Mitophagy Impairment Aggravates Cisplatin-Induced Ototoxicity

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\u2009μM, 0, 8, 16, and 24\u2009h); cisplatin (10\u2009μM, 24\u2009h) post transfection with small-interfering (si)RNAs targeting mitophagy-associated mRNAs; cisplatin (10\u2009μM, 24\u2009h) succeeding pretreatment with the mitophagy suppressor, 3-methyladenine (3-MA; 5 or 10\u2009mM, 6\u2009h); or cisplatin (30\u2009μM, 24\u2009h) following pretreatment with the mitophagy promoter, carbonyl cyanide m-chlorophenylhydrazone (CCCP; 1 or 2\u2009μM, 2\u2009h). 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.