Tumoricidal Activity of Endothelial Cells

The mechanism of NO- and H2O2-induced tumor cytotoxicity was examined during B16 melanoma (B16M) adhesion to the hepatic sinusoidal endothelium (HSE) in vitro. We used endothelial nitric-oxide synthetase gene disruption andNG-nitro-l-arginine methyl ester-induced inhibition of nitric-oxide synthetase activity to study the effect of HSE-derived NO on B16M cell viability. Extracellular H2O2was removed by exogenous catalase. H2O2was not cytotoxic in the absence of NO. However, NO-induced tumor cytotoxicity was increased by H2O2due to the formation of potent oxidants, likely ⋅OH and −OONO radicals, via a trace metal-dependent process. B16M cells cultured to low density (LD cells), with high GSH content, were more resistant to NO and H2O2than B16M cells cultured to high density (HD cells; with ∼25% of the GSH content found in LD cells). Resistance of LD cells decreased using buthionine sulfoximine, a specific GSH synthesis inhibitor, whereas resistance increased in HD cells using GSH ester, which delivers free intracellular GSH. Because NO and H2O2were particularly cytotoxic in HD cells, we investigated the enzyme activities that degrade H2O2. NO and H2O2caused an ∼75% (LD cells) and a 60% (HD cells) decrease in catalase activity without affecting the GSH peroxidase/GSH reductase system. Therefore, B16M resistance to the HSE-induced cytotoxicity appears highly dependent on GSH and GSH peroxidase, which are both required to eliminate H2O2. In agreement with this fact, ebselen, a GSH peroxidase mimic, abrogated the increase in NO toxicity induced by H2O2.