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 H(2)O(2)-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 and N(G)-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 H(2)O(2) was removed by exogenous catalase. H(2)O(2) was not cytotoxic in the absence of NO. However, NO-induced tumor cytotoxicity was increased by H(2)O(2) due to the formation of potent oxidants, likely ( small middle dot)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 H(2)O(2) than B16M cells cultured to high density (HD cells; with approximately 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 H(2)O(2) were particularly cytotoxic in HD cells, we investigated the enzyme activities that degrade H(2)O(2). NO and H(2)O(2) caused an approximately 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 H(2)O(2). In agreement with this fact, ebselen, a GSH peroxidase mimic, abrogated the increase in NO toxicity induced by H(2)O(2).
Carretero
J
J
Departamento de Fisiologia, Universidad de Valencia, and the Servicio de Anatomia Patológica, Hospital Universitario La Fe, Valencia, Spain.
Obrador
E
E
Esteve
J M
JM
Ortega
A
A
Pellicer
J A
JA
Sempere
F V
FV
Estrela
J M
JM
D002450:Cell Communication; D016923:Cell Death; D002465:Cell Movement; D018920:Coculture Techniques; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009362:Neoplasm Metastasis; D009569:Nitric Oxide / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D009569:Nitric Oxide; D019331:NG-Nitroarginine Methyl Ester