be reduced by various antioxidants by counteracting this response [6]. Silymarin is a lipophilic extract from the seeds of the milk thistle (Silybum marianum) and composed of three isomers of flavonolignans (silybin, silydianin, and silychristin), and two flavonoids (tamoxifen and quercetin) [7,8]. Silymarin has been
used in the treatment of liver diseases such as cirrhosis, viral hepatitis due to its hepatoprotective effect. The effect is mediated by scavenging of free radicals, decreasing formation of ROS and inhibition of fatty acid peroxidation. Another mechanism involves anti-apoptotic actions and anti-inflammatory actions [9,10]. Silymarin has no significant adverse reactions in human studies and has been reported to be safe in animal models [11]. In addition, silymarin has been shown to exert anti-neoplastic effects in a variety of cancer models [12,13]. Anti-oxidant and anti-apoptotic properties of silymarin may also have protective role against cisplatin-induced ototoxicity. In this study, we aimed to investigate the effect of silymarin on cisplatin-induced ototoxicity in an auditory cell line and establish potential application for prevention of ototoxicity after cisplatin chemotherapy. Materials and methods Cell culture
* Corresponding author at: Department of Otolaryngology-Head and Neck Surgery, Chosun University Hospital, 365 Pilmun-daero, Dong-gu, Gwangju 501717, South Korea. Tel.: +82 62 220 3207; fax: +82 62 225 2702. E-mail address: [email protected] (S.I. Cho). 0165-5876/$ – see front matter ß 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijporl.2013.12.024
The establishment of House Ear Institute-Organ of Corti 1 (HEIOC1) cell line was derived from postnatal organ of Corti of a transgenic immortomouse. The auditory cell line is extremely sensitive to ototoxic drug and express molecular markers, which
S.I. Cho et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 474–478
are characteristic of organ of Corti cells [14]. HEI-OC1 cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; GIBCO BRL, NY, USA) supplemented with 10% fetal bovine serum (FBS; Lonza Walkersvile, MD, USA) at 33 8C in a humidified incubator with 5% CO2. MTT assay Cell viability was determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. HEI-OC1 cells were seeded at 3 104 cells per well of a 24-well plate and were incubated in DMEM with 10% FBS at 33 8C with 5% CO2. Cells were divided into four groups: control, silymarin, cisplatin, silymarin plus cisplatin. Silymarin was purchased from Sigma (Saint Louis, MO, USA). Control group, silymarin, and cisplatin treated groups were incubated for 48 h. A 30 mM concentration of cisplatin for 48 h was used as adequate concentration to study cisplatininduced cytotoxicity on the HEI-OC1 cells [15]. In order to investigate the effects of silymarin on cisplatin ototoxicity, the cells were pretreated with silymarin (50 mM) for 1 h, and exposed to cisplatin (30 mM) for 48 h. For the MTT assay, 5 mg/ml of MTT solution (Sigma, Saint 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 dissolved by the addition of 500 mL/well of DMSO. Absorption was measured using a spectrophotometer (BioTek, VT, USA) at 570 nm. Hoechst 33258 staining Apoptotic cell death was determined by evaluating the nuclear morphology using Hoechst 33258 staining. Cells were incubated with 10 mg/mL of the Hoechst 33258 (Sigma, Saint Louis, MO, USA) for 30 min. Membrane-permeable Hoechst 33258 was a blue fluorescent dye and stained the cell nucleus. After washing twice with phosphate buffered saline (PBS), the cells were detached by trypsinization and fixed with 4% paraformaldehyde for 10 min at room temperature (RT). The cells were placed over the slides and mounted with glycerol after drying with air. The cells were observed under a fluorescence microscope (DM5000, Leica, Wetzlar, Germany).
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Propidium iodide staining The floating and trypsin-detached HEI-OC 1 cells were collected and washed once with ice-cold PBS, followed by fixing in 70% cold ethanol for 30 min at 4 8C. The cells were then stained in PBS and propidium iodide (50 mg/ml), RNase A (50 mg/ml), and 0.05% Triton X-100 for 45 min at RT. The DNA content of the HEI-OC1 cells was analyzed by fluorescent-activated cell sorting (FACSort, Becton Dickinson). At least 10,000 events were analyzed, and the percentage of cells in sub-G1 population was calculated. Aggregates of cell debris at the origin of histogram were excluded from the sub-G1 cells. Statistical analysis Statistical analysis of the results was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). The Student’s t-test was used for pairs of data. A p < 0.01 was considered statistically significant. Results MTT assay To investigate whether silymarin was able to prevent apoptosis induced by cisplatin, cell viability in HEI-OC1 cultures was determined by MTT assay. HEI-OC1 cells were treated with various concentrations of silymarin and with 30 mM of cisplatin for 48 h, the cell viability of HEI-OC1 cells was maximally protected at 50 mM of silymarin. Silymarin in concentrations over 100 mM had cytotoxic effects on the HEI-OC1 cells. Therefore, a 50 mM concentration of silymarin was used as the optimal experimental concentration for this study (Fig. 1). Fig. 2 shows cell viability of HEI-OC1 cells. When the cells were exposed with 30 mM of cisplatin for 48 h, the cell viability was 23 0.9%. The cell viability of the HEI-OC1 cells was not affected by a 50 mM concentration of silymarin and it was 98 2.8%. After pre-treatment with 50 mM of silymarin for 1 h, the cells were exposed with 30 mM of cisplatin for 48 h. The cell viability was 70 6.8% (Fig. 2). Pretreatment of HEI-OC1 cells with silymarin significantly prevented the loss of cell viability induced by cisplatin treatment.
Western blot analysis 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 X-100, 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 contents were measured using a Bio-Rad dye binding microassay (Bio-rad, Hercules, CA, USA), and heated at 98 8C for 5 min in Laemmli sample buffer and were subjected to SDS-PAGE on gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes. The membranes were blocked for 2 h in 5% skim milk with TBST (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween-20) at RT and then incubated overnight at 4 8C with primary antibodies at appropriate dilutions; actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase 3 and PARP (Cell Signaling Technology, Danvers, MA, USA). Unbound antibody was removed by four washes for 15 min with TBST. The membranes were then incubated with the appropriate secondary antibodies (1:4000; Santa Cruz Biotechnology, Inc.) in blocking buffer for 2 h, and washed again. The protein bands were detected using the Super Signal West Pico chemiluminescence kit (Thermo Scientific, Waltham, MA, USA) and signals were acquired by image analyzer (LAS-3000 Imaging System, FujiFilm, Tokyo, Japan).
Fig. 1. The effect of silymarin on cisplatin-induced cell viability in HEI-OC1 cells. Cell viability using MTT assay was not significantly affected until a 50 mM concentration silymarin was reached. However, silymarin in concentrations over 100 mM had cytotoxic effects. The maximal protective effect against cisplatin cytotoxicity was observed at a concentration of 50 mM of silymarin on the HEI-OC1 cells. * Optimal experimental concentration of silymarin.
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Hoechst 33258 stain Apoptosis was examined by the appearance of nuclei on Hoechst 33258 staining. The nuclei of the normal control cells showed round-shaped with homogenous intensity (Fig. 3A). After HEI-OC1 cells were exposed to 30 mM of cisplatin for 48 h, the reduction of cell viability was shown. Cisplatin induced apoptotic morphology, condensation and fragmentation with heterogenous intensity in the nuclei (Fig. 3B). The cells treated with 50 mM of silymarin for 48 h showed the normal appearance like the nuclei of control cells (Fig. 3C). Pre-treatment of cells with 50 mM silymarin for 1 h prior to 30 mM cisplatin exposure for 48 h reduced the apoptotic features of nuclei (Fig. 3D). Therefore, silymarin appeared to protect against cisplatin-induced apoptotic features. Western blot analysis of caspase-3 and PARP
Fig. 2. MTT assay. HEI-OC1 cells were exposed 30 mM of cisplatin for 48 h. Cisplatin group shows decrease of cell viability. Cells were pretreated with 50 mM silymarin for 1 h prior to 48 h cisplatin exposure. The group (cisplatin + silymarin) shows increase of cell viability compared with cisplatin alone treated group (*p < 0.01 by Student’s t-test).
To evaluate whether silymarin inhibited cisplatin-induced apoptotic cascades, Western blot analysis was done. Cisplatin caused marked increase of the cleavage of caspase-3 and PARP. When cisplatin was pre-treated with silymarin, the expression of cleaved caspase-3 and PARP was reduced (Fig. 4). These results suggest that silymarin is able to protect HEI-OC1 cells from cisplatin-induced apoptosis by inhibiting activation of apoptotic effectors.
Fig. 3. Hoechst 33258 stain. (A) Control cells show round-shaped neclei with homogenous intensity. (B) Cells were treated with cisplatin for 48 h. The loss of HEI-OC1 cell was noted. Arrows indicate apoptotic nuclear profiles of pyknotic and condensed nuclei. (C) Cells treated with silymarin alone show normal features like control. (D) Inhibitory effect of silymarin on apoptotic features in cisplatin-treated HEI-OC1 cells is seen. Silymarin reduces the cisplatin-induced cell death.
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Fig. 4. Western blotting of caspase-3 and PARP. Cisplatin increased cleaved caspase3 and PARP. Those expression was inhibited by pre-treatment of silymarin prior to cisplatin exposure.
Propidium iodide staining To investigate the effects of silymarin on the cell cycle, the cellular DNA content was analyzed. Cells with sub-G1 DNA content were quantified and classified as apoptotic cells. Control cells and silymarin-treated cells had no significant change in cell cycle phase. The proportion of sub-G1 were 0.21 0.03% and 0.51 0.15% respectively. The ratio of apoptotic cells significantly increased after the exposure of 30 mM cisplatin for 48 h. (p < 0.01) The proportion of sub-G1 was 25.97 2.1%. After 1 h of pretreatment with 50 mM silymarin prior to cisplatin exposure for 48 h, the ratio of apoptotic cells significantly decreased to 6.53 1.12% (p < 0.01). Fig. 5 is representative histograms showing that silymarin inhibits the cisplatin-induced cell cycle arrest. Discussion Cisplatin is a highly effective chemotherapeutic agent commonly used in the treatment of several malignant tumors. However, the clinical use of cisplatin is limited by its side effects
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including ototoxicity [16]. Cisplatin induces monoadducts at nucleophilic sites such as guanine or adenine and can subsequently lead to intrastrand and interstrand crosslinks in the DNA. These lesions can prevent normal cell cycle progression and activate apoptotic cascades via two main pathways: extrinsic (cytoplasmic) and intrinsic (mitochondrial) pathway [17]. Caspase cascades are activated and result in DNA fragmentation and cell apoptosis. When DNA is damaged, the cell cycle is suspended at the G1 phase. Moreover, cisplatin generates free radicals, specifically ROS. The generation of ROS can deplete cochlear tissue of antioxidant protective molecules such as glutathione and antioxidant enzymes and increase lipid peroxidation, and then lead to calcium influx and apoptosis in cells of the cochlea [18,19]. Antioxidants compound can make upstream protection of the cochlea before death pathways and may function as free radical scavengers [6]. Therefore, the development of therapies to prevent cisplatin-induced ototoxicity has focused on administration of antioxidants. Silymarin has been shown to have antioxidative activity and capability to protect normal cells [20]. Moreover, previous studies have reported that silymarin exerts antitumor activities in some cancer cells [21–25]. Silymarin has been revealed to have potent scavenging activity against hydroxyl radicals and consequently protect lipid peroxidation of the cell membrane [26,27]. It has besides antifibrotic, immunomodulating, and antiinflammatory effects [28]. Cell viability was assessed using MTT assay, which is an important way for measuring cell viability by detecting damage of the function of mitochondria, cisplatin led to increased loss of HEIOC1 cells, while the pre-treatment of silymarin prior to cisplatin exposure prevented cell loss, suggesting silymarin may protect against cisplatin-induced damage to auditory cells. The apoptosis was investigated by utilizing Hoechst 33258 staining. The morphological characteristics of apoptosis involve nuclear fragmentation, chromatin condensation, cell shrinkage [29]. In this study, the results of Hoechst 33258 staining showed that cisplatin promoted apoptotic changes like nuclear condensation, fragmentation and cell death, and silymarin effectively protected against cisplatin-induced apoptosis in the HEI-OC1 cells. Two apoptotic pathways converge to produce caspase 3 activation, resulting in cell death via cleaving proteins necessary for cell
Fig. 5. Propidium iodide staining. PI florescent intensity was measured by flow cytometry. The ratio of cells with sub-G1 DNA content (M1 fraction) significantly increases after exposure of 30 mM of cisplatin for 48 h. The proportion of sub-G1 DNA content is markedly decreased in pretreated cells with 50 mM silymarin for 1 h prior to 48 h cisplatin exposure.
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survival, including Bcl-2, inhibitors of deoxyribonucleases and cytoskeletal proteins [30,31]. And, caspase activates cleavage of PARP which is known to detect and repair the DNA strand breaks generated by toxic agents [32]. We demonstrated that cisplatin enhanced caspase-3 and PARP activity, while silymarin attenuated the activation of caspase-3 and cleavage of PARP in HEI-OC1 cells. This results suggest that silymarin serves important functions in anti-apoptosis through inhibition of caspase-dependant pathway. The interaction between cisplatin and DNA leads to cell cycle arrest. Endonuclease action at a late stage in the apoptotic cascade makes small fragments of DNA. When cells are fixed in ethanol and subsequently rehydrated, some of the lower-molecular-weight DNA leaches out and lessen the DNA content. These cell can be analyzed as a sub-G1 peak in a DNA histogram. Therefore, an increase of sub-G1 population indicates the apoptosis of cells [33,34]. In cell-cycle phase distribution, cisplatin significantly increased the number of cells in the sub-G1 phase, which represented the apoptotic cells. Silymarin decreased the number of cells in the sub-G1 phase compared with the cisplatin-treated cells. Silymarin successfully inhibited the cell cycle arrest by cisplatin. In this study, we analyzed the protective effects of silymarin in cultured HEI-OC1 cells and demonstrated that silymarin significantly suppresses cisplatin-induced apoptosis and increases cell viability in the HEI-OC1 cells. Silymarin can play a preventive role against cisplatin-induced ototoxicity by inhibiting apoptotic cascade that leads to cell death, and it may be a potential candidate drug to eliminate cisplatin induced ototoxicity. Further studies may reveal potential clinical usefulness of silymarin as a chemopreventive agent 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 research fund from Chosun University, 2013. References [1] N. Pabla, Z. Dong, Cisplatin nephrotoxicity: mechanisms and renoprotective strategies, Kidney Int. 73 (2008) 994–1007. [2] J.T. Hartmann, H.P. Lipp, Toxicity of platinum compounds, Expert Opin. Pharmacother. 4 (2003) 889–901. [3] N. Dehne, J. Lautemann, 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. [4] E. Pigeolet, P. Corbisier, A. Houbion, D. Lambert, C. Michiels, M. Raes, et al., Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals, Mech. Ageing Dev. 51 (1990) 283–297. [5] W.J. Clerici, D.L. DiMartino, M.R. Prasad, Direct effects of reactive oxygen species on cochlear outer hair cell shape in vitro, Hear. Res. 84 (1995) 30–40. [6] L.P. Rybak, C.A. Whitworth, Ototoxicity: therapeutic opportunities, Drug Discov. Today 10 (2005) 1313–1321. [7] V. Kren, D. Walterova´, Silybin and silymarin – new effects and applications, Biomed. Pap. Med. Fac. Univ. Palacky Olomouc. Czech Repub. 149 (2005) 29–41. [8] L. Abenavoli, R. Capasso, N. Milic, F. Capasso, Milk thistle in liver diseases: past, present, future, Phytother. Res. 24 (2010) 1423–1432.
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