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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Hear Res. 2022 Jan 10;415:108430. doi: 10.1016/j.heares.2022.108430

A Combinatorial Approach to Protect Sensory Tissue Against Cisplatin-Induced Ototoxicity

Nicole K Febles 1,4, Mark A Bauer 1,4, Bo Ding 3,4, Xiaoxia Zhu 4, Nathan D Gallant 2,*, Robert D Frisina 1,3,4,*
PMCID: PMC8810742  NIHMSID: NIHMS1772502  PMID: 35051751

Abstract

Sensorineural Hearing Loss (SNHL) is a highly prevalent disorder involving permanent damage or loss to the inner ear’s mechano-sensory hair cells and nerve fibers. Major contributing causes are ototoxic drugs, loud noises, and aging. Drug-induced hearing loss (DIHL), affects over 25% of patients treated with common therapeutics such as aminoglycoside antibiotics, loop diuretics or chemotherapeutics. A commonly used chemotherapeutic agent, cisplatin, is very effective for treating malignant tumors, but results in a majority of patients experiencing irreversible hearing loss and/or tinnitus. Additionally, since there is currently no FDA-approved treatments for SNHL, attenuation of ototoxicity is a major area of investigation in oncology, otolaryngology and hearing research. Several potential otoprotective agents have been investigated at the clinical trial stage, but none have progressed to a full FDA-approval. In this study, we investigated a combinatorial approach comprised of an antioxidant, a p53 inhibitor and a neurotrophin, as a multifactorial otoprotective treatment for cisplatin exposure. In vitro, HEI-OC1 cells, an immortalized organ of Corti epithelial cell line, pre-treated with this biotherapeutic cocktail had significantly reduced cisplatin-induced cell death, DNA fragmentation, and apoptotic activation. In an ex vivo study, rat pup D2-D3 organ of Corti explants, significant protection against cisplatin-based hair cell and neuronal loss was achieved by delivery of the same combinatorial pretreatment. Interestingly, the hair cell protection was localized to the basal and middle regions of the organ of Corti. Together, these findings highlight a novel approach to attenuate cisplatin ototoxicity and potentially prevent DIHL by addressing biological mechanisms of cisplatin ototoxicity.

Keywords: Sensorineural hearing loss, D-methionine, NT3, PFT-alpha, Combinatorial agents

Introduction

Sensorineural hearing loss (SNHL) is a highly prevalent sensory impairment, resulting in structural degeneration and dysfunction of one or more cellular components of the peripheral auditory system [1–3]. SNHL affects tens of millions of Americans and is primarily caused by ototoxic drugs, environmental factors, loud noises, and aging [4, 5]. Cisplatin, a well-known chemotherapeutic agent has proven very effective in treatments of various malignant tumors throughout the body since its clinical introduction in the late 1970s, but a major side effect is irreversible bilateral SNHL [6–8]. The percentage of patients suffering from cisplatin ototoxicity (i.e., inner ear cell toxicity), is between 11% and 97% with an average incidence of 62% of adult patients, and 60% of pediatric patients being affected overall [6, 9]. Despite this high incidence rate among cancer survivors, there is currently no FDA-approved prevention or treatment, making attenuation of ototoxicity a major area of focus at the intersection of oncology, otolaryngology, and hearing research [2, 3, 6, 10].

The mechanisms of action associated with cisplatin-induced ototoxicity have been widely reported to involve the overpowering of the cochlear anti-oxidant defense systems and the activation of inner ear apoptotic pathways [11, 12]. Specifically, anti-oxidant agents such as glutathione and its regenerating enzymes become depleted due to cisplatin’s high-reactivity to thiol-containing molecules like glutathione [13]. Following this depletion, the cellular redox status shifts, resulting in the accumulation of reactive oxygen species (ROS) [13–16]. Excessive ROS further overwhelms the cochlea’s compromised anti-oxidant defense mechnisms, leading to activation of apoptotis [12, 16, 17]. Cisplatin-induced apoptotic activation causes increased rates of lipid peroxidation, oxidative modifications of proteins and nucleic acid damage in healthy proliferating cells (i.e., non-cancerous cells) [6, 7, 18, 19]. DNA damage resulting from cisplatin ototoxicity upregulates tumor suppressor protein p53 when the p21 DNA repair pathway is irreparable (p21 kinase inhibitor is also upregulated) [16, 20, 21]. Since some apoptotic pathways are dependent on p53 expression level changes, this upregulation ultimately promotes inhibition of anti-apoptotic BCL-2 proteins and consequently activates the cascades of initiator caspases: Caspase-3, -6, -7, and -9 [17, 20]. Due to the complex biological pathways involved in cisplatin-induced ototoxicity, the drug discovery and local delivery mechanism research efforts have been diverse, and none clinically acceptable yet.

A range of compounds has been investigated for their potential to prevent cisplatin ototoxicity, including (1) anti-oxidants, (2) apoptotic inhibitors, (3) anti-inflammatory drugs and (4) neurotrophic factors [12, 14, 22–25]. Among these, anti-oxidants are often utilized as otoprotective agents for their free radical scavenger abilities to prevent associated cellular death [25]. One noteworthy example, D-methionine (D-met), has demonstrated protection against cisplatin ototoxicity in several animal studies, without interference to cisplatin anti-tumor activity [22, 26]. D-met’s reversible oxidizing properties allows it to serve as an effective free radical scavenger and protector against cisplatin toxicity [22, 26]. Alternatively, since changes in p53 levels are a contributing factor to the associated inner ear cisplatin-induced apoptosis, inhibitors of this pathway have also been investigated. Significant suppression of p53 has been reported with a water-soluble lipophilic p53-inhibitor known as Pifithrin-alpha treatment, resulting in the maintenance of a normal number of inner and outer hair cells (i.e., IHCs and OHCs, respectively) in cisplatin-treated rat organotypic organ of Corti explant samples [21]. Similarly, the protection against cisplatin-associated spiral ganglion neuron (SGNs) death has also been investigated by genetically inducing cells to produce neurotrophic factors (e.g., BDNF, NT3) with some success [24, 27].

To address the complexity of the mechanisms that lead to cisplatin-induced SNHL, we investigated the novel approach using a cocktail of these therapeutic agents targeting multiple pathways to disrupt apoptotic cell loss and promote protection against damage. Specifically, the HEI-OC1 cell line, characterized by important markers of cochlear hair cells, and cochlear tissue explants, were treated with the combination of Pifithrin-alpha (PFT-α), D-met, and Neurotrophin-3 (NT3) prior to cisplatin exposure. Otoprotection against cisplatin-induced toxicity was analyzed with a cellular viability assay based on MTT metabolism, a DNA fragmentation assay, Western blotting for apoptotic pathway proteins, and fluorescent staining of F-actin and β-3 Tubulin proteins in organ of Corti explants to count preserved hair cells and assess the structural integrity of stereocilia and neuronal fibers.

Methods

Cisplatin Ototoxic Cellular Model

HEI-OC1 cells were utilized for our cellular experiments due to the ability of these cells to possess (1) cellular proliferation and differentiation controllability via the interferon-γ-promoter element of the temperature-sensitive mutant of the SV40 large T antigen gene in these cells, (2) sensitivity to ototoxic drugs, and (3) biomarkers specific to auditory sensory cells making them favorable when studying auditory ototoxicity mechanisms and prevention [28]. HEI-OC1 cells were given to us directly from Dr. Federico Kalinec (Immortomouse, California USA) and grown in permissive conditions (i.e., proliferative condition) of 33°C and 10% CO2 with high glucose DMEM (hg-DMEM; Gibco 11965092, USA), 10% FBS (Fetal Bovine Serum; Gibco 26140079, USA), and 50 U/mL of IFN-γ (IFN-γ; Millipore IF005, USA) as previously described [29].

Otoprotective Agents and Cisplatin

Otoprotective agents D-met, PFT-α, and NT3, were purchased from Sigma Aldrich in powder form, respectively, Sigma catalog numbers: M9375, P4236, and M9375, and prepared following manufacturer’s protocols, as previously published [27, 30, 31]. D-met was dissolved to a 2.5 mg/mL stock solution in filtered distilled water and administered at a working solution of 2.5 mg/mL in appropriate growth media used for both the cellular (in vitro) and organ of Corti (ex vivo) experiments. PFT-α was dissolved in 500 uL of DMSO to make a 20 mg/mL stock solution, and a 100 mM working concentration within appropriate media was used in the respective cellular experiment. NT3 was dissolved in filtered distilled water to a final stock solution of 10,000 ng/mL and used at 50 ng/mL working concentration within serum-free growth media for each respective experiment. Stock solutions were aliquoted out and placed in a −20 °C freezer for future usage. Working concentrations were in high glucose DMEM growth media for cellular experiments and in serum -free Basal Medium Eagle ((BME) Gibco 2010–046; 2g BSA, Sigma A-4919; 2 mL Serum-Free Supplement, Sigma I-1884; 20% Glucose, Teknova G2020; 0.4 mL Penicillin G, Alfa Aesar J67315; 200 mM L-Glutamine, Gibco 25030–081) for organ of Corti explant experiments as previously published [27, 30, 31]. Before incubation with cells or explants, the homogenous cocktail solution was warmed to 37 °C shielded from light before applying the solution to each cell culture well or plate for 24 hours incubation in protective conditions before cisplatin incubation.

Cisplatin, cis-diammineplatinum (II) dichloride, was purchased from Santa Cruz in powder form (i.e., Santa Cruz SC-200896) and dissolved in 0.9 % w/w of sodium chloride (Fisher Brand) with distilled water to a final stock solution of 2 mM. Solutions were sterile filtered before usage in cellular and explant experiments at varying concentrations. Stock cisplatin solution was kept at 4°C for a short-term duration and used for immediate cellular experiments. Stock cisplatin solutions were diluted to 200 μM concentration in growth media, and 10 μM concentration in serum-free BME media (i.e., organ of Corti explants) and the homogenous solution was applied to each cell culture well for 24 hr.

Cellular Assays: Therapeutic Protection Assessment

MTT Viability Assays

HEI-OC1 cells were collected after passaging for otoprotective studies, administering a PBS – magnesium - calcium wash, and treating with Trypsin-EDTA (0.25% Trypsin-EDTA 1X; Gibco 25200–056, USA) for 3 min, via centrifugation and reconstitution with fresh-growth media. Cells were re-seeded with a growth media void of IFN-γ at a final seeding density of 20,000 cells per well in a 96 well plate and incubated in permissive conditions for 24 hours. After 24 hr of cellular recovery, pre-treatment of otoprotective agents (i.e., 2.5 mg/mL of D-methionine, 50 ng/mL of NT3, and 100 μM of PFT-α) or IFN-γ-free growth media was added (See Table 1) and allowed to incubate for another 24 hr. On D3 (i.e., 72 h from initial cell seeding), respective wells received cisplatin treatment and incubated for another 24 hr. On D4 (i.e., at 96 h from initial cell seeding), ATCC MTT Cell Proliferation Assay (ATCC 30–1010K, USA) was initiated on these 96 well plates according to manufacturer’s protocol(American Type Culture Collection, 2011 #97). Viability of experimental conditioned cellular absorbances were measured at 570 nm with a BioTek microplate reader (BioTek® Synergy™ HT, USA) along with a reference wavelength of 650 nm. Upon averaging triplicate experimental readings, each conditional value was subtracted from the background and normalized to the control. Statistical analysis was then completed on these experimental readings.

Table 1.

Detailed Experimental Conditions for Cisplatin Otoprotective Cellular MTT Viability, TUNEL Western Blots and Organotypic Culture Immunohistochemistry Studies.

Experimental Conditions Agent/Media Added with Respective Concentrations and Days of Administration
Control Cells Only Cells Seeded on D1, Media change on D2 and D3
Cisplatin Only Cells Seeded on D1, Media change on D2 On D3 Cisplatin added at:

  • 200 μM final concentration for Cellular studies, incubated for 10 hr (MTT), 10 or 20 hr (TUNEL), and 10, 16, 20 hr (WB) studies.

  • 10 μM final concentration for Organotypic IHC Cultures, incubated for 24 hr.
All Otoprotective Agents + Cisplatin Cells Seeded on D1
On D2, All Otoprotective Agents Added for 24 hr at Following Concentrations:

  • NT3 at final concentration of 50 ng/mL

  • PFT-α at final concentration of 100 μM

  • D-methionine at final concentration of 2.5 mg/mL


 Incubated for 24 hr.
On D3, Cisplatin added at:

  • 200 μM final concentration for Cellular studies, incubated for 10 hr (MTT), 10 or 20 hr (TUNEL), and 10, 16, 20 hrs (Cellular WB) studies.

  • 10 μM final concentration for Organotypic IHC Cultures, incubated for 24 hr.
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TUNEL Staining: DNA Fragmentation Studies

For TUNEL staining of DNA Fragmentation studies, HEI-OC1 cells were seeded on 35-mm tissue-culture treated dishes with growth media void of IFN-γ (i.e., hgDMEM; Gibco 11965092 and 10% FBS; Gibco 26140079, USA) in permissive conditions on D1, at 122,000 cells per plate, and grown for 24 hr. On D2, media was removed and replaced with pre-treatment of experimental conditions (i.e., otoprotective agents; See Table 1 for details) or IFN-γ-free growth media was added for control and cisplatin-only conditions. Respective otoprotective conditions were incubated for a 24-hr time frame (D2). On Day 3, media was removed again and replaced with 200 μM cisplatin for a final 10 hr or 24-hr cisplatin incubation period. Control conditions had media changed on D3 as well. Finally, on D4, samples were fixed with 4% formaldehyde in Dulbecco’s Phosphate Buffered Saline (Fisher BioReagents BP31–500; dPBS Ca++, Mg++, Gibco 1040–133, USA) for 15 minutes at room temperature and permeabilized with 0.25% Triton X-100 (Fisher BioReagents BP 151–500; filtered dH20) for 20 minutes at respective 10 and 24 hr time points.

Invitrogen Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay kit (Invitrogen C10245, USA) was used on samples via prepared TdT reaction and Click-iT Reaction solutions, to visually image TUNEL activity in all experimental conditions. Following removal of reaction cocktail, a 3% BSA wash for 5 minutes was done to prepare samples for the nuclei staining with Hoechst 33342 at 1:1000 dilution. Fixed and stained samples were then analyzed using a Nikon Eclipse Ti-U fluorescent microscope (Nikon Instruments, Melville, NY). Using excitation of 350 nm for Hoechst and 488 nm for TUNEL, resulted in two individual and one merged image per condition. NIS Elements Advanced Research software was used to quantify the apoptotic behavior occurring in our combinatorial otoprotective agents + cisplatin conditions, compared to control, and cisplatin only conditions. Following image capturing, NIS Elements AR software’s automated measurement counter parameters were adjusted (i.e., the intensity ranges for each channel, separation and size of cells were adjusted), for each image to ensure proper cell counting per channel. Following these adjusted parameters, counted nuclei and positive TUNEL-green cells were determined automatically for each binary threshold. Four fields per condition were used to obtain the number of positive counts. These counted object values for TUNEL cells in each field were normalized to the counted object values for Hoechst positive cells in same field. The Average of four fields was taken then multiplied by 100 to receive a percentage of TUNEL positive stained cells. Statistical analysis was then completed on these percentages of positive TUNEL cells for each condition to determine significant differences between groups and time points.

Western Blot Cellular Studies

For protein expression studies utilizing western blotting (WB), HEI-OC1 cells were seeded on 100 mm treated tissue-culture dishes with growth medium void of IFN-γ in permissive conditions on D1, at 122,000 cells per plate and grown for 24 hr. After 24 hr, media was removed and replaced with otoprotective agents or media change in the control and cisplatin only conditions. Respective combinatorial otoprotective conditions (Refer to Table 1) were incubated for a 24-hour time frame (D2) when media was removed again and replaced with 200 μM cisplatin for a final 10-, 16-, or 20- hour cisplatin incubation period. Control conditions had media changed on D2 as well. On D3, conditioned and control cells were collected using a modified RIPA buffer (i.e., RIPA lysis and Extraction Buffer with protease inhibitor cocktail 1:100 dilution; Thermo Scientific 89901; Sigma P8340) on ice for approximately 5 min by scraping and placing in labeled Eppendorf tubes. Samples were agitated in a cold room for two and a half hr and sonicated to isolate proteins of interest into the supernatant.

Following isolation of supernatant, sample protein concentrations were determined via a Pierce™ BCA protein assay (Thermo Fisher Scientific 23225) for equally loading into the 10% Thermo NuPAGE Bis-Tris Gel (Thermo Fisher Scientific NP0315Box) within the XCell SureLock Mini Cell System (Thermo Fisher Scientific EI0002) used to WB gel electrophoresis and transfer. The gels were loaded with a ladder (BioRAD Precision Kaleidoscope 160375) and 9 conditions with equally loaded 30 μg of protein per gel lane for Caspase-3 blots and 20 μg per lane of gel for Caspase-8 and Caspase-9 blots. Upon completion of each run, samples were transferred using XCell II Blot Module to a nitrocellulose paper at 30V for 4 h at room temperature. Upon completion of the transfer, blotted nitrocellulose membranes were blocked with 5% Non-Fat-Milk (NFM)/Tris-Buffered Saline with 0.1% Tween (Non-Fat Milk: Lab Scientific M0841; TBST: Tris Base Fisher Scientific BP152, NaCl Fisher Chemical S271–3, Tween-20 Fisher Scientific BP 337–500) blocking buffer for 1 h on a shaker at room temperature. Following blocking, membranes were washed one time with TBST, then probed overnight with respective Cell Signaling (CS) primary rabbit antibody for equal loading control of β-Actin or GAPDH on a shaker at 4°C (CS Technology: β-Actin 4967, Santa Cruz GAPDH 25778; 1:1000 in 5% w/v Bovine Serum Albumin (BSA)/TBST mixture or 1:1000 5% w/v NFM/TBST respectively). After 24 hr, primary antibodies were collected, and membrane were washed three times with TBST, probed at room temperature with secondary anti-rabbit IgG HRP-linked antibody (CS Technology 7074 1:1000 in 5% NFM/TBST mixture) for 1 hr in a dark box. Following removal of the secondary antibody, blots were then washed three times with TBST and activated with Cell Signaling Technologies 20X LumiGLO (Cell Signaling Technology 7003S). Imaging of blots were obtained by using Analytik-jena UVP ChemStudio SA2 Bioimager.

After probing for equal loading control of β-Actin or GAPDH, respective proteins of Caspase-3, -8 and -9(Cas-3: CS 9662, Cas-8: CS 8592, Cas-9: CS 9504), were probed with primary rabbit antibody (1:1000 in 5% NFM/TBST or 5% BSA/TBST mixtures) for 24 hour at 4°C and respective anti-rabbit secondary antibodies (1:1000 in 5% NFM/TBST or 5% BSA/TBST mixtures) for 1 hr at RT. Membranes were then activated and imaged as mentioned above. After three replicable experiments were achieved for each protein of interest, NIH ImageJ software was utilized to perform densitometry measurements and quantification of each blot. More specifically, analysis of each experimental conditioned band was evaluated using NIH ImageJ to obtain an intensity value that was subtracted from background intensity values and compared to control 10 hr for normalization and quantification purposes. These final quantified measurements were then statistically analyzed using SigmaPlot for significant differences in protein expressions between experimental conditions.

Explant Studies: Protection following Combinatorial Agents

Organotypic Cultures

Organotypic cultures have been advantageous ex vivo models in multiple research areas, because of their success in overcoming traditional cellular model limitations. More specifically, these ex vivo models can grow, manipulate, and maintain the cell-to-cell interactions, and 3-D mechano-electric transductive atmosphere similar to their natural environment [32]. This in turn, has advanced experimental findings in cellular stress and death pathways, novel regenerative pathways, ototoxicity studies and screening of potential otoprotective agents, to name a few [15, 31–33]. Thus, for our ex vivo otoprotective studies against cisplatin ototoxicity, we investigated protective effects via organotypic organ of Corti rat explants. Postnatal Day 2–3 old Sprague Dawley rat pups were surgically decapitated and entire cochlea isolation was completed following previous literature [27, 30, 31]. After organ of Corti isolation, samples were placed on pre-gelled Collagen type-1 rat-tail matrices, with 100–200 uL of serum -free Basal Medium Eagle (see otoprotective agents and cisplatin for more details) on top of samples and allowed to equilibrize to gels for 2–3 hours in a 37°C, 5% CO2 incubator. Following equilibrating, samples were covered with 1 ml of Serum-Free Basal Medium, placed at 37°C and 5% CO2 and allowed to recover for 24 hours. On D2, serum-free medium was removed, and pre-treated with a 1.2 ml solution of otoprotective agents or fresh serum-free media for control and cisplatin only conditions and re-incubated for 24-hours. Following an additional 24 hr (D3), pre-treatment or serum-free medium were removed and replaced with serum-free medium for control or cisplatin 10 μM for protective or cisplatin-only treated conditions for a 24-hr incubation. On D4, each condition of 3 samples of organ of Corti was either prepared for visual quantification of hair cells and spiral ganglion protection from cisplatin-induced damage via immunocytochemistry (IHC) procedures and confocal imaging or protein expression evaluation by western blotting experiments.

Immunohistochemistry

For immunohistochemistry evaluation, organotypic samples were prepared for confocal image analysis by carefully washing samples with dPBS (++) prior to fixation in 4% formaldehyde in PBS (PBS; Mg+Ca+) for 30 minutes at room temperature. Samples were then carefully washed three times in PBS (Mg+Ca+) for five minutes each wash, and permeabilized with 0.01% Triton-X-100 in PBS ++ solution for 15 minutes at room temperature. Next, these samples were blocked in 5% normal horse serum (NHS) in PBS (Mg+Ca+) with an addition of 0.01% sodium azide, at room temperature for 1 hr on shaker. Samples were kept at 4° Celsius, until staining was initiated. Samples were then washed with PBS and incubated with a combination of conjugated Phalloidin-Alexa Fluor-568, β3-Tubulin-Alexa Fluor-488 antibodies and Hoechst 33342 stain (i.e., Invitogen A12380 1:200, Fisher Scientific 50–112-4599 1:50 and Component F from Invitrogen C10245 1:1000) respectively in 5% NHS at room temperature shielded from light for 1 hour. Samples were then washed with 5% NHS Blocking buffer twice, rinsed with PBS once and final rinse was made with dH20. Three organotypic samples of same conditions were mounted carefully onto labeled microscope slides, covered with Invitrogen ProLong Gold Antifade reagent (Invitrogen P10144) and gently covered with a glass cover slip. Mounted samples were kept at room temperature shielded from light, to allow for curing of the mounting reagent. Upon curing completion, samples were placed in a black box until confocal imaging was performed or placed in −80 °C Freezer for long term storage.

Laser Scanning Confocal Imaging

Confocal imaging of cochlea was taken using a Nikon A1 HD25 Multi-Photon & Laser Scanning Confocal combined with a Nikon Eclipse Ti2 inverted microscope. A 1024 × 1024-pixel image at 20X was obtained via excitation at 405, 488, 561 nm wavelength for DAPI, Phalloidin and B3-Tubulin respectively, at a scan speed of 0.5 frames/second and digital zoom of 4.789 for each experimental condition. Z-stack images were taken at 0.8 μM steps to ensure accuracy through the different planes of cochlea samples. Each stack was exported into individual images, used for cell counting of the IHCs and OHCs of the apex, middle and basal regions for each condition.

Organ of Corti Hair Cell Counting

For organ of Corti hair cell counting, each individual z-stack image of an experimental condition was separated into blue and red fields, representative of blue-labeled DNA within the nuclei of each cell, and the red-labeled F-Actin for the organ of Corti organotypic explants. Using FIJI Image-J software, NIS images were visually evaluated, and object count measurements of each individual hair cell in the Inner and Outer Hair Cell rows, within the apex, middle and basal regions for each condition were taken. A total of three to four fields per condition were averaged with a total of three samples per condition. The outer hair cell averages of each row were added together and analyzed for significant differences in inner and outer hair cell counts per mm length using SigmaPlot.

Statistical Analysis

All statistical analysis was done with a one-way analysis of variance and Holm-Sidak pairwise multiple comparison procedure using SigmaPlot software. p ≤ 0.05 was considered a significant difference for all statistical comparisons in this article (denoted by * in the Figures). All error bars reported standard deviation values for averages of three samples of each condition.

Results

In Vitro Toxicity and Protection

HEI-OC1 viability was examined for each control, cisplatin 200μM only, and combinatorial agent + cisplatin treated condition, using MTT proliferation assays. The MTT viability assay confirmed that cisplatin-only conditions at high concentration (i.e., ≥200 μM) for 24-hr administration, induced approximately 99.8% cellular death for HEI-OC1 cells, which is significantly decreased viability when compared to the control condition without cisplatin (P<0.001; Figure 1). Whereas pre-treatment with the combinatorial approach of agents for 24 hr before cisplatin-administration, showed significantly increased cellular viability when compared to cisplatin-only conditions (DF = 7, t= 4.086, P=0.011; Figure 1).

Figure 1. MTT Cell Viability Experimental Results Using HEI-OC1 Cells Shows Protection with Combinatorial Therapy (n=3).

Figure 1.

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A combinatorial pre-treatment usage of otoprotective agents: NT3, PFT-α, and D-methionine 24 h prior to cisplatin high dosage of 200 μM administration for 24 h shows a higher cellular viability compared to cisplatin alone at 200 μM for 24 h incubation (DF =7, t = 4.086, P = 0.011). Cisplatin 200 μM administration for 24 hr, results in significantly lower cellular viability compared to control (DF =7, t =10.148, P < 0.001). P-values ≤ 0.05are represented by *; Standard Deviations for each group are reported.

In Vitro DNA Fragmentation

DNA Fragmentation, a classic hallmark of apoptosis, was verified for HEI-OC1 cells using TUNEL staining. Control cells had negligible DNA fragmentation at 24 hr; represented by the low amount, approximately averaging 3.0 % of TUNEL green staining (presented in Figure 2B), relative to the high amount of intensely fluorescent nuclei, Hoechst blue staining, found in Figure 2A. Whereas, cisplatin-only treated HEI-OC1 cells showed a notable decrease in the overall amount of viable cells compared to controls after 24 hr incubation, represented by the low amount of blue Hoechst staining displayed in Figure 2C. Additionally, primarily all Hoechst blue-stained HEI-OC1 cells in cisplatin-only conditions at 24 hr found in Figure 2C, are also stained green indicative of DNA Fragmentation (i.e., an average of 71% TUNEL Positive HEI-OC1 cells; visibly represented in Figure 2D; quantified in Figure 2G). These positive TUNEL stained cisplatin-only treated cells at 24 hr, were found to be significantly different from control cells at 24 hr (DF=11, t = 6.246, P<0.001; Figure 2G). Conversely, the combinatorial pre-treatment for 24 hr before cisplatin-treatment reveals negligible DNA fragmentation, at an average of 4.4 % TUNEL positive; visible representation found in Figure 2F. Thus, combinatorial pre-treated cells act similarly to control 24 hr cells. Additionally, the combinatorial pre-treatment approach shows significant protection against cisplatin-induced DNA fragmentation at 24 hr, when compared to cisplatin-only conditions at 24 hr (DF=11 , t = 5.884, P<0.001; Shown in Figure 2G).

Figure 2. TUNEL Positive DNA Fragmentation Representation and Quantification Show Declines due to the Combinatorial Therapy (n=3).

Figure 2.

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HEI-OC1 cells were pre-treated with combinatorial agents for 24 hr prior to cisplatin (at 200 μM) which was administered for 10 or 24 hours. Cells were fixed and prepared with Invitrogen Click iT TUNEL (B, D, F) and Hoechst nuclei staining (A, C, E) for visualization of DNA Fragmentation or absence thereof. (B) Control cells at 24 hr (i.e., not treated; media changed only), show negligible amounts of DNA fragmentation. Whereas cisplatin at longer duration of time, induces DNA fragmentation at 24 hr with HEI-OC1 cells, as represented by green, TUNEL stained DNA (D). The combinatorial pre-treated cells show inhibition of Cisplatin-induced DNA fragmentation, performing similarly as control cell (F). (G) One-way ANOVA revealed significant findings in TUNEL stained cisplatin 200 μM compared to control and protective conditions (DF= 11, t = 6.246, P-value ≤ 0.001 for cisplatin vs. control andP-value ≤0.001 for cisplatin vs. combinatorial treated group). Whereas, combinatorial therapy resulted in significant decline of DNA Fragmentation following cisplatin incubation for 24 hr. P-values ≤0.05 represented by *; Standard Deviations for each group are reported.

In Vitro Apoptotic Pathways

The activation (cleavage) of Caspase-3, an executioner caspase, was investigated in our otoprotective cellular studies because of its role in cisplatin-induced auditory cellular apoptosis (Elmore 2007; Han et al. 2016). Control HEI-OC1 cells expressed the inactive whole Caspase-3 protein, like the combined otoprotective agents with the cisplatin conditioned group. However, both control and combinatorial pre-treated conditions revealed no cleaved-Caspase-3 protein presence represented by the absence of bands for cleaved Caspase-3 in Figure 3A (Quantified in Figure 3C). In comparison, all cisplatin-only treated groups revealed high levels of cleaved-Caspase-3 protein presence in WB experiments. The most and significantly different cleaved Caspase-3 protein expression occurred at 16 hr, compared to control and combinatorial agents + cisplatin treatment groups, as seen in Figure 3A (Quantified in 3C and significant findings listed in Table 2). Additionally, all cisplatin-only treated groups showed slightly lower whole Caspase-3, with significant differences seen at the 16- and 20-hr time points than control and combinatorial agents with cisplatin administration (See Figure 3A, quantified in Figure 3B and significant findings listed in Table 2). Notably, the extended exposure of these cells to cisplatin resulted in overall protein degradation, most likely related to the extensive cell death, as indicated by reduced β-Actin and whole Caspase-3 as the duration of treatment increased.

Figure 3. Caspase-3 and Cleaved Caspase-3 Indicate Combinatorial Therapy Reduces Apoptosis in HEI-OC1 by Western Blotting (n=3).

Figure 3.

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(A) HEI-OC1 cells were either: pre-treated with combinatorial agents for 24 hours and then treated with one high dosage of cisplatin (200 μM) for 10, 16 or 24 hours, treated with cisplatin only (200 μM cisplatin-treated) for 10, 16 or 24 hours, or had no treatment at all (i.e., control; media change only) for 10-, 16-, or 20-hour time points. Western blotting techniques revealed that cisplatin-only conditions induced apoptotic activity in HEI-OC1 cells at all experimental time points, demonstrated by the cleaved Caspase-3 bands (Quantified in Figure 3B and C). Our combinatorial approach protects against this cleaved Caspase-3 activity (represented by no band above in 10, 16 and 20 hr conditions; Quantified in Figure 3B). Each western blot had a loading control of β-Actin and represents one blot of three reproducible blotting experiments. BCA quantification was utilized to ensure equal protein loading per gel column. One-Way ANOVA revealed significant differences with (DF=8). P-values ≤ 0.05 represented by *. t and P values can be found in Table 2. Standard Deviations for each group are reported.

Table 2.

Significant Findings from Western Blot Caspase-3 and Cleaved Caspase-3 Protein Presence Using One-Way Analysis of Variance

Protein Presence Comparison t Value P Value P < 0.050
Whole Caspase 3 35 kDa Control 20 hr vs. Cisplatin 200 μM 20 hr 7.351 <0.001 Yes
Control 16 hr vs Cisplatin 200 μM 16 hr 6.734 <0.001 Yes
All Otoprotective Agents + Cisplatin 200 μM 16 hr vs. Cisplatin 200 μM 16 hr 4.408 0.009 Yes
All Otoprotective Agents + Cisplatin 200 μM 20 hr vs Cisplatin 200 μM 20 hr 3.971 0.023 Yes
Cleaved Caspase-3
19 kDa 		Cisplatin 200 μM 16 hr vs Control 16 hr 4.613 0.008 Yes
Cisplatin 200 μM 16 hr vs All Otoprotective Agents + Cisplatin 200 μM 16 hr 4.249 0.015 Yes
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Initiator Caspases -8 and -9, both upstream of Caspase-3, have been implicated in activating mitochondria and cellular destruction as critical mediators in the intrinsic and extrinsic apoptotic pathways. Thus, we also investigated the protective effects of our cocktail agents against both pathways. Combinatorial agents + cisplatin conditioned cells revealed no cleaved Caspase-8 activity, similar to control cells (See Figure 4A). Conversely, all varying time points of cisplatin-only treated conditions exhibited significant cleaved Caspase-8 activity when compared to control and combinatorial conditions (Refer to Figure 4A and quantification in B and C). More specifically, the p18 and p43 subunit of cleaved Caspase-8 increased significantly at 16 at 24 hr and 16 hr respectively, as shown in Figure 4A (quantified in Figure 4B and C, with significant findings listed in Table 3). For Caspase-9 protein presence, whole Caspase-9 protein presence existed in all experimental groups, with significant differences seen in cisplatin-only treated groups compared to control and combinatorial + cisplatin groups. However, the reduction seen in whole Caspase-9 protein expression showed similar protein reduction trends to that of the β-Actin control. This reduction indicates protein degradation in cisplatin-treated groups where observation of total cellular death also occurred (See Figure 5A; quantified in Figure 5B with significant findings listed in Table 4). Remarkable, cells treated with the otoprotective agents had little-to-no visible cleaved Caspase-9 protein expression seen in any experimental conditions.

Figure 4. Caspase-8 and Cleaved Caspase-8 Western Blot Representation and Quantification Supports Combinatorial Therapy Oto-Protective Effects (n=3).

Figure 4.

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HEI-OC1 cells were treated the same way as described above. Results show that the cisplatin-only conditions induce apoptotic activity in HEI-OC1 cells at 16 and 20 h experimental time points, demonstrated by the cleaved Caspase-8 bands (quantified Figure B and C). Our combinatorial approach protects against this cleaved Caspase-8 activity at all experimental time points (Represented by no band in A; quantified in Figure B and C). Each western blot had a loading control of GAPDH and represents one blot of three reproducible blotting experiments. BCA quantification was utilized to ensure equal protein loading per gel column. The western blot representation image was contrast-enhanced to report GAPDH more clearly. (B and C) One-Way ANOVA revealed significant cleaved Caspase-8 activity in cisplatin only condition at 24 hr when compared to control and combinatorial conditions. (DF=8, t and P-values can be found in Table 3 for all significant findings. P-values ≤ 0.05 represented by *; Standard Deviations for each group are reported.

Table 3.

One-Way Analysis of Variance of Western Blot Cleaved Caspase-8 Protein Presence Significant Findings.

Protein Presence Comparison t Value P Value P < 0.050
Cleaved Caspase-8 43 kDa Cisplatin 200 μM 16 hr vs. Control 16 hr 10.9 <0.001 Yes
Cisplatin 200 μM 16 hr vs All Otoprotective Agents + Cisplatin 200 μM 16 hr 10.548 <0.001 Yes
Cisplatin 200 μM 20 hr vs. Control 20 hr 4.277 0.011 Yes
Cisplatin 200 μM 20 hr vs. All Otoprotective Agents + Cisplatin 200 μM 20 hr 4.087 0.016 Yes
Cleaved Caspase-8 18 kDa Cisplatin 200 μM 16 hr vs Control 16 hr 4.536 0.009 Yes
Cisplatin 200 μM 16 hr vs All Otoprotective Agents + Cisplatin 200 μM 16 hr 4.459 0.010 Yes
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Figure 5. Caspase-9 and Cleaved Caspase-9 Western Blotting Representation and Quantification Indicates No Intrinsic Activation (n=3).

Figure 5.

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HEI-OC1 cells were treated the same way as mentioned above. (A) As a result, WB reveals cisplatin only conditions did not induce cleaved Caspase-9 activity in HEI-OC1 cells at any experimental time points, demonstrated by the absence of cleaved Caspase-9 bands. Our combinatorial approach inhibited cleaved Caspase-9 activity at all experimental time points (Represented by no band). Each western blot had a loading control of β-Actin and represents one blot of three reproducible blotting experiments. BCA Quantification was utilized to ensure equal protein loading per gel column. WB representation image was contrast enhanced to report β-Actin more clearly, but quantification of three experiments involving intrinsic apoptotic pathway and statistical analysis was done on un-enhanced blots. (B) Combinatorial treated HEI-OC1 cells revealed no cleaved Caspase-8 activity, similar to control cells. Cisplatin treated conditions had a significant decrease in whole Caspase-9 protein presence, suggestive of cleavage occurring, but no significant cleavage was found in this condition. t and P-values can be found in Table 4 for all significant findings. P-values ≤ 0.05 are represented by *; Standard Deviations for each group are reported.

Table 4.

One-Way Analysis of Variance of Western Blot Whole Caspase-9 Protein Presence Significant Findings.

Comparison t Value P Value P < 0.050
Whole Caspase-9 49 kDa Cisplatin 200 μM 20 hr vs. Control 20 hr 5.673 <0.001 Yes
Cisplatin 200 μM 16 hr vs. Control 16 hr 4.761 <0.005 Yes
Cisplatin 200 μM 20 hr vs. All Otoprotective Agents + Cisplatin 200 μM 20 hr 3.792 0.039 Yes
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Ex Vivo Inner and Outer Hair Cell Toxicity and Protection

Confocal imaging allowed for visual examination and hair cell counting quantification of phalloidin-stained within the apex, middle and basal regions of the organotypic organ of Corti rat explants. Our study revealed cisplatin-induced cellular damage in cisplatin 10 μM only 24 hours, while otoprotective effects were visually seen in combinatorial + cisplatin 10 μM 24 hr conditions. More specifically, 24 hr incubation of cisplatin at 10 μM resulted in visual hair cell structural disorganization, reduced nucleus size, and hair cell loss in specific regions of interest. At the same time, protective effects resulted in the organ of Corti samples pre-treated with combinatorial agents + cisplatin 10 μM for 24 hr as seen in Figure 6E and Figure 8E for images of middle and basal regions of the organ of Corti results. Intact hair cell counts per mm length resulted in significantly decreased amounts within cisplatin 10 μM 24 hr conditions than control and combinatorial agents + cisplatin 10 μM 24 hr conditions as visually seen in Figure 6C for middle and Figure 8C for basal regions (quantified in Figure 7). Further analysis revealed significant decreases in outer hair cell counts per mm length in the middle and basal regions of cisplatin μM 24 hr conditions than control and combinatorial agents + cisplatin 10 μM 24 hr conditions (See Figure 9). There was some loss of IHCs amongst the cisplatin 10 μM 24 hr conditions, and a trend of our combinatorial agents for protection (see Figure 9), no statistically significant differences were seen in inner hair cell counts per mm length in the apex, middle or basal regions of the cochlea, for all experimental conditions. The significant IHC survival of the cisplatin treated groups, was an expected trend, as previous literature has reported that cisplatin induces cellular death primarily in OHCs of the organ of Corti [34]. Additionally, literature has suggested that the abundance of G-protein coupled receptors found in IHCs (amongst other protected areas of the organ of Corti) coupled with activation affords these cells protection from cisplatin toxicity [35]. β3-Tubulin staining allows for the assessment of neuronal fiber loss or protected dependent on the conditions applied. Visual significant losses of β3-Tubulin staining resulted in cisplatin 10 μM at 24 hr conditions, indicating the loss of neuronal innervation to IHCs and OHCs. Whereas, combinatorial pre-treated conditions showed minimal to no loss of β3-Tubulin staining (See Figure 6F and Figure 8F) similar to control. The lack of neuronal fiber loss in our combinatorial approach indicates protection against SGN loss.

Figure 6. 20X Phalloidin and β3-Tubulin Confocal Images of the Middle Turn of D2-D3 Rat Organ of Corti Organotypic Cultures Following Experimental Conditions, Representing Loss or Protection of IHCs, OHCs, and β3-Tubulin Neuronal Fibers (n=3).

Figure 6.

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Control organ of Corti explants stained with F-Actin Phalloidin staining (A) has little to no variation in structure and β3-Tubulin staining (B) showing innervation of IHC and OHCs representative of a normal control condition. (C) Cisplatin 10 μM for 24 hrs shows significant loss of structural organization, IHC and OHC counts, and significant loss of innervation to IHC and OHC of organ of Corti sample (D). Pre-treated organ of Corti samples with the three-agent approach for 24 hrs prior to cisplatin 10 μM for 24 hrs indicates protection against cellular disorganization and loss of IHCs and OHCs as seen in the Phalloidin stained image (E). Additionally, innervation of the three rows of OHC and one row of IHC is visually intact (F).

Figure 8. 20X Phalloidin and β3-Tubulin Confocal Images of the Basal Turn of D2-D3 Rat Organ of Corti Organotypic Cultures Following Experimental Conditions, Representing Loss or Protection of IHCs, OHCs and β3-Tubulin Neuronal Fibers.

Figure 8.

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Control organ of Corti explants stained with F-Actin Phalloidin staining (A) has little to no variation in structure and β3-Tubulin staining (B) showing innervation of IHC and OHCs representative of a normal control condition. cisplatin 10 μM for 24 hrs shows significant loss of IHC and OHC counts, (C) and significant loss of innervation to OHCs of organ of Corti sample (D). Pre-treated organ of Corti samples with the three-agent combinatorial approach for 24 hrs prior to cisplatin 10 μM for 24 hrs indicates protection against cellular disorganization and loss of IHCs and OHCs as seen in the Phalloidin stained image (E). Additionally, innervation of the three rows of OHC and one row of IHC is visually intact (F).

Figure 7. Hair Cell Counts per mm Length in Rat D2-D3 Samples Are Significantly Higher in Combinatorial Pre-treated Groups Prior to Cisplatin 10 μM Incubation of 24 hrs (n=3).

Figure 7.

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All Confocal Images were analyzed and IHC and OHCs were combined for each control, cisplatin 10 μM and combinatorial pretreated + cisplatin 10 μM 24 hr conditions. Significant differences in hair cell counts of cisplatin 10 μM 24 hr conditions were revealed when compared to control (DF = 2, t = 5.737, p = 0.004). Additionally, significant differences in cells per mm length of combinatorial agents + cisplatin 10 μM condition was found when compared to cisplatin 10 μM 24 hr condition (DF = 2, t = 4.622, p = 0.007). P-values ≤ 0.05 represented by *; Standard Deviations for each group are reported.

Figure 9. Combinatorial Pretreatment Revealed Significant Protection from OHC Loss in Middle and Basal Turns of Organotypic Rat D2-D3 Organ of Corti Hair Cell Confocal Imaging Counts per mm Length (n=3).

Figure 9.

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Organotypic Otoprotective Hair Cell Counts per mm Length of Apex, Middle and Basal Regions in Rat D2-D3 Samples of control, cisplatin 10μM and combinatorial agents + cisplatin 10 μM at 24 hr conditions are represented. Comparing regional hair cell counts per mm length, statistically quantifiable loss is seen in cisplatin 10 μM conditions in middle and basal regions when compared to control 24 hrs (i.e., middle: t =5.53, p ≤ 0.001; basal: t =5.585, p ≤0.001 respectively). Whereas significant protection of OHCs in middle and basal regions of combinatorial agents + cisplatin 10 μM 24 hr conditions was found when compared to cisplatin 10 μM conditions (i.e., middle: t = 4.543, p = 0.005; basal: t = 4.583, p =0.004). No significant findings of OHCs or IHCs loss was found in apical region, despite visual confirmation of some loss. P-values ≤ 0.05 represented by *; Standard Deviations for each group are reported.

Discussion

Attenuation of cisplatin ototoxicity is essential for future improvements in a cancer survivor’s speech, communication abilities, hearing, and patient’s quality of life. Although several studies attempted utilization of otoprotective agents to diminish cisplatin ototoxicity, to date, the FDA still has no approved prevention or treatment option for cisplatin-induced SNHL. The motivation of this study was to examine the protective effects of a combination of agents, more specifically, an anti-oxidant, a p53 inhibitor, and a neurotrophic factor together, against cisplatin-induced cellular death. To the best of our knowledge, the present study is the first to address cisplatin ototoxicity in a combinatorial fashion with three otoprotective agents in an in-vitro cellular model for cochlear hair cells and ex vivo animal cochlear explant model. This current study has confirmed significant protection from cisplatin-induced cellular death, DNA fragmentation, and apoptosis in the HEI-OC1 cellular model while confirming significant cellular protection from cisplatin-induced death in an ex vivo explant model.

A 200 μM dosage of cisplatin in this study induced significant reproducible in vitro cellular DNA fragmentation and death via apoptosis. More specifically, HEI-OC1 cells treated with 200 μM of cisplatin for 24 hours resulted in a significantly decreased amount of cell viability than HEI-OC1 untreated cells (i.e., control) at 24 hr, in agreement with previous reports [36]. Additionally, HEI-OC1 cells treated with cisplatin at 200 μM for 24 hr resulted in significant DNA fragmentation compared to control, confirming previous findings of cisplatin-induced fragmentation in the HEI-OC1 cellular model [36]. Devarajan’s study indicated the upregulation of p53 activity, leading to activation of Caspase-8 in HEI-OC1 cells treated with cisplatin. Our western blot findings suggest a similar activation of the extrinsic pathway with cleaved Caspase-8 activity in our cisplatin 200 μM 24 hr conditions. Thus, our study supports previous findings that cisplatin induces cellular DNA fragmentation and death via apoptosis in the HEI-OC1 cellular model. Moreover, a high dose of cisplatin was used in these in vitro studies to maximally challenge the protective effects of the combination of D-met, PFT-α, and NT3.

Key mediators of apoptosis were further evaluated to determine the pathways regulated by our combinatorial approach in cochlear cells. In our study, Caspase-3 activation, an ultimate executioner of apoptosis [37, 38], was prevented when cells were pre-treated with our cocktail of agents before cisplatin administration. Additionally, Caspase-8, a primary mediator of apoptotic cellular destruction in the absence of mitochondrial participation (i.e., extrinsic apoptotic pathway), was also blocked via pre-treatment with our cocktail of therapeutic agents given for 24 hr [38]. Conversely, the molecular mechanisms of cisplatin-induced HEI-OC1 cell death in our studies revealed activation of Caspase-3 and -8 (i.e., presence of cleaved Caspase-3 and -8). The activation of Caspase-3 and -8 seen in our study supports previously associated activation of Caspase-3 and -8 activity in the presence of cisplatin at low and high concentrations, for previous in vitro studies [36, 38]. Overall, the inhibition of cleaved Caspase-3 and -8 activity in our study demonstrates the combined protective ability of NT3, PFT-α, and D-met against the activation of the extrinsic apoptotic pathway; protection from cellular shrinkage, DNA fragmentation, and death.

Previous literature has reported that cisplatin can induce the intrinsic and extrinsic pathways, but our findings show no evidence for an intrinsic activation [21]. Western blots displayed the presence of the intrinsic apoptotic initiator Caspase-9 (i.e., whole Caspase-9; 49 kDa in Fig 5) in all conditions, and whole Caspase-9 protein expression decreased with cisplatin administration over longer incubation times. Additionally, no active caspase-9 was observed, as cleaved Caspase-9 protein was not present in any of the conditions. Although the concept of interconnection between intrinsic and extrinsic apoptotic pathways has been reported due to the extrinsic initiator Caspase-8 amplifying the death signal within the intrinsic pathway [39, 40], there have been no reports of the possibility of an extrinsic activation blocking the intrinsic pathway. Thus, our findings leave open the possibility that the intrinsic pathway is not always activated in the cochlea by cisplatin exposure, which opposes previous reports of cisplatin inducing both the extrinsic and intrinsic apoptotic pathways [12, 21].

Our ex vivo findings support the well-known phenomenon of the cisplatin-induced organ of Corti hair cell damage and loss [6, 12, 30]. More specifically, our organ of Corti cisplatin explant condition revealed significant OHC damage and loss relative to IHC damage. Additionally, our findings indicate considerable damage in cochlear basal and middle regions of cisplatin-treated groups (with an intermediate dose, 10 μM), supporting previous findings that cisplatin induces damage starting in the cochlear base and then moving towards apical regions with higher concentrations and longer durations of cisplatin incubation [34, 41]. Of note, pre-treatment with our cocktail of biotherapeutic agents provided significant protection from cisplatin-induced cellular damage in the same middle and basal regions. Furthermore, most stereocilia bundles remain visibly intact in the otoprotective group similar to the control, which is in contrast to the complete loss in the cisplatin-only 24 hr samples.

β3 Tubulin staining results showed a loss of neuronal innervation in cisplatin-only treated groups and protection in combinatorial + cisplatin-treated explants, respectively. Reduced β3 Tubulin positive neurons was consistent with hair cell loss in cisplatin-only explants, indicating that cisplatin at 10 μM for 24 hours induces loss of hair cells and neuronal innervation. This trend of neuronal fiber loss has previously been shown to result from cisplatin ototoxicity [22]. Conversely, our combinatorial approach resulted in the protection of neuronal fibers following cisplatin treatment, seen in all regions of the organ of Corti-treated samples. These β3 Tubulin results could be due to previous reports of NT3 transduction causing SGN fiber innervation density increases for inner hair cells [22].

Overall, these ex vivo findings suggest that a unique combination of otoprotective agents can have a substantial protective impact against cisplatin-induced hair cell and neuronal damage and death. The focus of this study was to investigate the potential effectiveness of NT3, PFT-α, and D-met in combination, an approach never evaluated before. Similar antioxidant, neurotrophin, and anti-apoptotic drugs have been investigated individually before with mixed results. For example, studies performed by Zhang and Campbell provide a qualitative suggestion that individual agents have a lesser ability of hair cell protection when compared to the organotypic hair cell protection seen in our ex vivo study [21, 22]. Thus, we did not investigate them individually, in pursuit of testing the hypothesis that the combination would be more potent. However, the lack of singular agent comparisons is a limitation of this study because synergistic interactions or relative benefits cannot be detected. Based on the exciting new combinatorial results of the present study, further preclinical and in vivo investigations of this combination protective treatment is warranted and should include single agent controls to elucidate synergistic mechanisms and advantages. These additional data could lead to the findings necessary for the first successful clinical trial for cisplatin otoprotectants.

Summary and Conclusions

For the first time, the current study confirms a unique 3-agent combinatorial otoprotective approach of D-met, PFT-α, and NT3 to prevent cisplatin-induced hair cell damage, death via apoptosis, and spiral ganglion neuronal fiber loss. The in vitro findings in this study found significant protection of cellular viability and prevention of DNA fragmentation in pre-treated HEI-OC1 cells with the 3-agents approach, for cisplatin toxicity prevention, suggesting that an immediate local administration may promise clinical prevention. Additionally, the apoptotic findings in this study suggest that cisplatin may induce the extrinsic apoptotic pathway independent of the intrinsic activation for cochlear cells. The protection from extrinsic apoptotic activation with the combinatorial approach in this study suggests cisplatin-induced hearing loss prevention should incorporate a combined approach rather than traditional unitary methodologies in the future. For the first time, the ex vivo findings in this study show a unique 3-agent approach to protecting hair cell death and the protection of spiral ganglion neuron loss. Together the 3-agents have provided insight into addressing the overwhelmed cochlear antioxidant system, loss of neurotrophic support, and cellular activated death associated with cisplatin usage for chemotherapy. Thus, our findings suggest that a combinatorial approach may play a vital role in the future prevention of clinical SNHL and deafness.

Highlights.

  • A combinatorial approach to prevent cisplatin ototoxicity was investigated.

  • It consisted of an antioxidant, neurotrophin and anti-apoptotic.

  • This therapy reduced cell death, DNA fragmentation, and apoptotic activation.

  • Also, significant protection against hair cell and neuronal loss was achieved.

  • These findings highlight a novel approach to attenuate cisplatin ototoxicity.

Acknowledgements

We thank Dr. Shannon Salvog for project support. This work was partially supported by NIH Grant P01 AG009524.

Disclosure Statement:

There are no actual or perceived conflicts for the authors of this manuscript in regard to funding source agencies, for the research reported in this manuscript. We have filed a patent related to the results reported here, which has not yet been approved. The first, corresponding and senior authors have had full access to all of the data. I take responsibility for the integrity of the data and the accuracy of the data analysis.

Footnotes

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