Tumoricidal activity of endothelial cells - Inhibition of endothelial nitric oxide production abrogates tumor cytotoxicity induced by hepatic sinusoidal endothelium in response to B16 melanoma adhesion in vitro

The mechanism of NO- and H2O2-induced tumor cytotoxicity was examined durin g B16 melanoma (B16M) adhesion to the hepatic sinusoidal endothelium (HSE) in vitro. We used endothelial nitric-oxide synthetase gene disruption and N O-nitro-L-arginine methyl ester-induced inhibition of nitric-oxide syntheta se activity to study the effect of HSE-derived NO on B16M cell viability. E xtracellular H2O2 was removed by exogenous catalase, H2O2 was not cytotoxic in the absence of NO, However, NO-induced tumor cytotoxicity was increased by H2O2 due to the formation of potent oxidants, likely (OH)-O-. 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 H2 O2 than B16M cells cultured to high density (HD cells; with similar to 25% of the GSH content found in LD cells). Resistance of LD cells decreased usi ng buthionine sulfoximine, a specific GSH synthesis inhibitor, whereas resi stance increased in HD cells using GSH ester, which delivers free intracell ular GSH, Because NO and H2O2 were particularly cytotoxic in HD cells, we i nvestigated the enzyme activities that degrade H2O2, NO and H2O2 caused an similar to 75% (LD cells) and a 60% (HD cells) decrease in catalase activit y without affecting the GSH peroxidase/GSH reductase system. Therefore, B16 M resistance to the HSE-induced cytotoxicity appears highly dependent on GS H and GSH peroxidase, which are both required to eliminate H2O2, In agreeme nt with this fact, ebselen, a GSH peroxidase mimic, abrogated the increase in NO toxicity induced by H2O2.