Nimodipine Treatment Protects Auditory Hair Cells from Cisplatin-Induced Cell Death Accompanied by Upregulation of LMO4

Ototoxicity is one of the main dose-limiting side effects of cisplatin chemotherapy and impairs the quality of life of tumor patients dramatically. Since there is currently no established standard therapy targeting hearing loss in cisplatin treatment, the aim of this study was to investigate the effect of nimodipine and its role in cell survival in cisplatin-associated hearing cell damage. To determine the cytotoxic effect, the cell death rate was measured using undifferentiated and differentiated UB/OC−1 and UB/OC−2 cells, after nimodipine pre-treatment and stress induction by cisplatin. Furthermore, immunoblot analysis and intracellular calcium measurement were performed to investigate anti-apoptotic signaling, which was associated with a reduced cytotoxic effect after nimodipine pre-treatment. Cisplatin’s cytotoxic effect was significantly attenuated by nimodipine up to 61%. In addition, nimodipine pre-treatment counteracted the reduction in LIM Domain Only 4 (LMO4) by cisplatin, which was associated with increased activation of Ak strain transforming/protein kinase B (Akt), cAMP response element-binding protein (CREB), and signal transducers and activators of transcription 3 (Stat3). Thus, nimodipine presents a potentially well-tolerated substance against the ototoxicity of cisplatin, which could result in a significant improvement in patients’ quality of life.


Introduction
Nimodipine belongs to the group of 1,4-dihydropyridines and exerts its effects by binding to the alpha1 subunit of L-type calcium channels, thereby decreasing calcium influx via negative allosteric inhibition [1,2]. Although nimodipine is one of the first calcium channel antagonists to be developed, it has been the focus of medical and scientific attention again [1,[3][4][5][6]. Due to its good cerebrospinal fluid penetrability [2], which distinguishes it from other calcium antagonists of its substance class, nimodipine acts in the central nervous system. Because of this, nimodipine is routinely used in the clinic for the prophylaxis of cerebral vasospasm after subarachnoid hemorrhage by relaxing the smooth muscle of cerebral blood vessels, causing vasodilatation [1,3]. Nimodipine has also been administered in the area of ischemic stroke, traumatic brain injury, and migraine to investigate whether treatment leads to a positive outcome, but with heterogeneous results [1]. However, nimodipine has already shown a neuroprotective tendency and a beneficial effect on hearing preservation after vestibular schwannoma surgery [4,5,7,8].
Mentionable previous studies of our group showed a protective effect of nimodipine [9][10][11] pre-treatment on Schwann cells and neuronal cells, which was associated with increased phosphorylation of Akt and CREB and decreased activation of effector caspases [6]. Activation of Akt [12] and CREB [13] signaling is known to be involved in neuroprotection, leading to inhibition of caspase 3 activation and consequent prevention of apoptosis [14][15][16]. Specific hair cell markers were detected to verify differentiation by performing qPCR. An increase in the brain-specific homeobox/POU domain protein 3.1 (Brn3.1), Myosin 6 (Myo6), Myosin 7a (Myo7a), and also 9 acetylcholine receptor ( 9AChR) gene expression was shown in both types of hair cells compared to its level in the undifferentiated state (set to 1.0, Table 1). Table 1. Upregulation of hair cell markers through cell differentiation.

Nimodipine Decreases Cisplatin-Induced Cytotoxicity in Differentiated Hair Cells
After differentiation, a similar reduction in cytotoxicity was determined for both cell lines dependent on nimodipine pre-treatment (Figure 3a,b). No cytotoxic effect was measured in cells treated with 0.9% NaCl, whereas the untreated cells and the solvent EtOH treated cells showed nearly no LDH activity in the cell culture supernatant (Figure 3a). However, UB/OC−1 cells treated with 20 µM cisplatin and nimodipine showed a decrease in cell death from 36.3% ± 4.3% to 20.4% ± 5.6% (10 µM nimodipine, p < 0.05) and 14.1% ± 5.6% (20 µM nimodipine, p < 0.05). For cells treated with 50 µM cisplatin and nimodipine a non-significant reduction in cell death from 46.3% ± 13.2% to 32.7% ± 12.7% (10 µM nimodipine, n.s.) and 23.5% ± 7.1% (20 µM nimodipine, n.s.) (Figure 3a) was determined. A reduction at 100 µM cisplatin from 74.3% ± 9.3% to 48.4% ± 13.9% (10 µM nimodipine, n.s.) and 37.7% ± 4.9% (20 µM nimodipine, p < 0.05) for UB/OC−1 (Figure 3a) after nimodipine pre-treatment was measured. The differentiated nimodipine pre-treated UB/OC−2 cells showed lower cytotoxicity under stress induced by 20 µM, 50 µM, and 100 µM cisplatin (Figure 3b) than the untreated cells. The 5 × 10 4 cells were seeded and treated with nimodipine, and cisplatin as described in the methods and materials section. To investigate the cell death rate, the LDH activity in the culture supernatant was measured 24 h after stress induction. A reduction in cell death induced by cisplatin treatment by pre-treatment with 10 µM and 20 µM nimodipine was visible in both UB/OC−1 (a) and UB/OC−2 (b) cells. Between cells treated with EtOH (0.1%, solvent) and untreated cells, there was no reduction in cytotoxicity. p values < 0.05 (* p < 0.05) compared to cells treated with EtOH death rates were accepted as significant. The mean values and standard deviations of three independent biological replicates are shown.

Nimodipine Counteracts the Downregulation of LMO4 by Cisplatin in Undifferentiated and Differentiated State of Hair Cells
Immunoblot analysis showed a strong reduction in LMO4 after cisplatin treatment ( Figure 4). Under 20 µM cisplatin, an increase in the amount of LMO4 with 10 µM nimodipine and 20 µM nimodipine (Figure 4a

Activation of Anti-Apoptotic Pathways by Nimodipine under Chemotherapy with Cisplatin in Undifferentiated and Differentiated State of Hair Cells
Undifferentiated UB/OC−1 and UB/OC−2 cells treated with 10 µM and 20 µM nimodipine showed increased phosphorylation of Akt at serine residue 473 and phosphorylation of CREB at serine residue 133, without and during stress conditions (Figure 5a,b) [6]. Increased activation of Stat3 through phosphorylation at tyrosine residue 705 by nimodipine pre-treatment is only evident after induction of stress with 20 µM cisplatin, while there is no altered phosphorylation detected without stress (Figure 5a,b). The total Fifty µg protein per sample was loaded and separated via sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto 0.2 µm nitrocellulose membranes as described in materials and methods section. Detection was performed by using specific antibodies. While the amount of the protein LMO4 was strongly reduced by cisplatin, there was an increase by pre-treatment with 10 µM and 20 µM nimodipine during 20 µM cisplatin.

Activation of Anti-Apoptotic Pathways by Nimodipine under Chemotherapy with Cisplatin in Undifferentiated and Differentiated State of Hair Cells
Undifferentiated UB/OC−1 and UB/OC−2 cells treated with 10 µM and 20 µM nimodipine showed increased phosphorylation of Akt at serine residue 473 and phosphory-lation of CREB at serine residue 133, without and during stress conditions (Figure 5a,b) [6]. Increased activation of Stat3 through phosphorylation at tyrosine residue 705 by nimodipine pre-treatment is only evident after induction of stress with 20 µM cisplatin, while there is no altered phosphorylation detected without stress (Figure 5a,b). The total amount of Akt and CREB were not affected by either nimodipine or cisplatin treatment, whereas Stat3 protein level was reduced through stress induction with 20 µM cisplatin and increased through nimodipine pre-treatment without and with cell stress.

Figure 5. Detection of anti-apoptotic pathways activated by nimodipine during cisplatin treatment for undifferentiated UB/OC−1 (a) and UB/OC−2 (b) cells as well as for differentiated UB/OC−1 (c) and UB/OC−2 (d) cells.
After transfer of the proteins separated by SDS-PAGE (30 µg) onto 0.45 µm nitrocellulose membranes, the phosphorylation and total protein amount of anti-apoptotic cell signaling components were determined by specific antibodies. As a loading control, GAPDH protein level was used. The Western blot shown is representative of the results from three independent biological replicates.
In the differentiated state, the immunoblots detected an increase in phosphorylation of Akt at serine residue 473 and CREB at serine residue 133 under 20 µM cisplatin, which increased after 10 µM and 20 µM nimodipine pre-treatment for both UB/OC−1 ( Figure 5c) and UB/OC−2 ( Figure 5d). The total amount of Akt and CREB were not affected by either nimodipine or cisplatin treatment. Stress induction by 20 µM cisplatin detected again a reduction in Stat3 activation, with 10 µM and 20 µM nimodipine pre-treatment leading to an increase in phosphorylation. The total amount of Stat3 was decreased by stress induction and showed an increase by nimodipine treatment without and during stress conditions in both UB/OC−1 and UB/OC−2 (Figure 5c,d).

Figure 5. Detection of anti-apoptotic pathways activated by nimodipine during cisplatin treatment for undifferentiated UB/OC−1 (a) and UB/OC−2 (b) cells as well as for differentiated UB/OC−1 (c) and UB/OC−2 (d) cells.
After transfer of the proteins separated by SDS-PAGE (30 µg) onto 0.45 µm nitrocellulose membranes, the phosphorylation and total protein amount of anti-apoptotic cell signaling components were determined by specific antibodies. As a loading control, GAPDH protein level was used. The Western blot shown is representative of the results from three independent biological replicates.
In the differentiated state, the immunoblots detected an increase in phosphorylation of Akt at serine residue 473 and CREB at serine residue 133 under 20 µM cisplatin, which increased after 10 µM and 20 µM nimodipine pre-treatment for both UB/OC−1 (Figure 5c) and UB/OC−2 ( Figure 5d). The total amount of Akt and CREB were not affected by either nimodipine or cisplatin treatment. Stress induction by 20 µM cisplatin detected again a reduction in Stat3 activation, with 10 µM and 20 µM nimodipine pre-treatment leading to an increase in phosphorylation. The total amount of Stat3 was decreased by stress induction and showed an increase by nimodipine treatment without and during stress conditions in both UB/OC−1 and UB/OC−2 (Figure 5c,d).

Discussion
Cisplatin is widely used in antitumor therapy of solid tumors such as testicular, ovarian, bladder, non-small cell lung carcinoma, and head and neck tumors [17][18][19]. The main side effects of cisplatin treatment include neuropathy and hearing loss [17,20]. As a calcium channel antagonist with lipophilic properties [2], nimodipine has the possibility of acting centrally by crossing the blood-brain barrier and has already shown evidence of a neuroprotective effect in clinical trials [6,9,10,35] including a positive benefit on the preservation of hearing after surgery [6,8]. In parallel, in vitro studies on neuronal and Schwann cells showed a lower cytotoxic effect after nimodipine pre-treatment under different stress conditions [6,36]. The use of nimodipine has been established over the last 40 years, and its good tolerability, already proven in vivo, suggests that in addition to the standard

Discussion
Cisplatin is widely used in antitumor therapy of solid tumors such as testicular, ovarian, bladder, non-small cell lung carcinoma, and head and neck tumors [17][18][19]. The main side effects of cisplatin treatment include neuropathy and hearing loss [17,20]. As a calcium channel antagonist with lipophilic properties [2], nimodipine has the possibility of acting centrally by crossing the blood-brain barrier and has already shown evidence of a neuroprotective effect in clinical trials [6,9,10,35] including a positive benefit on the preservation of hearing after surgery [6,8]. In parallel, in vitro studies on neuronal and Schwann cells showed a lower cytotoxic effect after nimodipine pre-treatment under different stress conditions [6,36]. The use of nimodipine has been established over the last 40 years, and its good tolerability, already proven in vivo, suggests that in addition to the standard treatment of vasospasm following subarachnoid hemorrhage, the use of nimodipine in clinical practice can be further extended [1][2][3]. In this study, the effect of nimodipine on cisplatin-induced apoptosis of inner ear hair cells was investigated for the first time. Increasingly with the concentration of nimodipine, we also confirmed a significant reduction in cytotoxicity in both cochlear pre-cursor cell lines UB/OC−1 and UB/OC−2 during cisplatin stress. While previous studies observed a dose-independent effect [6,36,37], this study showed an increase in the protective effect with increasing nimodipine concentration. The formation of reactive oxygen and nitrogen species leads to the downregulation of the transcription regulator LMO4, during chemotherapy with cisplatin ( Figure 7a) [18,23,26,32]. This is associated with a negative influence on cell survival of cisplatin treatment [26]. As one of the main side effects of chemotherapy with cisplatin, ototoxicity is also related to the downregulation of LMO4 [18,26,32]. This protein is found in three regions of the cochlea: the spiral ganglion, the organ of Corti, and the stria vascularis [32]. It also plays an important role in the development of the inner ear and knock-out leads to a malformation of the organ of Corti in mice [30]. Since a physiological protein level of LMO4 causes intact cell function in terms of anti-apoptotic effect [18], our study focused on the influence of nimodipine under cisplatin on LMO4. Pre-treatment of immortalized hair cells with 10 µM and 20 µM nimodipine counteracts the downregulation of LMO4 and thus leads to an increase under cisplatin stress (Figures 4 and 7b). This positive regulation of LMO4 through nimodipine treatment has been shown also for the first time and could represent a key factor in nimodipine's otoprotective mechanism of action. Further studies should shed light on whether there is a protective effect of nimodipine even after the knock-out of LMO4 and whether this also counteracts the negative effects of cisplatin to the same extent in vivo.

Figure 7. Nimodipine pre-treatment could protect hair cells from cisplatin-induced apoptosis via upregulation of LMO4 and anti-apoptotic pathways.
Cisplatin leads to the downregulation of the transcription factor LMO4 via the production of intracellular reactive oxygen and nitrogen species. This causes an increase in apoptosis of auditory hair cells and hearing loss, one of the most common side effects of this chemotherapy, via downregulation of the anti-apoptotic cell signaling pathways (a). Nimodipine counteracts this process and reduces auditory hair cell death via the upregulation of LMO4 and the associated activation of Akt, CREB and Stat3 (b). The scheme was created with BioRender.com.
As in previous studies on neuronal and Schwann cells, nimodipine pre-treatment led to increased activation of Akt at the serine residue 473 as well as increased activation of the transcription factor CREB at serine residue 133 by phosphorylation [6]. Both cell signaling proteins are known to interact with LMO4 [38] and to promote an anti-apoptotic effect ( Figure 7) [12,13,39]. Increased expression of LMO4 leads to higher activation of Akt, which exerts a neuroprotective effect via its various anti-apoptotic pathways [12,38,39]. A higher protein level of LMO4 also increases the phosphorylation of transcription factor CREB [40], resulting in neuronal cell survival during the interaction with Akt [13,14] as well as with LMO4 via the formation of a transcription complex [28]. CREB exerts its neuroprotective effect via starting gene expression with anti-apoptotic activity, such as B-cell lymphoma 2 (Bcl2) acting via regulation of intrinsic apoptosis induction and promoting DNA damage repair [40]. Therefore, these results clearly demonstrate the positive effect of nimodipine on cell survival of hair cells, which is accompanied by upregulation of LMO4 and activation of Akt and CREB (Figure 7b). Further functional studies should elucidate whether LMO4 plays a modulatory role in the activation of CREB and Akt or the mechanism of action of nimodipine.
Furthermore, higher expression of LMO4 can lead via the stabilization of glycoprotein 130 to increased activation of its downstream target Stat3 via the Jak/Stat pathway. This is known to cause increased expression of anti-apoptotic genes and thus downregulation is associated with apoptosis and thus ototoxicity [18,33]. Rosati et al. 2019 already showed a strong decrease in Stat3 and less activation by cisplatin. In our results, nimodipine was shown not only to increase LMO4 but also to increase the activation of Stat3 at As in previous studies on neuronal and Schwann cells, nimodipine pre-treatment led to increased activation of Akt at the serine residue 473 as well as increased activation of the transcription factor CREB at serine residue 133 by phosphorylation [6]. Both cell signaling proteins are known to interact with LMO4 [38] and to promote an anti-apoptotic effect (Figure 7) [12,13,39]. Increased expression of LMO4 leads to higher activation of Akt, which exerts a neuroprotective effect via its various anti-apoptotic pathways [12,38,39]. A higher protein level of LMO4 also increases the phosphorylation of transcription factor CREB [40], resulting in neuronal cell survival during the interaction with Akt [13,14] as well as with LMO4 via the formation of a transcription complex [28]. CREB exerts its neuroprotective effect via starting gene expression with anti-apoptotic activity, such as Bcell lymphoma 2 (Bcl2) acting via regulation of intrinsic apoptosis induction and promoting DNA damage repair [40]. Therefore, these results clearly demonstrate the positive effect of nimodipine on cell survival of hair cells, which is accompanied by upregulation of LMO4 and activation of Akt and CREB (Figure 7b). Further functional studies should elucidate whether LMO4 plays a modulatory role in the activation of CREB and Akt or the mechanism of action of nimodipine.
Furthermore, higher expression of LMO4 can lead via the stabilization of glycoprotein 130 to increased activation of its downstream target Stat3 via the Jak/Stat pathway. This is known to cause increased expression of anti-apoptotic genes and thus downregulation is associated with apoptosis and thus ototoxicity [18,33]. Rosati et al. 2019 already showed a strong decrease in Stat3 and less activation by cisplatin. In our results, nimodipine was shown not only to increase LMO4 but also to increase the activation of Stat3 at tyrosine residue 705 under stress after it was strongly reduced by the influence of cisplatin (Figure 7). In addition, an increased nimodipine-dependent total protein level of Stat3 was detected independent of the stress condition.
The maintenance of intracellular calcium homeostasis plays an essential role in a lot of important cell functions and cell survival and is regulated by a complex system [41,42]. Nimodipine regulates the calcium influx as an L-type calcium channel inhibitor [1,2]. Since it has already been shown that the calcium concentration increases under cisplatin [18,20], it was hypothesized that the neuroprotective effect of nimodipine is achieved by preventing calcium overload of the cell and stabilizing calcium homeostasis. In contrast, previous studies suggested a toxic effect of incoming calcium triggered via N-methyl-daspartate (NMDA) receptor, an ionotropic glutamate receptor, rather than L-type calcium channels [42,43]. Our study showed an opposite effect in immortalized hair cells, where divergent results between the different cell lines indicate a more receptor-independent effect. While UB/OC−1 cells showed a decrease in intracellular calcium concentration by nimodipine under cisplatin stress, no significant change in intracellular calcium was observed in UB/OC−2 cells, which was also confirmed for the differentiated cells (data not shown). The assumption that reduced intracellular calcium concentration is caused by a reduced cell quantity must be rejected since otherwise there would be no increase in undifferentiated UB/OC−2 or in differentiated cells under stress, while the cell number has been reduced by apoptosis. Since it is also known that LMO4, in addition to its own regulation by calcium, also has an influence on calcium concentration via the expression of ryanodine receptors [28,31], further investigations should aim to determine whether a protective effect occurs in a calcium-dependent manner and in combination with nimodipine pre-treatment.
In summary, it can be concluded that nimodipine reduces the cytotoxic effect induced by cisplatin accompanied by the upregulation of LMO4 and the associated activation of anti-apoptotic pathways in vitro (Figure 7).
Clinical trials focusing on the treatment of ototoxic effect triggered by cisplatin have already been carried out numerous times in recent years and, since no standard therapy has yet been approved by the Federal Drug Administration, many studies with a potentially good outcome are currently underway [19]. Otoprotection by SENS-401 (R-azasetron besylate) was observed in both in vitro and in vivo models with no effect on the chemotherapeutic potential of cisplatin [44,45]. Amifostine initially showed otoprotective potential in average-risk medulloblastoma patients [46,47], but this was found to be insufficient upon further investigation [48]. Similarly, the promising inhibition of OCT2 [21] by the proton pump inhibitor pantoprazole only leads to an insufficient reduction [49]. The intratympanic injection of dexamethasone, in contrast to systemic administration [50], showed a protective effect in cisplatin-induced ototoxicity [51]. Further studies on new non-invasive forms of administration observed the same effect [52,53]. For nimodipine, too, with the same otoprotective efficacy in vivo, studies should be conducted on the best possible form of administration. Substances such as the antioxidant N-acetylcysteine also counteract hearing loss during chemotherapy with cisplatin [54,55] and, like ginkgo [56] and other substances, are the focus of current clinical studies. In particular, sodium thiosulphate stood out as a potential agent that is both well tolerated [57] and, in a phase III clinical trial, showed a significant reduction in the incidence of hearing loss during chemotherapy with cisplatin in children with standard-risk hepatoblastoma without compromising the chemotherapeutic potential [58]. Despite the large number of substances that have already been investigated, there is still no established standard therapy against cisplatin-based ototoxicity. The results in neuroprotection of nimodipine, as well as its good tolerability, identifies it as a potential new medication.
By reducing the side effects of cisplatin through nimodipine pre-treatment, a significant improvement in patients' quality of life and better utilization of the chemotherapeutic effect could be achieved, if this protective effect can also be demonstrated in vivo. However, the most effective therapy against the side effects of cisplatin is not only the molecular inhibition of intracellular apoptosis mechanisms but also the optimization of the tumorcentered effect and blockade of the formation and impact of the toxic degradation products of cisplatin [59]. In addition to the reduced compliance of patients with the increase in side effects under chemotherapy, these also lead to a deterioration in mental health. Likewise, the removal or reduction in the dose limitation leads to greater potential in the total utilization of the chemotherapeutic effect of platinum derivatives. Nimodipine could thus play a potential role in the improved use of chemotherapeutic agents such as cisplatin by improving the quality of life of patients under and after oncological disease.
Int. J. Mol. Sci. 2022, 23, x  1 ever, the most effective therapy against the side effects of cisplatin is not only the m ular inhibition of intracellular apoptosis mechanisms but also the optimization of t mor-centered effect and blockade of the formation and impact of the toxic degra products of cisplatin [59]. In addition to the reduced compliance of patients with crease in side effects under chemotherapy, these also lead to a deterioration in m health. Likewise, the removal or reduction in the dose limitation leads to greater po in the total utilization of the chemotherapeutic effect of platinum derivatives. Nimo could thus play a potential role in the improved use of chemotherapeutic agents s cisplatin by improving the quality of life of patients under and after oncological dis

Cell Treatment
Cells were seeded and pre-treated with 10 µM and 20 µM nimodipine (Tokyo C ical Industry, Zwijndrecht, Belgium) diluted in EtOH   Simultaneously, nimodipine was given in the same amount as the day before, respectiv ( Figure 9b). NaCl served as solvent control of cisplatin.

Cytotoxicity Measurement
The 5 × 10 4 UB/OC−1 or UB/OC−2 were seeded in 24-well plates (Techno Plastic Pr ucts, TPP, Trasadingen, Switzerland) and cytotoxicity was measured by the activit LDH as a marker for cell death using Cytotoxicity Detection Kit (Roche, Basel, Swit land) according to manufacturer's instructions. In brief, 100 µL cell culture supernatan triplicates per sample and 100 µL reaction mix were incubated in the dark for 30 m Absorbance was measured at 492 nm with Tecan Reader F2000 Pro (Tecan, Männed Switzerland) at four definite points of the wells. Absorbance of cells lysed with 2% Tr X-100 (Carl Roth, Karlsruhe, Germany) served as positive control (100% cell death) w medium signal without cells served as background signal. The calculation of the cell de rate was performed as described before [6].

Western Blot
The 2 × 10 6 cells were seeded in Petri dishes (TPP, Trasadingen, Switzerland) treated as shown in Figure 9 schematically. At 24 h after cisplatin treatment, cells w washed two times with ice-cold Dulbecco's Phosphate Buffered Saline (PBS, Ther Fisher Scientific, Waltham, MA, USA) and harvested in PBS containing Halt TM Prote and Phosphatase Inhibitor Single-Use Cocktail (1:100, Thermo Fisher Scientific, Walth MA, USA). The proteins were extracted with 1x LDS Sample Buffer (Invitrogen, Ther Fisher Scientific, Waltham, MA, USA) and heated at 70 °C for 10 min, followed by pro concentration measurement performing bicinchoninic acid (BCA) assay using Pierc BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to m ufacturer's instructions. After 5% -mercaptoethanol (Carl Roth, Karlsruhe, Germa was added, the samples were again heated at 70 °C for 10 min. To separate protein, S PAGE was performed using NuPAGE TM 4-12% Bis-Tris Gels (1.5 mm × 10 well) (Invi gen, Thermo Fisher Scientific, Waltham, MA, USA) and 1× NuPAGE ® MES SDS Runn

Cytotoxicity Measurement
The 5 × 10 4 UB/OC−1 or UB/OC−2 were seeded in 24-well plates (Techno Plastic Products, TPP, Trasadingen, Switzerland) and cytotoxicity was measured by the activity of LDH as a marker for cell death using Cytotoxicity Detection Kit (Roche, Basel, Switzerland) according to manufacturer's instructions. In brief, 100 µL cell culture supernatant in triplicates per sample and 100 µL reaction mix were incubated in the dark for 30 min. Absorbance was measured at 492 nm with Tecan Reader F2000 Pro (Tecan, Männedorf, Switzerland) at four definite points of the wells. Absorbance of cells lysed with 2% Triton X-100 (Carl Roth, Karlsruhe, Germany) served as positive control (100% cell death) while medium signal without cells served as background signal. The calculation of the cell death rate was performed as described before [6].

Western Blot
The 2 × 10 6 cells were seeded in Petri dishes (TPP, Trasadingen, Switzerland) and treated as shown in Figure 9 schematically. At 24 h after cisplatin treatment, cells were washed two times with ice-cold Dulbecco's Phosphate Buffered Saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) and harvested in PBS containing Halt TM Protease and Phosphatase Inhibitor Single-Use Cocktail (1:100, Thermo Fisher Scientific, Waltham, MA, USA). The proteins were extracted with 1x LDS Sample Buffer (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and heated at 70 • C for 10 min, followed by protein concentration measurement performing bicinchoninic acid (BCA) assay using Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer's instructions. After 5% β-mercaptoethanol (Carl Roth, Karlsruhe, Germany) was added, the samples were again heated at 70 • C for 10 min. To separate protein, SDS-PAGE was performed using NuPAGE TM 4-12% Bis-Tris Gels (1.5 mm × 10 well) (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and 1× NuPAGE ® MES SDS Running Buffer (Novex, Thermo Fisher Scientific, Waltham, MA, USA) followed by blotting onto 0.2 µm or 0.45 µm nitrocellulose membranes (Amersham, GE, Healthcare, Freiburg, Germany) depending on molecular weight of the proteins to be detected (0.2 µm < 20 kDa). After blocking with 5% skim milk (Carl Roth, Karlsruhe, Germany) diluted in tris-buffered saline (TBS) with 0.1% Tween ® 20 (Sigma-Aldrich St. Louis, MO, USA, TBS-T), membranes were incubated overnight at 4 • C with primary antibodies (Table 2). Afterward, membranes were washed five times with TBS-T for 5 min, incubated with the secondary horseradish peroxidase (HRP)-linked antibody ( Table 2) for at least 60 min, and washed again three times with TBS-T and two times with TBS for 5 min each. Membranes were developed using Pierce TM ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and signals were detected with a CCD camera (ImageQuant LAS4000, GE, Healthcare, Freiburg, Germany).

Statistical Analysis
Statistical analysis of three independent replicates was performed with unpaired, two-sided student's t-test and one-way ANOVA followed by Tukey's post hoc test (SPSS version 28, IBM, Ehringen, Germany). Significance was accepted if p values were <0.05. Data were expressed as the mean ± S.D.

Conclusions
Nimodipine not only acts via vasodilation as a calcium channel antagonist but also shows a protective effect against cell death induced by cisplatin in auditory cells. This offers the possibility to extend the application of vasospasm prevention after subarachnoid hemorrhages to the treatment of prevention of cisplatin-mediated side effects or auditory cell damage associated with degenerative diseases.
The transcriptional regulator LMO4 and the accompanied increased activation of antiapoptotic pathways via Akt, CREB, and Stat3 seemed to be associated with the otoprotective effect. Future studies should aim to prove the obtained results in vivo in order to enable future application in patients.