Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells

Ángeles Rodríguez-Hernández ,G Suárez-Artacho,A Gila-Bohórquez,L Barrera-Pulido,M.A Torres-Nieto,J Serrano-Díaz-Canedo,J.M Álamo-Martínez,L.M Marín-Gómez,A Sarrias-Giménez,Francisco J Padillo ,C Bernal Bellido ,Lysandra Castro ,Dimitri E Pacheco ,Miguel Ángel Gómez‐Bravo ,Elena Navarro-Villarán ,Sheila M Pereira ,José Antonio Bárcena ,Raúl González ,C Alicia Padilla ,Alejandro Serrablo-Requejo ,Gerardo Blanco‐Fernández ,Á Nogales-Muñoz ,Jordi Muntané

doi:10.1016/j.redox.2015.07.010

Abstract

Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.

Highlights

Nitric oxide (NO) is a lipophilic, highly diffusible, and shortlived physiological messenger [1]
Cells were routinely maintained in EMEM (Eagles Minimum Essential Medium) (Sigma-Aldrich Chemical Co) pH 7.4 supplemented with 10% fetal bovine serum (F7524, Sigma-Aldrich, Lot No: 022M3395, endotoxin o0.2 EU/ml), 2.2 g/l HCO3Na, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin and the corresponding selection antibiotic in 5% CO2 in air at 37 °C
Sorafenib dose-dependently regulated apoptotic cell signaling in hepatoblastoma cells

Summary

Introduction

Nitric oxide (NO) is a lipophilic, highly diffusible, and shortlived physiological messenger [1]. NO is synthesized by three different gene-encoded NO synthases (NOS) in mammals: neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2) and endothelial NOS (eNOS or NOS-3). NO regulates a variety of important physiological responses, including vasodilation, respiration, cell migration, immune response and apoptosis. Different clinical trials have demonstrated that the infusion of NO donors increases the effectiveness of chemotherapy and radiotherapy in patients with cancer [3,4]. NO appears to induce genotoxic lesions, and promotes angiogenesis, tumor cell growth, and invasion [5]. NO is able to exert antitumoral properties, as well as increase susceptibility to chemotherapy in different experimental in vivo and in vitro models [6]

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