FDA-Approved Tedizolid Phosphate Prevents Cisplatin-Induced Hearing Loss Without Decreasing Its Anti-tumor Effect

Abstract

Purpose

Cisplatin is a low-cost clinical anti-tumor drug widely used to treat solid tumors. However, its use could damage cochlear hair cells, leading to irreversible hearing loss. Currently, there appears one drug approved in clinic only used for reducing ototoxicity associated with cisplatin in pediatric patients, which needs to further explore other candidate drugs.

Methods

Here, by screening 1967 FDA-approved drugs to protect cochlear hair cell line (HEI-OC1) from cisplatin damage, we found that Tedizolid Phosphate (Ted), a drug indicated for the treatment of acute infections, had the best protective effect. Further, we evaluated the protective effect of Ted against ototoxicity in mouse cochlear explants, zebrafish, and adult mice. The mechanism of action of Ted was further explored using RNA sequencing analysis and verified. Meanwhile, we also observed the effect of Ted on the anti-tumor effect of cisplatin.

Results

Ted had a strong protective effect on hair cell (HC) loss induced by cisplatin in zebrafish and mouse cochlear explants. In addition, when administered systemically, it protected mice from cisplatin-induced hearing loss. Moreover, antitumor studies showed that Ted had no effect on the antitumor activity of cisplatin both in vitro and in vivo. RNA sequencing analysis showed that the otoprotective effect of Ted was mainly achieved by inhibiting phosphorylation of ERK. Consistently, ERK activator aggravated the damage of cisplatin to HCs.

Conclusion

Collectively, these results showed that FDA-approved Ted protected HCs from cisplatin-induced HC loss by inhibiting ERK phosphorylation, indicating its potential as a candidate for preventing cisplatin ototoxicity in clinical settings.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10162-024-00945-2.

Introduction

Since its approval for clinical use by the Food and Drug Administration (FDA) in 1978, cisplatin has provided an effective treatment for life-threatening solid tumors, including lung cancer and nerve tumors [1, 2]. However, cisplatin is associated with significant side effects such as dose-related ototoxicity and nephrotoxicity, which greatly affects the quality of life [3, 4]. Whereas renal damage caused by cisplatin is reversible, the damage to the inner ear hair cells (HCs) is permanent [5]. Cisplatin-induced ototoxicity usually manifests as a progressive, bilateral, and irreversible hearing loss [6]. Studies have reported that 40–80% of adults and at least 50% of children develop permanent hearing loss after receiving cisplatin treatment [7, 8]. Despite its numerous side effects, cisplatin is still the mainstay of cancer treatment in many developing countries with limited medical resources because of its low cost [2, 5]. Therefore, it is crucial to find ways to prevent cisplatin-related hearing loss.

Cisplatin-induced ototoxicity mainly affects the Corti organs, stria vascularis, and spiral ganglia neuron [9]. In the cochlear Corti organ, outer hair cells (OHCs) are more susceptible to damage than inner HCs. Damage in OHC starts at higher frequencies and gradually extends to lower frequencies [10, 11]. The current study suggests that cisplatin-induced ototoxicity is caused by several factors, including inflammatory response, production of reactive oxygen species (ROS), cellular uptake of cisplatin, endoplasmic reticulum stress, and DNA damage, leading to HC death [12, 13]. Several transporters act synergistically to translocate cisplatin into target cells in the cochlea where it remains for a long time. Cisplatin retention in cochlear hair cells can lead to DNA damage, inhibit protein synthesis, and cause the accumulation of excessive ROS. In turn, excessive ROS promotes lipid peroxidation, protein nitration, and DNA damage, reduces NAD⁺/NADH ratio, and finally activates a series of signaling pathways that induce the release of cytokines, leading to cell inflammation and death of inner ear cells, causing irreversible sensorineural hearing loss. However, the precise mechanism of cisplatin ototoxicity is not fully understood [14], which hinders the clinical search for a better target for preventing cisplatin-induced ototoxicity.

To alleviate the ototoxicity of cisplatin, various therapeutic strategies have been used in previous studies [1, 12]. Among them, reducing the release of pro-inflammatory cytokines within the cochlea has shown potential to protect against hearing loss [1, 15, 16]. In addition, flunarizine, a T-type Ca²⁺ channel antagonist, showed an ear-protective effect by significantly reducing levels of serum and cochlear pro-inflammatory cytokines through the activation of Nrf2/HO-1[17]. Another potential approach to cisplatin-induced hearing loss (CIHL) prevention is to reduce the accumulation of intracellular ROS [18–20] using antioxidant treatments such as sodium thiosulfate [21]and N-acetylcysteine [22]. For example, sodium thiosulfate successfully reduced hearing loss in children with hepatoblastoma by 48% but also reduced the antitumor effect [23, 24]. Reducing cellular uptake of cisplatin is also an effective strategy for reducing cisplatin ototoxicity [25, 26]. When the organic cation transporter (OCT) blocker cimetidine and cisplatin were administered together, then the cochlea toxicity was reduced [27, 28]. None of the available strategies to prevent cisplatin damage has been approved for clinical use. Therefore, it is urgent to find effective drugs and explore their mechanism of action to create more reasonable clinical treatment strategies [12, 29, 30].

At present, cell lines and zebrafish are used in large-scale drug screening to identify new hearing-protective drugs [10, 31, 32], and many compounds have shown promising results in vivo and in vitro [33, 34]. Using an immortalized cell line (HEI-OC1) from the cochlea, Zuo et al. [35] conducted a high-throughput screening of a library of 4385 compounds and found that CDK2 inhibitors-Kenpaullone showed significant protection both in vitro and in vivo experiments. Kros et al. extensively screened more than 400 compounds using a zebrafish model and identified a promising protective drug, ORC-13661, which reduced the toxicity of cisplatin by blocking the mechanoelectrical transducer (MET) channels in outer hair cells [30]. Recently, FDA-approved sodium thiosulfate (Pedmark®), a chemoprotectant/antioxidant, was reported to reduce the risk of cisplatin-related ototoxicity in children [36]. However, it is still necessary to discover more effective FDA-approved drugs for rapid clinical application to alleviate CIHL. Repurposing the use of drugs approved by the FDA has recently become a particularly attractive and effective alternative in drug development, which has the advantages of short development time and low cost. Therefore, in this study, we conducted an unbiased high-throughput screening of 1967 FDA-approved drugs. Due to the large number of drugs, we used mouse immortalized inner ear cell line (HEI-OC1) to screen the library and identified the small molecules with a potential to protect against CIHL. Screening analysis, revealed Tedizolid Phosphate (Ted) as a compound, with the best protective effect. However, Ted has not been previously tested for treating inner ear diseases. Ted has three advantages as an otoprotective therapeutic candidate: (1) Ted is a second-generation oxazolidinone antimicrobial with a similar mechanism as the first-generation oxazolidinone antimicrobial, linezolid [37] and has been approved for the treatment of multiple drug-resistant gram-positive infections [38]; (2) Ted has the potential to cross the blood labyrinth barrier in the inner ear. On the one hand, the BBB in the inner ear is similar to the blood labyrinth barrier [39]. On the other hand, Ted was found in cerebrospinal fluid both in animal experiments and patients with relapsed acute myeloid leukemia [40, 41]; (3) As a new antibiotic, Ted is unlikely to be an antioxidant in itself and thus may not interfere with the antitumor effect of cisplatin but may rather improve it. Further, we evaluated the protective effect of Ted against CIHL in mouse cochlear explants, zebrafish, and adult mice. The mechanism of action of Ted was further explored using RNA sequencing analysis. The results showed that Ted did not interfere with the anti-tumor effect of cisplatin in vitro and in vivo. Our experiments revealed and identified Tedizolid Phosphate as a promising ototoxicity-protective agent for clinical use, which provided hearing protection without interfering with the anti-tumor efficacy of cisplatin.

Materials and Methods

Study Approval

All experimental procedures involving animals were approved by the Tianjin Medical University General Hospital Experimental Animal Administrative Committee and were in accordance with animal welfare principles.

Animal Models

SPF BALB/c nude mice (male, 5 weeks old) and wild-type C57BL/6 mice (male, 8 weeks old) were purchased from Beijing Vital River Laboratory Co., Ltd. and used for cisplatin treatment experiments. All animals were raised in the experimental animal center of Tianjin Medical University (constant temperature 25 °C, 12-h light/dark cycle). All experimental protocols that involved animals were approved by the Animal Ethics Committee of Tianjin Medical University. To maintain normal body temperature during the experiment, all anesthetized animals were placed on a heating pad. The Tg (brn3c:GFP) transgenic zebrafish was obtained from Shandong Provincial Hospital, which is affiliated with Shandong University. All fish were raised in a culture medium under standard laboratory conditions, as described previously [29].

Cell Viability Assay

Human lung cancer A549 and mouse colon cancer MC-38 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Mouse cochlear immortalized cell line HEI-OC1 was provided by Professor Chai Renjie at Southeast University. MC-38 and A549 and HEI-OC1 cells were cultured in a 37 °C incubator with 10% CO₂ and 33 °C in a 5% CO₂ atmosphere, respectively. All cells were cultured with DMEM-F12 medium (HyClone, SH30022.01, USA) containing 10% fetal bovine serum (Biological Industries, Israel) and 1% Penicillin/Streptomycin solution (Solarbio, 20,200,617, Beijing, China).

HEI-OC1 cells were plated in 96-well plates at 5000 cells per well overnight and then pre-incubated with indicated FDA-approved Drug Library from Selleck (L1300, USA). All test compounds were added to each well to a final concentration of 10 µM for 24 h and challenged with cisplatin to a final concentration of 50 µM for another 24 h, according to previous research methods and manufacturer’s instructions [35, 42, 43]. Then, cell viability was measured using CCK8 with a Cell Counting Kit-8 (CCK-8) assay (TargetMol, C0005). All cells were then incubated with 10 µl of CCK-8 at 37 °C for 45–60 min, and the absorbance (optical density, OD) at 450 nm was measured using a microtitration plate reader (Bio-Rad, Shanghai, China). MC-38 and A549 cells (5000 cells/well) were inoculated into 96-well plates overnight. The tumor cells were pretreated with Ted for 1 h and then incubated with 15 µM cisplatin for 48 h. Finally, cell viability was measured using CCK8 analysis. Cell viability = (OD of treatment − OD of blank control) / (OD of control − OD of blank control) × 100%.

Explants Culture

Cochlear specimens were dissected from P3-P4 (postnatal day, P) C57BL/6 mice and attached to a Petri dish coated with Cell-Tak (Corning, 354,242). Cochlear organs were cultured in DMEM/F12 medium (HyClone, SH30022.01) supplemented with N2/B27 (Stemcell, 07152; Invitrogen, Waltham, aMO, USA) and ampicillin (Beyotime, ST008, Shanghai, China) at 37 °C with 5% CO₂ for 24 h. The following day, cochlear organs were pre-incubated with medium with or without Ted (Selleck, S4641, Houston, USA) for 2 h and then exposed to 150 µm cisplatin for another 24 h. In the cisplatin-alone and control groups, the culture was pretreated with DMSO (Solarbio, D8371, Beijing, China) for 2 h.

Zebrafish Lateral Line Hair Cell Protection Assay

Zebrafish (Brn3c: GFP), on the 5th day after fertilization, were selected for the experiment. The larvae were pretreated with Ted for 1 h followed by a high dose of 600 µM cisplatin for 6 h, with or without Ted treatment, and allowed to recover in fresh water for 1 h, as previously described (Fig. 2A) [29, 44]. In brief, 5dpf (5 days after fertilization, 5dpf) GFP + zebrafish were pretreated with Ted (1–2 µM) for 1 h, followed by co-incubation with cisplatin (600 µM) for 6 h. The larvae (5 dpf) were imaged in vivo according to a previously published method, and nerve mast cells were identified using green fluorescent protein. Transgenic zebrafish Tg (Brn3c: GFP) expresses green fluorescent protein in HCs, allowing changes in HCs of the neuromasts to be observed in vivo. In our study, hair cells were counted in two lateral line neuromas: L1 and L3 (Fig. 2B). Only cells that looked intact without any distortion in morphology were considered to be viable and counted. The total number of HCs in all fish was calculated to obtain an average.

Fig. 2.

Tedizolid Phosphate protected against cisplatin-induced ototoxicity in zebrafish lateral lines. A The schedule of Ted and cisplatin administration to 5dpf zebrafish larvae. Five days after fertilization, Tg (brn3c: GFP) zebrafish were pretreated with Ted or solvent for 1 h, then exposed to cisplatin with or without Ted for 6 h, and recovered in fresh water for 1 h. B Schematic diagram of neuromasts in the middle and posterior parts of 5dpf zebrafish larvae. Green spots indicate the position of hair cells in the lateral line system. Yellow spots indicate the approximate location of neuromasts (L1 and L3). No additional neuromasts were seen in this schematic diagram. C HC counting per neuromast. D Zebrafish were treated as shown in A; GFP fluorescence of hair cells was examined under a confocal microscope. The average number of HCs at L1 and L3 was calculated from at least three animals. Scale bars = 20 µm. Data are presented as the means ± SD, ***P < 0.001; n = 10 zebrafish

Cisplatin Administration in Mice

Cisplatin was diluted to a specific concentration with sterile physiological saline. As described in Fig. 3A, C57BL/6 mice (male, 8 weeks old) were pretreated with Ted by intraperitoneal injection (i.p.) every day at a dosage of 8 mg/kg as described in a previous study [45], which was comparable to the daily dose approved for humans. C57BL/6 mice were intraperitoneally injected (i.p.) with cisplatin at a dose of 4 mg/kg. Mice were given initial treatment with Ted (8 mg/kg) the day before cisplatin administration and injected for 4 days. After resting for 2 days, the above administration was repeated once more. After cisplatin administration, all groups of mice received intraperitoneal injection of 1 mL of saline once a day for consecutive 12 days to reduce nephrotoxicity and dehydration. Auditory brainstem response (ABR) was performed, and cochlear HCs were counted 2 days after the last administration. No mice died in the cisplatin and combination group.

ABR Measurements

Mice underwent ABR audiometry measurement after receiving the last cisplatin treatment as described previously [46]. Briefly, mice were anesthetized with pentobarbital sodium (100 mg/kg, i.p.) and kept warm during the experiments. The ABR waveform was recorded in the sound booth, and the hearing threshold was evaluated at 8, 12, 16, 24, and 32 kHz with TDT System III (Tucker-Davis Technologies Inc., USA). At each frequency, the stimulus intensity is reduced from 90 to 20 dB to determine the hearing threshold of this frequency. The thresholds were determined at 4, 8, 16, 24, and 32 kHz and for a broadband click. ABR score, defined as the lowest response that could demonstrate a reproducible waveform, refers to the previous research [47]. If there was any doubt about the result, the test would be repeated the next day. All thresholds of each mouse were determined independently by two experimenters to exclude human interference.

Immunofluorescence

Cochleae tissue paraffin sections and cochlear explants were used for immunofluorescence staining as previously described (30). Briefly, 0.2% Triton-X100 (Solarbio, T8200, Beijing) was added dropwise to the samples and incubated at room temperature for 30 min and blocked with 5% goat serum for 1 h. Next, the samples were incubated with a primary antibody against Myosin7a (Proteus bioscience, #25–6790, 1:400) and p-ERK1/2 (1:200; 9101S, Cell Signaling Technology) overnight at 4 °C. After three washes with PBS buffer, the specimen were incubated with secondary antibody (A11034, Invitrogen, 1:400) at 37 °C for 1 h and then labeled with phalloidin (A12380, Invitrogen, 1:1000). Secondary antibody controls were set for each experiment to exclude non-specific staining, which refer to the previous method [48]. DAPI (Beyotime-Biotechnology) was used to stain the nucleus for 10 min. All images were captured by a confocal laser scanning microscope (LSM 900, Carl Zeiss; OLYMPUS, Invitrogen) and were analyzed using the ZEN lite 2020 software package. All pictures’ average fluorescence intensity was performed using ImageJ software (1.52v, USA)[35]. And the average fluorescence intensity of the intervention group and control group was normalized. The relative fluorescence intensity of MitoSOX-Red was determined by a method the same as above. All experiments were repeated at least three times.

RNA-seq and qRT-PCR

The cochlear tissues were inoculated in 3.5 cm culture dishes. After 24 h of culture, they were pretreated with Ted (20 µM) for 1 h and then co-incubated with cisplatin (150 µM) for 24 h. The basement membrane tissue was digested using the TRIzol reagent and then sent to the Annoroad Gene Technology Corporation (Beijing) for next-generation sequencing. Briefly, RNA was identified using the BioAnalyzer 2100, and Sequencing libraries were generated with the use of NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA). The index-coded samples were clustered according to the manufacturer’s instructions. Genes with q ≤ 0.05 and |log2_ratio|≥ 1 were considered to be differentially expressed genes (DEGs). After measuring the concentration of RNA extracted from cochlear tissues by a Nanodrop 2000, cDNA was obtained by reverse transcription kit (Vazyme, Nanjing, China). According to the manufacturer’s suggestion, the ChamQ Universal SYBR qPCR Master Mix (Vazyme #Q711) was used to perform real-time PCR on Gentier96E real-time PCR system (Tianlong, Gentier 96e, Xian, China). The reaction proceeded as follows: 95 °C for 30 s; 95 °C for 3–10 s, 60 °C for 10–30 s, × 40 cycles; then, use the instrument default dissolution curve program. The primer sequences are listed in Table S1. GAPDH was used as a standardized control. More details are provided in the supplementary information.

Western Blot Assay

HEI-OC1 cells cultured in 6-well plates were lysed with the RIPA lysis buffer (PP109, Protein Biotechnology) containing PMSF and phosphorylase inhibitors (04693132001, Roche). The total protein concentration in the supernatant was quantified with the BCA protein detection kit (Beyotime, Shanghai, China). The protein was then separated by SDS-PAGE and electrotransferred to a PVDF membrane and incubated with the corresponding primary antibodies. The following antibodies were used: anti-pERK (9101S, Cell Signaling Technology), anti- ERK (4695, Cell Signaling Technology), and anti-GAPDH (C4; SC-47778, Santa Cruz). The primary antibodies were diluted at 1:1000, and the secondary antibody was diluted at 1:5000. Finally, protein bands were visualized using a chemiluminescent substrate kit (4600SF, Tanon). The quantitative analysis of blot bands was performed with ImageJ software.

Establishment of Tumor Model and Cisplatin Exposure

Male BALB/c nude mice aged 5 weeks (Vital River Laboratory Animal Technology Co., Ltd., Beijing) were housed in a cage and kept in a room with a 12-h light/dark cycle. The nude mice were tested after 1 week of adaptation. A549 cells were inoculated subcutaneously into nude mice at a density of 1 × 10⁶ cells per mouse. After 30 days, when the tumor volume reached about 100 mm³, the mice were randomly divided into three groups shown as the control group, Con; cisplatin alone group, Cis; combined treatment group, Cisplatin + Ted, n = 8 in each group; the mice were treated with vehicle or Ted (8 mg/kg, i.p., once daily for 3 days) with or without cisplatin (4 mg/kg, i.p., once every 3 days) treatment, and then the mice rest for 1 day. The above administration was then repeated for 3 times (Fig. 7A). The tumor size and body weight were measured every 2 days, and tumor volume was calculated as volume (mm³) = length × width² / 2. After the last measurement, all mice were sacrificed, and tumor tissues were harvested and photographed [49].

Fig. 7.

Ted did not affect the tumor-killing effect of cisplatin. A Administration regimen of Ted and cisplatin in BALB/c nude mice (5 weeks old). B, C Colon and lung cancer cell lines were pretreated with Ted for 1 h and then treated with cisplatin for 48 h. The cell survival rate was detected by CCK8 assay. Data shown as means ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001 compared to cisplatin alone; n = 5–6. D Representative images of xenograft tumors. Ted pre-treatment does not affect cisplatin’s chemotherapeutic ability in the mouse xenograft model. Xenograft model was established by inoculating nude mice with A549 cells. When tumors reached an average of 100 mm³, nude mice were treated with cisplatin (4 mg/kg, i.p., once every 4 days) and Ted (8 mg/kg, i.p., once every 1 day). E Tumor sizes were measured every other day from days 0 to 16. F The weight of nude mice undergoing treatment was measured every other day. Means ± SD. ***P < 0.001, **P < 0.01, and * P < 0.05 vs cisplatin alone; n = 8 mice

Statistical Analysis

Data analysis was performed using GraphPad Prism 8 software. Differences between groups were compared with Student’s t-test and one-way analysis of variance (ANOVA). Data were presented as the means ± SD, P < 0.05 indicated statistical significance. For the cochlear tissue culture experiments, “n” represents the number of independent cochlea (Fig. 4C, “n” represents the number of repeated experiments); for cell culture experiments, “n” represents the number of replicates. For animal experiments, “n” represents the number of independent animals.

Fig. 4.

The effect of Ted on the production of mitochondrial ROS and inflammatory factors in hair cells of cochlear explants after cisplatin injury. A Confocal images of P3 C57BL/6 mouse cochlear explants in the middle turn were labeled with MitoSOX-Red probe with different treatments. B The relative fluorescence intensity of MitoSOX under each condition in A. At least three different samples were analyzed. Data are presented as the means ± SD. *P < 0.05, **P < 0.01; n = 3. Scale bars = 20 µm. C qRT-PCR results showing mRNA level of inflammatory-related factors after cisplatin and Ted treatment, normalized to GAPDH mRNA expression. Means ± SD. *P < 0.05, **P < 0.01; n = 3. RNA of each sample was extracted from at least 8 cochleae (4 mice) of P3 C57BL/6 mice. D KEGG analysis showing the top 20 most affected pathways with or without Ted treatment in cochlear explants. Means ± SD. *P < 0.05, **P < 0.01. Scale bars = 20 µm

Results

Screening of the FDA-Approved Library for Protection Against Cisplatin-Induced Ototoxicity

HEI-OC1 cells are widely used as a cellular model to study ototoxicity and are well-suited for high-throughput screening for hearing-protective drugs [50]. In this study, 1967 compounds from the FDA-approved library at a primary concentration of 10 µM were screened for their ability to protect the HEI-OC1 cell line from the damage induced by 50 µM cisplatin. To compare the effectiveness of different drugs to prevent cisplatin-induced cell death, survival rate ratio was used. Specifically, this ratio was used to compare the number of viable cells after treatment with the combination of compound with cisplatin and cisplatin alone, with a value greater than 1 considered to be protective. Of the 1967 compounds screened, 819 showed potential protection against cisplatin-induced ototoxicity, with Ted showing the highest effectiveness (survival rate ratio = 1.703, P < 0.001, n = 3) (Fig. 1A). The molecular structure of Ted is shown in Fig. 1B. No toxicity was found using cell viability assay with different concentrations of Ted alone (Fig. S1). To further determine the optimal conditions for Ted to exert its cytoprotective effects, we tested the cell survival rate at different concentrations of Ted. CCK-8 results showed that pretreatment with Ted at a dose of 10–50 µM significantly reduced cisplatin-induced cell death (Fig. 1C), as indicated by the increased survival rate of hair cells from 45 to nearly 71% (P < 0.001, n = 6 vs. cisplatin alone).

Fig. 1.

Tedizolid Phosphate (Ted) pretreatment reduced cisplatin-induced cell death in HEI-OC1 cells and cochlear explants. A A library containing 1967 FDA-approved drugs. Notably, 819 drugs (above the red line) had a survival rate greater than 1, among which Ted was the most effective drug. B The molecular structure of Ted. C The CCK-8 assay was used to determine the protective effect of Ted on cisplatin-treated HEI-OC1 cell injury. Means ± SD. *P < 0.05 and ***P < 0.001; n = 3–6. At least three independent experiments were performed. D Confocal images of immunofluorescence staining of P3 C57BL/6 mouse cochlear explants HCs with different treatments (Myo7a, green). Scale bars = 20 µm. The number of HCs in apex (E), middle (F), and basal (G)-turn of (D). Data are presented as the means ± SD. *P < 0.05 and ***P < 0.001, vs. cisplatin-alone; n = 3–4 cochleae

Tedizolid Phosphate Protects Against CIHL in Mouse Cochlear Explants

Mouse cochlear explants are widely accepted as an alternative method for modeling cochlear disease in vivo and a practical material for studying ototoxicity [51, 52]. This model is advantageous as it allows evaluation of the response of hair cells in the inner ears of mammals. Cochlear explants of mammals, even in the developing state, provide more reliable results compared with other assays [53]. Ted’s protective effect was characterized using P3-P4 C57BL/6 mouse cochlear explants pretreated with Ted for 2 h followed by 150 µM cisplatin for 24 h. Figure 1D shows representative immunofluorescence images of the HCs from the apical, middle, and basal turn of the cochlea. Previous studies have reported that about 40% of HCs are lost after treatment of cochlear explants with 150 µM cisplatin for 24 h [29, 35], which was consistent with our result. Hair cell counting result showed that cisplatin alone caused a significant loss in cochlear hair cells (48.7% lost, P < 0.01, vs. control). The number of HCs in cisplatin combined with Ted (5–20 µM) treatment group was significantly higher (77.3–99.3% recovery, P < 0.01, vs. cisplatin) compared with the cisplatin alone group (Fig. 1F). Meanwhile, the same trend was observed in the apical and basal turn (Fig. 1E, G). Collectively, these results reveal that Ted has a significant protective effect on CIHL.

Protective Effect of Tedizolid Phosphate on CIHL in Zebrafish Lateral Hair Cells

Considering the promising results of Ted in vitro, we wondered whether its protective effects could be replicated in vivo. Zebrafish lateral-line HCs are structurally homologous to mammalian HCs, making it a good model for testing drug protective activity against cisplatin toxicity in vivo [32, 54]. Therefore, we assessed whether Ted could protect lateral line hair cells of zebrafish from cisplatin damage. Previous studies have reported that more than half of the lateral HCs of zebrafish are lost after treatment with 600 µM cisplatin for 6 h [13], which was consistent with our results (63% lost, P < 0.001, vs. control). As shown in Fig. 2B, we examined 2 neuromasts (L1 and L3) in zebrafish larvae for evidence of protection by Ted as previously described [44, 54]. Evaluation of the average cell number of each zebrafish lateral line showed that 2 µM of Ted had significant protection against cisplatin in larvae, with a cell number nearly equivalent to that of the control group (P < 0.001, vs. cisplatin group) (Fig. 2C, D). Thus, it indicates that Ted is an optimal protective drug against cisplatin-induced hair cell death in nonmammalian vertebrates.

Tedizolid Phosphate Protects Against CIHL in Mice In Vivo

As Ted showed excellent protection against cisplatin ototoxicity in mouse cochlear explants, HEI-OC1 cell line, and Zebrafish lateral-line HCs, we tested it in mice. The results showed that compared with the control group, the increase in the threshold of ABR in mice treated with cisplatin alone was significantly higher (12 to 32 kHz and click) (P < 0.05, vs. control). However, the ABR threshold of mice injected with the combination of Ted and cisplatin was significantly lower compared with mice treated with cisplatin alone at 12, 16, 24 kHz and click (P < 0.05, vs. cisplatin) but not different from that of mice in the control group (P > 0.05, vs. control) (Fig. 3B, D). Similar to the ABR results, more OHC loss was found in cisplatin-alone–treated animals, especially in their middle and high-frequency regions, compared with controls (P < 0.01, vs. control). However, fewer OHCs were lost in mice treated with cisplatin combined with Ted than cisplatin alone (P < 0.01, vs. cisplatin) (Fig. 3C, E). These results indicate that Ted could alleviate cisplatin-induced hearing impairment in mice by reducing the loss of outer hair cells.

Fig. 3.

Ted prevents hair cell loss and hearing loss following cisplatin treatment in mice. A Schematic diagram of the experimental design of Ted and cisplatin administration and ABR test in adult C57BL/6 mice (8 weeks old). B and D Effects of Ted (8 mg/kg, ip) and DMSO treatment on ABR threshold in mice. ABR measurements were conducted using a broadband click and 8, 12, 16, 24, and 32 kHz stimulation (n = 9–10 mice). C Confocal images of immunofluorescence staining of Myo7a (green) and phalloidin (red) in middle and basal turn. Scale bars = 20 µm. E The number of cochlear HCs per 160 µm in middle and basal turn. Data represented as the means ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001 vs. cisplatin alone; n = 4–5 cochleae

Tedizolid Phosphate Does Not Play a Protective Role Through Anti-Inflammatory and Antioxidant Mechanisms

After verifying Ted’s protective effect against cisplatin ototoxicity in vivo and in vitro, we further sought to explore its mechanism of action. Previous studies showed that abnormal oxidative stress could mediate cisplatin ototoxicity [55–57]. ROS level is an important indicator of oxidative stress in HCs, and the production of mitochondrial ROS is related to cisplatin-related ototoxicity [58]. Therefore, we first analyzed ROS production in mitochondria after cisplatin treatment with MitoSOX-Red fluorescent probe in hair cells of explants. The level of cisplatin-induced mitochondrial ROS was significantly higher (P < 0.05, vs. control) compared with the control group, and pretreatment with Ted did not reverse this trend (P > 0.05, vs. cisplatin) (Fig. 4A, B). At present, it is believed that cisplatin induces the production of pro-inflammatory cytokines in cochlea, with studies indicating that in various hearing-related cell models, cisplatin promotes the release of cytokines, including IL-6, TNF-α, and IL-1β [59, 60]. As an antibiotic, Ted might protect CIHL through an anti-inflammatory mechanism. We therefore measured mRNA expression of pro-inflammation mediators and found that pro-inflammatory cytokines IL-6 was significantly upregulated after cisplatin treatment compared with the control group (P < 0.05, vs. control, n = 3). However, compared with the control group, no significant difference in the expression level of these pro-inflammatory mediators was found after Ted pretreatment (P > 0.05, vs. cisplatin, n = 3) (Fig. 4C), indicating that Ted’s protection is not through inhibition of cisplatin-induced inflammatory process. Collectively, these results suggest that Ted did not exert its protective effects through classical antioxidant and anti-inflammatory pathways.

Tedizolid Phosphate Prevents Cisplatin-Induced MAPK Signal Activation

To comprehensively explore the effect of Ted on cisplatin-induced hair cell loss, we performed transcriptome sequencing. KEGG analysis revealed the top 20 regulated signaling pathways between the cisplatin and Ted + cisplatin treatment groups (Fig. 4D). Among them was a pathway known to play an important role in cisplatin-induced ototoxicity [18, 61] and several MAPK pathway inhibitors that have been proved to alleviate HC loss caused by cisplatin [62, 63]. We subsequently verified the expression of marker genes in this signal pathway using Western blotting analysis (Fig. 5A, B). Although cisplatin-induced phosphorylated ERK protein levels were higher compared with control cells, Ted attenuated these changes (Fig. 5A, B). We also found that the change in MAPK signaling in Ted combined with cisplatin group was lower compared with cisplatin alone, suggesting its essential role in CIHL. Therefore, we hypothesized that Ted plays protective roles through MAPK/ERK signaling. To test this hypothesis, we first used two ERK signal agonists Bortezomib (Boz) [64] and Tert-Butylhydroquinone (TBHQ) [65] to intervene in HEI-OC1 cells and to observe the viability of cells. HEI-OC1 cells were then pretreated with the ERK signaling activators for 2 h before cisplatin treatment. As shown in Fig. 5 E, F, Boz and TBHQ blocked the protective effect of Ted on cisplatin-injured HEI-OC1 cells, resulting in significantly lower cell viability compared with cells not treated with Boz and TBHQ. Subsequently, we validated the protective effect of Ted via the MAPK/ERK signal in explant conditions and completely in vivo. Cochlear explants were treated with Ted (20 µM) for 2 h followed by cisplatin for 1 h. We observed a significantly high signal of ERK phosphorylation in the cisplatin alone group, which was consistent with a previous study [29]. Notably, pretreatment with Ted attenuated ERK phosphorylation (Fig. 5C, D). In addition, cochlear explant models showed that Boz and TBHQ treatment also effectively abolished the protective effects of Ted in HCs after cisplatin challenge (Fig. 6A–D). To further verify whether ERK inactivation is the key point in mediating the protective effects of Ted in vivo, we performed fluorescence staining of paraffin sections of cochlear tissue from adult C57BL/6 mice. The results confirmed that Ted treatment downregulated ERK in cisplatin-treated cochlear HCs as indicated by reduced levels of phosphorylated fluorescence signal of ERK (Fig. 6E, F). Taken together, these results indicate that Ted treatment attenuates CIHL via preventing the activation of MAPK/ERK pathway.

Fig. 5.

The MAPK/ERK signaling pathway mediates the inhibitory effect of Ted against cisplatin-induced hair cell injury. A, B Relative expression levels of ERK and ERK phosphorylation in HEI­OC1 cells as determined by Western blot analysis. (n = 3) D Whole-mount middle turn cochlear explants of P3 C57BL/6 mouse were treated with Ted for 1 h and cisplatin for 24 h. Representative confocal images of phalloidin (red) and phosphorylated ERK (green). C Quantification of the relative fluorescence intensity of p-ERK from each group in D. Data are presented as means ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001. n = 4 cochlea. E, F Effects of ERK activators: Boz, TBHQ on Ted mediated protection on HEI-OC1 cells following cisplatin challenge. HEI-OC1 cells were pretreated with Boz, TBHQ, and Ted for 24 h and then treated with 30 µM cisplatin for 24 h. The cell survival rate was determined by CCK-8 colorimetric method. Means ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001; n = 5–6

Fig. 6.

ERK pathway activator counteracted the protective effect of Ted on cochlear explants. A and C: Boz and TBHQ weakened the protective effect of Ted on P3 C57BL/6 mouse cochlear HCs induced by cisplatin (Myo7a, green). Cochlear explants were treated with Ted (20 µM) for 2 h followed by cisplatin for 1 h. B and D: HCs counting chart from A and C. Data are presented as means ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001; n = 4–5 cochlea. E Representative cochlear slices from C57BL/6 adult mice (8 weeks old) stained by p-ERK (green) and DAPI (blue) immunofluorescence and enlarged images. After ABR test, the cochlea of mice was embedded, and the paraffin sections were used for staining. The mode of administration into mice is shown in Fig. 3A. F Quantification of the relative fluorescence intensity of p-ERK from each group in E. Means ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001; n = 3 mice

Ted Has No Influence on the Anti-Tumor Activity of Cisplatin

Although Ted demonstrated a protective effect on cisplatin ototoxicity both in vivo and in vitro, its potential for clinical use depended on not affecting the anti-tumor effect of cisplatin. To determine whether Ted affected cisplatin’s tumor-killing efficacy, we used murine colon adenocarcinoma–MC-38 and human lung cancer cell lines–A549 in in vitro experimental. Cells pretreated with different concentrations of Ted for 1 h were treated with 15 µM cisplatin for 48 h, and then cell viability was measured using the CCK8 method. The results showed that Ted (10 µM) had no effect on cisplatin-induced tumor cell death (P > 0.01, vs. cisplatin-alone) compared with cisplatin alone (Fig. 7B, C). Notably, increasing the concentration of Ted to 50 µM significantly enhanced the killing effect of cisplatin on these two tumor cells (P < 0.01, vs. cisplatin-alone). Because in vivo experiments could provide better methods for testing the effects of drugs on tumors, we further established a xenograft model by inoculating nude mice with A549 cells. When tumors reached an average of 100 mm³, nude mice were treated with cisplatin (4 mg/kg, i.p., once every 4 days) and Ted (8 mg/kg, i.p., once every 1 day) (Fig. 7A). The results showed that cisplatin significantly decreased the tumor size in nude mice compared with the control (P < 0.05, vs. control). Notably, the combined treatment (Ted + cisplatin) showed a tumor response similar to that of the cisplatin-alone treatment group (P > 0.01, vs. cisplatin-alone) (Fig. 7D, F). This demonstrates that Ted reduced Cis-induced ototoxicity without accelerating tumor development in these murine models.

Discussion

Cisplatin is a highly effective chemotherapy drug used to treat many types of cancers, including lung cancer, nasopharyngeal cancer, malignant lymphoma, and other tumors [2, 66]. Nevertheless, its use is limited by its serious side effect of ototoxicity, which is characterized by progressive, irreversible neurosensory hearing loss [9, 11]. Therefore, it has become particularly urgent to develop ideal treatment or preventive measures to halt the progression of this disease.

In the present work, 1967 compounds in an FDA-approved drug library were screened to identify a compound that could protect against cisplatin ototoxicity in HEI-OC1 hair cells. Ted, an antibiotic drug, showed the best protective effect in our cell line screening against CIHL. Our results showed that Ted significantly reduced cisplatin-induced cell loss in both HEI-OC1 and cochlear HCs. In addition, its protective effect was found to be dose-dependent both in vitro and in cochlear explants. Ted’s protection of HCs from cisplatin-induced ototoxicity in vitro prompted us to further test whether it could induce the same effect in zebrafish and mouse models. As expected, Ted protected zebrafish and mouse HCs from cisplatin-induced cell loss in vivo. These characteristics suggest that Ted may be superior to many other drug candidates currently under clinical trials for ear protection and can be quickly applied in the clinical treatment of CIHL because of its ability to cross the blood labyrinth barrier [12, 67, 68]. It is noteworthy that the areas affected by cisplatin ototoxicity were limited to hair cells treated with cisplatin. No significant changes were observed in stria vascularis and spiral ganglion neurons, which were previously reported to be damaged after treatment [69, 70]. One possible explanation for this difference is that our cisplatin-treated concentration was not enough to cause damage to these two cells. Nonetheless, our result indicated that Ted was not harmful to stria vascularis and spiral ganglion neurons, but further research is needed to confirm the exact effect of the Ted on these cells.

In this study, we tested the effect of Ted on the anti-tumor effects of cisplatin. As cisplatin has been widely used to treat lung and colon cancer, we chose two tumor cell lines, A549 and MC38, to explore Ted’s antitumor effects. As previously reported, cisplatin alone inhibited the survival of cancer cells [71, 72]. Our results demonstrated that Ted did not interfere with this effect but rather showed a synergistic tumor-killing effect with cisplatin in these two tumor cell lines. Further, using a xenograft model of nude mice, we tested the effect of Ted on the antitumor effects of cisplatin in vivo. No difference was found in tumor size between the Ted plus cisplatin group and the cisplatin alone group. Together, these results strongly suggest a protective effect of Ted against cisplatin-induced ototoxicity. However, the most optimal administration time and dosage of Ted have not yet been determined and will be the focus of our follow-up study.

Traditionally, cisplatin-induced ototoxicity has been associated with changes in antioxidant activity in hair cells [73]. Among them, mitochondrial dysfunction and free radical (e.g., ROS) accumulation are thought to be the primary causes of CIHL [46]. To test whether Ted protects CIHL by inhibiting oxidative stress, we first evaluated the mitochondrial ROS production in explants after cisplatin treatment. Mito-SOX staining result showed that compared with the control, there was a significant increase in cisplatin-induced mitochondrial ROS production but did not decrease after pretreatment with Ted. These results suggest that Ted’s protective role is not through inhibition of oxidative stress. As an effective antibiotic, Ted has been used in clinical treatment of refractory infections [38]. Cisplatin induces the production of pro-inflammatory cytokines in the cochlea, including TNF-α, IL-6, and IL-1β [59, 60]. Therefore, we examined the levels of inflammatory cytokines in mouse cochlear tissues cultured in vitro. The results showed that Ted did not affect these levels, suggesting that its function is not through a non-inflammatory pathway.

Since Ted’s protective role in hearing was not through anti-inflammation and anti-oxidation, we further conducted RNA-sequencing using cochlear explants to explore the specific mechanism. RNA-seq analysis showed that MAPK signaling was one of the 20 KEGG pathways significantly upregulated in HCs, proving its essential role for the formation and long-term survival of the HCs pathway [74]. MAPK signaling includes several different groups, with extracellular signal regulatory proteins (ERK)1 and 2(ERK1/2) being the most widely studied in hearing research [75]. As cisplatin treatment can activate MAPK pathway in HEI-OC1 cells, inhibition of this signaling activity can alleviate cisplatin-induced ototoxicity [76]. Previous studies have shown that Dapranafenil protects cisplatin-induced hearing loss by inhibiting ERK phosphorylation in cochlear cells and alleviating cisplatin-induced cochlear hair cell death [29]. Another study showed that U0126, a specific inhibitor of ERK signaling pathway, exerts a hearing protective effect by reducing cisplatin-induced oxygen free radical production and mitochondrial membrane potential [63]. In the present study, Western blot result showed that phosphorylated ERK was upregulated in HEI-OC1 cells and in the adult cochlea after cisplatin treatment. However, after Ted pretreatment, the expression of p-ERK in HEI-OC1 cells and cochlear tissues was significantly lower compared with the controls. Another evidence demonstrating that Ted exerts its protective effect via the MAPK/ERK signaling pathways is that two ERK activators, Boz and TBHQ, blocked the protective effect of Ted on cisplatin-damaged HEI-OC1 cells and cochlear explants. The decrease in cell survival after activation of ERK signaling pathway by Boz and TBHQ further supports this conclusion. It is noteworthy that the level of p-ERK was significantly increased in cochlear hair cells, and further studies are needed to clarify how ERK signals and related upstream and downstream genes are regulated in different parts of the cochlea treated with cisplatin. In addition, the mechanism of cisplatin-induced ototoxicity mainly involves passive diffusion and active uptake of certain membrane transporters [77], such as copper transporter 1(CTR1) and MET channel [78, 79]. However, whether these are involved in Ted’s resistance to cisplatin, ototoxicity is still unclear.

Conclusions

In summary, our results suggest that Ted can protect cochlear hair cells from CIHL by inhibiting ERK activation, making it a promising therapeutic candidate for the prevention of cisplatin-induced hearing loss without affecting its antitumor effect.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

Ted

Tedizolid Phosphate

FDA

Food and Drug Administration

HCs

Hair cells

OHCs

Outer hair cells

ROS

Reactive oxygen species

CIHL

Cisplatin-induced hearing loss

MET

Mechanoelectrical transducer

BBB

Blood-brain barrier

CCK8

Cell Counting Kit-8

Author Contribution

Xiaolong Fu, Daqing Sun, Lei Xu, and Wen Li designed the research. Zhiwei Yao, Yu Xiao, Wen Li, Hailong Tu, Shuhui Kong, Ruifeng Qiao, Lushun Ma, and Siwei Guo conducted experiments and analyzed data. Song Wang and Miao Chang contributed to writing the manuscript. Zhiwei Yao, Xiaolong Fu, Wen Li, and Daqing Sun wrote the manuscript and interpreted the data. Xiaoxu Zhao and Yuan Zhang revised the article.

Funding

This research was supported by grants from the National Natural Science Foundation of China (no. 82271175, 82192863, 82171162, 81900937, 82201296, 82201294, 82001204), the Natural Science Foundation from Shandong Province (no. ZR2021QH269 and ZR2022MC216), the Natural Science Foundation from Henan Province (no. 232300420259), and the Scientific and Technological Innovation Plan Project of Medical System Staff of Shandong Province (no. SDYWZGKCJHLH2023096) and Shandong Province medical health science and technology project (no. 202307010965).

Availability of Data and Material

The data sets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics Approval and Consent to Participate

All experimental procedures involving animals were approved by the Tianjin Medical University General Hospital Experimental Animal Administrative Committee and were in accordance with animal welfare principles.

Competing Interests

The authors declare no competing interests.