Cisplatin-induced apoptosis in auditory, renal, and neuronal cells is associated with nitration and downregulation of LMO4.

R Rathinam,S Jamesdaniel,Wl Neumann,S Ghosh

doi:10.1038/cddiscovery.2015.52

Abstract

Cytotoxic effects of cisplatin occur primarily through apoptosis. Though several pro- and anti-apoptotic signaling molecules have been identified to play an important role in mediating the ototoxic, nephrotoxic, and neurotoxic side effects of cisplatin, the underlying mechanism is yet to be fully characterized. We reported that nitration of LIM domain-only 4 (LMO4), a transcriptional regulator, facilitates cochlear apoptosis in cisplatin-induced ototoxicity. However, its role in cisplatin-mediated nephrotoxicity and neurotoxicity is poorly understood. Therefore, HK2 and SH-SY5Y cells were used along with UBOC1 cells, to investigate the perturbations of LMO4 in cisplatin-induced cytotoxicity, in renal, neuronal, and auditory cells, respectively. Cisplatin induced an increase in the expression of active caspase-3, indicating cellular apoptosis, and increased the nitration of proteins, 24 h post treatment. Immunostaining with anti-nitrotyrosine and anti-LMO4 indicated that nitrotyrosine co-localized with LMO4 protein in cisplatin-treated cells. Immunoblotting with anti-LMO4 indicated that cisplatin induced a decrease in LMO4 protein levels. However, a corresponding decrease in LMO4 gene levels was not observed. Inhibition of protein nitration with SRI110, a peroxynitrite decomposition catalyst, attenuated cisplatin-induced downregulation of LMO4. More importantly, overexpression of LMO4 mitigated the cytotoxic effects of cisplatin in UBOC1 cells while a dose-dependent decrease in LMO4 protein strongly correlated with cell viability in UBOC1, HK2, and SH-SY5Y cells. Collectively, these findings suggested a potential role of LMO4 in facilitating the cytotoxic effects of cisplatin in auditory, renal, and neuronal cells.

Highlights

Ototoxicity, nephrotoxicity, and neurotoxicity are among the major side effects of cisplatin, a highly effective anti-neoplastic drug used in the treatment of solid tumors.[1]
Specificity of neuronal cells based on the differences in their resilience to the immunoreaction with anti-LIM domain-only 4 (LMO4) was indicated by a faint cisplatin treatment
The sensitivity of the neuronal cells to cisplatin protein band in extracts derived from LMO4 knockout HAP1 cells

Summary

INTRODUCTION

Ototoxicity, nephrotoxicity, and neurotoxicity are among the major side effects of cisplatin, a highly effective anti-neoplastic drug used in the treatment of solid tumors.[1] Upon entering the cell, cisplatin is converted into a highly reactive intermediate by an aquation reaction, which eventually leads to the generation of reactive oxygen species and DNA damage, resulting in apoptosis and cell death. These processes facilitate a reduction in tumor size and/or prevent tumor growth, they adversely affect the normal cells in the inner ear, kidney, and nervous system. Nitrotyrosine immunoreactivity was detected by immunocytochemical analysis with anti-nitrotyrosine, and the

RESULTS

DISCUSSION

MATERIALS AND METHODS