Cisplatin (CDDP, cis-diamminedichloroplatinum II) and other related platinum drugs used as antineoplastic agents, are widely applied for both adult and pediatric cancers. [1,2] However, these antineoplastic agents are associated with multiple side effects including nephrotoxicity, neurotoxicity and ototoxicity; thus far, effective approaches for attenuating these side effects are unavailable. [3–5] CDDP-induced ototoxicity is characterized as bilateral, irreversible sensorineural hearing loss. Notably, even mild levels of hearing loss contribute modifiable risk factors to dementia. [6,7] Moreover, the impact of CDDP-induced hearing loss can be great in children. [3,5,6] Thus, there is an urgent and unmet medical need to identify therapies for CDDP-induced ototoxicity.
Mitochondria are particularly sensitive to CDDP-induced damage. Cisplatin forms platinum (Pt)-DNA cross-links that destroy the structure of DNA, thus interfering with normal DNA transcription and replication mechanisms. [8–10] DNA damage, which is considered to play a core role in cell death, can be repaired in the nucleus because it contains machinery for nucleotide excision repair. However, as mitochondria do not have this repair machinery,  Pt-mitochondrial DNA (mtDNA) adducts remain within mitochondria, thus making them more sensitive to CDDP. CDDP-damaged mitochondria translocate cytochrome c, which leads to hair cell death, DNA fragmentation, and subsequent release. [11–13] Thus, the remaining hair cells require more mitochondria to generate ATP to protect against damage.  Therefore, we believe mitochondria are important in CDDP-induced hair cell death, because they are the major injured organelle that is required for cell survival.
Mitochondria are dynamic cytoplasmic organelles that maintain intracellular quality control by balancing several cellular processes, including biogenesis, fusion and fission, and mitophagy—the process by which mitochondria are degraded. Mitochondrial biogenesis, an important process by which new mitochondria are synthesized in the cell to maintain mitochondria quality control, [15,16] can be affected by mtDNA damage.  Impaired mitochondrial biogenesis is thought to be a primary mechanism of neurodegenerative diseases.  However, whether this process is involved in CDDP-induced ototoxicity remains unknown. Thus, we speculated that CDDP impairs mitochondrial biogenesis to lead to hair cell death, and activating mitochondrial biogenesis may enhance the survival of hair cells damaged by CDDP.
As such, the present study explored the relationship between mitochonrial biogenesis and CDDP-induced ototoxicity in the auditory cell line House Ear Institute-Organ of Corti 1 (HEI-OC1).
Materials and methods
Cell culture and drug treatments
HEI-OC1 cells are a widely used immortalized auditory cell line derived from a transgenic mouse organ of Corti, which was established by F. Kalinec (House Ear Institute, Los Angeles, CA, USA). Cells were cultured in high-glucose Dulbecco's Modified Eagle's Medium (Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco BRL) at 33°C and under 10% CO2 without antibiotics.  To examine the protective effect of an activator of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) against cisplatin-induced cytotoxicity, cells were seeded at proper density and allowed to attach overnight for 24 hours, then exposed to 20 μM CDDP (S1166, Selleck, Houston, TX, USA) together with 20 μM ZLN005, a specific activator of PGC-1α (S7447, Selleck), for 24 hours after pre-treatment with ZLN005 for 24 hours.
Cell viability assessment
Cell viability was quantified using a commercial Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, Shanghai, China) in accordance with the manufacturer's instructions. Briefly, 10 μL of CCK-8 was added in 100 μL of medium after drug treatment and incubated for 2 hours. Optical density values were measured at 450 nm using a Multiskan MK3 microplate reader (Thermo Labsystems, Tiilitie, Finland) to indicate cell viability. Relative cell viability of the negative control group was considered as 100% cell survival.
Western blot assay
HEI-OC1 cells incubated as described above were extracted by RIPA lysis buffer (Thermo Fisher, Waltham, MA, USA) with 1% protease inhibitors (Selleck). Protein extraction and western blot were performed as described in our previous study  using the following primary antibodies: rabbit anti-NRF1, anti-NRF2, anti-TFAM (1:1000, Abcam, Cambridge, UK), rabbit anti-β-actin, mouse anti-PGC-1α (1:1000, Proteintech, Wuhan, China), and rabbit anti-cleaved-caspase3 (1:1000, Cell Signaling Technology, Danvers, MA, USA). Finally, protein signals were visualized with a chemiluminescent substrate kit (WBKLS0010, Millipore, Darmstadt, Germany) and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Mitochondrial DNA copy number
Total DNA was isolated from HEI-OC1 cells using a Universal Genomic DNA Extraction Kit (Takara, BIO, Shiga, Japan) in accordance with the manufacturer's instructions. MtDNA copy number was measured by quantitative real-time PCR (qRT-PCR) as the relative number of mtDNA to nuclear DNA. The following primers for nuclear DNA were used: β-actin, forward 5′-GCT CCT CCT GAG CGC AAG-3′ and reverse 5′-CAT CTG CTG GAA GGT GGA CA-3′. For quantification of mtDNA, we used the following pair of primers: mtDNA, forward 5′-CCT ATC ACC CTT GCC ATC AT-3′ and reverse 5′-GAG GCT GTT GCT TGT GTG AC-3′, which target the D-loop gene. Relative mtDNA and nuclear DNA copy numbers were compared and normalized to controls.
Measurement of adenosine triphosphate content
Adenosine triphosphate (ATP) content was determined using a commercially available luciferin-luciferase assay kit (S0027, Beyotime Biotechnology) in accordance with the manufacturer's protocol. Briefly, lysed cells were centrifuged for 5 minutes at 4°C and 12,000×g, and the supernatant was collected. Before ATP detection, 100 μL of detection solution was added to each well of a 96-well plate for 5 minutes. The supernatant was then mixed quickly with the detection solution at room temperature, and measured using a microplate reader (Infinite M200; Tecan, Männedorf, Switzerland) within 30 minutes. After incubation, total ATP levels were calculated according to an ATP standard curve and normalized to protein concentrations.
All statistical analyses were performed using IBM SPSS Statistics V22.0 software (IBM Corp., Armonk, NY, USA), and all values are shown as mean ± standard error of the mean (SEM). One-way analysis of variance was used for statistical comparison between groups. A value of P < 0.05 was considered statistically significant.
Cisplatin reduced ATP levels and induced cell death in HEI-OC1 cells
To determine whether CDDP caused cytotoxicity, a CCK-8 assay was employed to monitor cell viability, and expression of the apoptosis-related protein cleaved-caspase 3 was examined in HEI-OC1 cells after CDDP exposure. HEI-OC1 cells began to exhibit a decreased survival rate after 12 hours of exposure to 20 μM CDDP, which became more aggravated during the following hours (Fig. 1A). Cleaved-caspase 3, a key activator of mitochondrial apoptosis pathways, was significantly increased after CDDP exposure in dose-dependent manner (Fig. 1B). Meanwhile, significantly decreased levels of mitochondrial ATP were observed to occur in a dose-dependent manner in HEI-OC1 cells after CDDP exposure, indicating that CDDP damaged mitochondrial function and caused cell death (Fig. 1C).
Mitochondrial biogenesis was impaired in cisplatin-exposed HEI-OC1 cells
CDDP exposure decreased levels of mtDNA (Fig. 2A), indicating decreased mitochondrial content in response to CDDP-induced damage. With respect to mitochondrial biogenesis, we examined four core regulators: PGC-1α, mitochondrial transcription factor A (TFAM), nuclear respiratory factor 1 (NRF1), and NRF2.  PGC-1α, NRF1, and NRF2 were decreased in response to CDDP exposure (Fig. 2B–F). Collectively, these results demonstrate that mitochondrial biogenesis was impaired in response to CDDP.
PGC-1α overexpression by ZLN005 protected HEI-OC1 cells against cisplatin-induced cytotoxicity
The small molecule ZLN005, also known as 2-(4-tert-Butylphenyl) benzimidazole, can activate the master regulator of mitochondrial biogenesis PGC-1α.  ZLN005 treatment did not lead to cytotoxicity (Fig. 3A). Additionally, western blotting analysis revealed increased PGC-1α expression after exposure to ZLN005 (Fig. 3B). Notably, ZLN005 rescued CDDP-induced HEI-OC1 cell death (Fig. 3C and D) and decreased ATP content (Fig. 3E).
PGC-1α overexpression by ZLN005 activated mitochondrial biogenesis in cisplatin-exposed HEI-OC1 cells
To determine whether PGC-1α overexpression could activate mitochondrial biogenesis after CDDP exposure, mtDNA contents were measured. PGC-1α overexpression by ZLN005 rescued CDDP-induced decreases in mtDNA contents (Fig. 4A). Moreover, mitochondrial biogenesis-related proteins NRF1 and NRF2 were increased in response to ZLN005, even after CDDP exposure (Fig. 4B–F), indicating that the recovery of mitochondrial biogenesis elicited by PGC-1α overexpression attenuated CDDP-induced mitochondrial dysfunction and HEI-OC1 cell death.
Mitochondria play a critical role in cell survival and death. Mitochondria, which are dynamic, maintain intracellular quality control by balancing several cellular processes, including biogenesis, fusion and fission, and mitophagy. [15,16] Mitochondrial biogenesis is an important process for regulating mitochondrial turnover and homeostasis. CDDP is transported into cells, where is forms Pt-mtDNA adducts that damage mitochondria. [12,13] Damaged and dysfunctional mitochondria are dissociated by fission and then degraded by mitophagy. Finally, mitochondrial biogenesis supplements the decrease in mitochondrial mass to maintain mitochondrial function and homeostasis. [15,16] CDDP inhibited mitophagy to affect mitochondria quality control.  Consistent with previous studies, [21,22] we observed decreased mitochondrial function and cell death in HEI-OC1 cells after CDDP exposure. We next examined whether CDDP decreased mitochondrial accumulation and biogenesis.
PGC-1α is a coactivator of mitochondrial biogenesis and function. PGC-1α modulates mitochondrial biogenesis in both direct and indirect manners. PGC-1α interacts with transcription factors and nuclear receptors NRF1 and NRF2 to positively regulate the expression of genes encoding proteins related to mitochondrial protein import and assembly, as well as mitochondrial translation. [23,24] In addition, PGC-1α indirectly activates the expression of TFAM, a regulator of mtDNA replication, via activation of NRFs.  Thus, PGC-1α is considered to be a key regulator of mitochondrial biogenesis. Our data revealed that mitochondrial biogenesis-related proteins PGC-1α, NRF1, and NRF1, as well as mtDNA accumulation, were downregulated in response to CDDP, suggesting mitochondrial biogenesis is inhibited after CDDP exposure. However, TFAM expression was increased. Thus, we considered TFAM activation in response to CDDP exposure to be independent of PGC-1α.
The small molecule ZLN005 is a specific PGC-1α transcriptional regulator that has been shown to elicit beneficial effects in diabetic mice.  Here, we found that ZLN005 increased PGC-1α protein expression. Moreover, it enhanced mitochondrial biogenesis after CDDP exposure by increasing not only PGC-1α, NRF1, and NRF2, but also TFAM. However, whether increased mitochondrial biogenesis resulted in functional benefits remains unknown, as a compensatory increase of mutated mtDNA instead of new healthy mitochondria could lead to further mitochondrial dysfunction.  Thus, we examined mitochondrial function to determine whether elevated mitochondrial biogenesis was a positive response to cell survival. Overexpression of PGC-1α improved mitochondrial function and attenuated cell death, suggesting that PGC-1α plays an important role in mitochondrial biogenesis during CDDP ototoxicity, and can rescue mitochondrial biogenesis to attenuate CDDP-induced hair cell death.
The present study has several limitations. First, the generation of excessive reactive oxygen species (ROS) is considered to be one of the major causes of CDDP-induced ototoxicity, and mitochondria are an important cellular organelle associated with ROS generation. The role that ROS play in activation of mitochondrial biogenesis to attenuate CDDP-induced HEI-OC1 cell death remains unknown. Second, although ZLN005 acts as a PGC-1α transcriptional regulator to enhance mitochondrial biogenesis, whether it is involved in mitophagy is unknown. Third, following up this study with an in vivo examination of the ability of ZLN005 to protect hair cells to demonstrate its clinical potential is lacking.
In summary, our present study demonstrated that CDDP inhibited mitochondrial biogenesis to damage mitochondrial function, leading to HEI-OC1 cell death. This is the first report of the protective mechanism by which auditory cells rescue themselves by enhancing mitochondrial biogenesis during CDDP exposure. Our results suggest that mitochondrial biogenesis, which modulates the quality control of mitochondria, is associated with CDDP ototoxicity; thus providing new understanding of the interplay between mitochondrial biogenesis and CDDP-mediated cell death.
Study conception and design, data analysis and interpretation, and experiment implementation: WJZ and HX. Manuscript writing: WJZ and JQP. Study conception and design, manuscript writing, final approval of manuscript: YQZ and HDY; data analysis and interpretation: LL, ZWS, HQL and BQJ.
This work was supported by the Project funded by China Postdoctoral Science Foundation (No. 2018M640863 to JP), the National Natural Science Foundation of China (No. 81771018, 81570935, 81570916, and 81873699 to HX and YZ), the Guangdong Natural Science Foundation of China (No. 2015A030313084 and 2017A030313585 to HX) and Yixian Research Launch Project of China (No. YXQH201807 to HX).
Conflicts of interest
The authors declare that they have no conflicts of interest.
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