Abstract
Cisplatin is an efficacious anticancer agent, but its use is limited by ototoxicity and resultant irreversible sensorineural hearing loss. Cisplatin ototoxicity is associated with cochlear cell oxidative stress and mitochondrial damage. However, mitophagy is vital for maintaining mitochondrial quality and cellular metabolism. Accordingly, we investigated the role of mitophagy in regulating cisplatin-induced ototoxicity using the auditory cell line HEI-OC1. In this study, HEI-OC1 cells were treated with either cisplatin alone (10 μM, 0, 8, 16, and 24 h); cisplatin (10 μM, 24 h) post transfection with small-interfering (si)RNAs targeting mitophagy-associated mRNAs; cisplatin (10 μM, 24 h) succeeding pretreatment with the mitophagy suppressor, 3-methyladenine (3-MA; 5 or 10 mM, 6 h); or cisplatin (30 μM, 24 h) following pretreatment with the mitophagy promoter, carbonyl cyanide m-chlorophenylhydrazone (CCCP; 1 or 2 μM, 2 h). The viability of cells, expression of mitophagy marker, and mitochondrial functions were then assessed in these cells. Cell viability was determined by a water-soluble tetrazolium assay; expression of mitophagy-associated proteins PINK1, Parkin, BNIP3, FUNDC1, p62, and LC3B was analyzed by Western blotting, mitochondrial membrane potential by flow cytometry, intracellular ATP by spectrophotometry, and mitochondrial degradation by dual staining for mitochondria and autophagosomes or lysosomes. Our results showed that cisplatin gradually reduced the viable cell number over time, induced mitochondrial depolarization, decreased intracellular ATP concentration, and enhanced the expression of PINK1, Parkin, BNIP3, p62, and LC3B. In addition, Parkin and BNIP3 knockdown accelerated cisplatin-induced loss of cell viability, mitochondrial membrane potential, mitophagosome/lysosome formation, and reduction in intracellular ATP production. Pretreatment with 3-MA aggravated the cisplatin-induced cytotoxicity, while that with CCCP reversed this effect. Overall, our findings indicate that mitophagy protects HEI-OC1 cells against cisplatin-induced cell death. Consequently, we strongly believe that targeted promotion of mitophagy may confer protection against cisplatin-induced ototoxicity.
Highlights
Cisplatin is the most widely used chemotherapeutic agent for both pediatric and adult cancer patients owing to its broad-spectrum antitumor efficacy [1]
Our results revealed that cisplatin treatment (10 μM) induces a time-dependent decline in the number of viable House Ear Institute-Organ of Corti 1 (HEI-OC1) cells as compared to the untreated controls (98:4% ± 0:8% vs. untreated controls after 8 h, 92:8% ± 0:6% after 16 h, and 85:3% ± 1:5% after 24 h) (Figure 1(a))
Relative expression levels of the mitophagy-associated proteins including PINK1, Parkin, and BNIP3 as well as the autophagosome markers LC3B and p62 increased in the mitochondrial protein extracts upon cisplatin exposure in a time-dependent manner (n = 5 independent experiments, p < 0:01, Figures 1(b) and 1(c))
Summary
Cisplatin (cis-diamminedichloroplatinum II) is the most widely used chemotherapeutic agent for both pediatric and adult cancer patients owing to its broad-spectrum antitumor efficacy [1]. Cisplatin chemotherapy causes bilateral, irreversible, and progressive hearing loss in 40%–80% of patients [2, 3]. This hearing loss results at least in part from excessive reactive oxygen species (ROS) generation in cochlear cells, leading to mitochondrial damage, metabolic disruption, and cell death [3]. Mitochondria generate adenosine triphosphate (ATP) required for most cellular activities via the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS). To prevent apoptosis and maintain cellular energy, the mitochondrial pool is subject to constant turnover through biogenesis and mitophagy, which is a mitochondria-specific form of autophagy that selectively degrades damaged mitochondria via the autophagosome-lysosome pathway. The maintenance of mitochondrial turnover is critical for long-term cellular fitness [6]
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