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PO1-8-44

SNP as a nitric oxide donor induces apoptosis of HepG2 cells by targeting the MAPK-mediated mitochondrial pathway

[Speaker] Zi-le Huang:1
[Co-author] Jian-Gang Wang:1, San-Qing Li:1, Ling Liu:1
1:Henan University of Science and Technology, Luoyang, China

BACKGROUND Sodium nitroprusside (SNP) was a nitric oxide donor, which had potent anti-neoplasticactivity in vitro. The present study aimed to investigate the apoptosis induction effects of SNP in the human hepatocellular carcinoma cell.
METHODS Cell proliferation and NO production were determined by MTT and Griess assays. JC-1 probe was used to examine the changes of mitochondrial membrane potential at the early stage of the cell apoptosis. Expression levels of apoptotic inducing factor (AIF), cytochrome c (Cyt c), Bax, Bcl-2, caspase- 3, 9 were determined by Western blot. In addition, HepG2 cells were treated with SP600125 (an inhibitor of JNK) or SB203580 (an inhibitor of P38) prior to SNP, and the protein expressions of c-Jun N-terminal kinase (JNK), p38 MAPK and ERK1/2 were measured by Western blot.
RESULTS Our results showed that SNP inhibited the growth of HepG2 cells in a time- and concentration-dependent manner, and the IC50 was 4 mM at 24 h. SNP can induce the release of NO in HepG2 cells, and induce the intracellular reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential, release of AIF and Cyt c from mitochondria, enhanced Bax-to-Bcl-2 ratio, and activation of caspase-3, 9, which indicated that SNP may induce apoptosis through a mitochondrial-mediated pathway. However, the cytotoxic effect and apoptosis can be prevented by Carboxy-PTIO (NO scavenger). Moreover, SNP could lead to the activation of the phosphorylation of JNK and p38 MAPK but not ERK1/2, which could be reversed by SP600125 and SB203580. Furthermore, antioxidant N-acetylcysteine (NAC) was found to block SNP-induced apoptosis and partly inhibited the activation of JNK and p38, up-regulation of Bax, down-regulation of Bcl-2 and the activation of caspase-3 in cells treated with SNP.
CONCLUSIONS The results suggested that SNP as a NO donor can significantly induce apoptosis through a MAPK-mediated mitochondrial pathway in HepG2 cells.
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