Peroxynitrite dominates sodium nitroprusside-induced apoptosis in human hepatocellular carcinoma cells.

Ying-Yao Quan,Tong-Sheng Chen,Xiao-Ping Wang,Chun-Mei Lin,Yu-Hong Liu

doi:10.18632/oncotarget.16164

Abstract

This study aims to explore which radicals dominate sodium nitroprusside (SNP)-induced cytotoxicity in human hepatocellular carcinoma (HCC) cells (HepG2 and Hep3B). Exposure of SNP to cell medium produced abundant nitric oxide (NO), superoxide anion (O2·−), hydrogen peroxide (H2O2) and iron ions. SNP potently induced caspases activation, mitochondrial membrane permeabilization and apoptosis in HCC cells. In Hep3B cells, pretreatment with NO scavenger (PTIO) did not prevent SNP-induced cytotoxicity. However, in HepG2 cells, SNP-induced cytotoxicity was prevented significantly by pretreatment with PTIO and O2·− scavenger, and especially was almost completely blocked by pretreatment with FeTPPS (peroxynitrite scavenger). In contrast, although H2O2 scavenger potently scavenged SNP-induced H2O2 production, it did not prevent SNP-induced cytotoxicity in HepG2 cells. In addition, pretreatment with DFO (iron ions chelator) and iron-saturated DFO respectively completely prevented SNP-induced cytotoxicity in HepG2 cells. Collectively, peroxynitrite from the reaction between NO and O2·− elicited from SNP dominates the SNP-induced apoptosis of HepG2 cells, in which both iron ions and H2O2 are not involved.

Highlights

Sodium nitroprusside (SNP) is a potent hypotensive agent widely used for treating hypertension-emergencies during surgery and improving heart function after infarction [1]
Cell Counting Kit-8 (CCK-8) assay showed that exposure of HepG2 cells to different concentration (0–1.5 mM) of sodium nitroprusside (SNP) for 24 h induced a dose-dependent cytotoxicity (Figure 1A), and exposure of cells to 1.25 mM of SNP for different times (0–48 h) induced a time-dependent cytotoxicity (Figure 1B). 1.25 mM SNP was adopted in following experiments without indication in HepG2 cells
We here found that SNP induced much more cytotoxicity (Figure 2B) than SNPex in HepG2 cells, further confirming the key role of nitric oxide (NO) in SNP-induced apoptosis in this cell line

Summary

Introduction

Sodium nitroprusside (SNP) is a potent hypotensive agent widely used for treating hypertension-emergencies during surgery and improving heart function after infarction [1]. It was reported that overexpression of gene-encoded NO synthase 2/3 (NOS2/3) potently increased the expressions of p53, CD95 and Rho kinase proteins [10, 11]. Excessive NO elicited from NO donor (GSNO, SNAP, or NONOate) increased the expression and activation level of proapoptotic Bax protein [12] and caspases [13, 14], and the loss of mitochondrial membrane potential [14]. Interaction of NO with superoxide anions (O2−) produces peroxynitrite (ONOO─) [15], a much more toxic oxidant than NO, that directly attacks DNA, triggers lipid peroxidation, dissipates mitochondrial membrane potential, and inactivates the complexes I, II, III and V of respiratory chain [16, 17]. ONOO─ can promote cell death via triggering both caspase-dependent (e.g., cytochrome c, APAF-1, Smac) and caspase-independent (especially apoptosis-inducing factor) apoptotic signal pathways [18]

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