Cisplatin-Induced Ototoxicity in Rats Is Driven by RIP3-Dependent Necroptosis.

Mi-Jin Choi,Seong Jun Choi,Jong-Seok Moon,Hyunsook Kang,Yun-Hoon Choung,Sung-Hee Park,Jeong Hun Jang,Oak-Sung Choo,Yun Yeong Lee

doi:10.3390/cells8050409

Mi-Jin Choi, Seong Jun Choi + Show 7 more

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https://doi.org/10.3390/cells8050409

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Journal: Cells	Publication Date: May 2, 2019
Citations: 33	License type: CC BY 4.0

Affiliation: Ajou University, Soonchunhyang University

Abstract

Cisplatin-induced early-onset ototoxicity is linked to hearing loss. The mechanism by which cisplatin causes ototoxicity remains unclear. The purpose of this study was to identify the involvement of receptor-interacting protein kinase (RIP)3-dependent necroptosis in cisplatin-induced ototoxicity in vitro and in vivo. Sprague–Dawley rats (SD, 8 week) were treated via intraperitoneal (i.p.) injection with cisplatin (16 mg/kg for 1 day), and their hearing thresholds were measured by the auditory brainstem response (ABR) method. Hematoxylin and eosin (H & E) staining, immunohistochemistry, and western blots were performed to determine the effect of cisplatin-induced ototoxicity on cochlear morphology. Inhibitor experiments with necrostatin 1 (Nec-1) and Z-VAD were also performed in HEI-OC1 cell line. H&E stains revealed that the necroptotic changes were increased in the organ of Corti (OC) and spiral ganglion neurons (SGNs). Moreover, immunohistochemistry and western blot analysis showed that cisplatin treatment increased the protein levels of RIP3 in both OCs and SGNs. The treatment of Nec-1, a selective RIP1 inhibitor, resulted in markedly suppression of cisplatin-induced cell death in HEI-OC1 cells, whereas Z-VAD treatment did not change the cisplatin-induced cell death. Our results suggest that RIP3-dependent necroptosis was substantial in cisplatin-induced ototoxicity; inner cochlear regions, the OCs, and SGNs were especially sensitive to necroptosis.

Highlights

Necroptosis is defined as a programmed form of necrosis and is executed by receptor-interacting protein kinase1 (RIP1) and receptor-interacting protein kinase3 (RIP3)
This regions of interest (ROIs) was chosen as the hair cell damage caused by cisplatin was most substantial at the mid-basal turn
Using immunohistochemistry and western blot analysis, we confirmed that RIP3-dependent necroptosis plays an important role in cisplatin-induced ototoxicity

Summary

Introduction

Necroptosis is defined as a programmed form of necrosis and is executed by receptor-interacting protein kinase (RIP1) and RIP3. Inhibition of caspase activity is necessary for necroptosis to occur and can be identified and characterized by the following: cellular rounding, an increase in cytosolic calcium ions, formation of reactive oxygen species (ROS), depletion of adenosine triphosphate (ATP), intracellular acidification, and, cellular swelling followed by cell membrane rupture, leading to release of damage-associated molecular patterns (DAMPs) [1,2]. Cells 2019, 8, 409; doi:10.3390/cells8050409 www.mdpi.com/journal/cells (TNFα) signal pathway has been studied for its influence in necroptosis; TNFα binds to tumor necrosis factor receptor (TNFR), leading to its internalization and subsequent formation of death-inducing signaling complex (DISC), known as complex II. In complex II, RIP1 and RIP3 are inactivated through their proteolytic cleavage by caspase-8. In the absence of caspase-8, the complex II signaling cascade leads to necroptosis [2]. Necroptosis is involved in various diseases, including stroke [3]

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