title: Walker carcinosarcoma cells damage endothelial cells by the generation of reactive oxygen species.
abstract: The passage of circulating tumor cells across vessel walls is an important step in cancer metastasis and is promoted by endothelial injury. Because Walker carcinosarcoma 256 (W256) cells generate oxygen-derived free radicals after cellular activation, the authors tested the hypothesis that these cancer cells can damage endothelial monolayers by producing such reactive oxygen species. To confirm that oxygen-derived radicals can damage endothelial cells, 3H-2-deoxyglucose-labeled human endothelial cell monolayers were exposed to xanthine oxidase in the presence of 0.2 mmol/l xanthine. 3H-2-deoxyglucose release was observed after the addition of xanthine oxidase in concentrations ranging from 6.5 x 10(-3) to 52 x 10(-3) units/ml. The extent of damage correlated with xanthine oxidase-dependent chemiluminescence (r = 0.91). Chemiluminescence assays in the presence of 5 x 10(-5) M luminol confirmed activation of the W256 cells by 1 x 10(-6) M chemotactic peptide fMLP. When fMLP-activated activated W256 cells were incubated with endothelial monolayers, concentrations of 2 x 10(6) to 6 x 10(6) W256 cells/ml were found to cause a 27% increase in the specific release of 2-deoxyglucose after a 90-minute incubation. A small but significant increase in 3H-2-deoxyglucose release also was observed in the absence of fMLP. Detection of 3H-2-deoxyglucose release in the presence of activated or unactivated tumor cells was dependent on preincubating the endothelial cell monolayer with 1 mM buthionine sulfoximine, an inhibitor of glutathione synthesis. Under these conditions, the specific release of 3H-2-deoxyglucose was increased from nondetectable levels to 21%, in the presence of 6.5 x 10(-3) units of the oxidase. Cultured W256 cells promoted isotope release from endothelial cell monolayers when activated with phorbol myristate acetate. Catalase (1000 units/ml) inhibited the tumor cell-induced release of 3H-2-deoxyglucose by 84% whereas superoxide dismutase, even at concentrations of 1 mg/ml, had no effect. A requirement for cell contact was shown because addition of cell-free supernatants from fMLP activated tumor cells did not cause 3H-2-deoxyglucose release and because pretreatment of W256 cells with 1 microM cytochalasin B inhibited their ability to promote isotope release even while increasing tumor cell-generated chemiluminescence threefold. Electron microscopy revealed that fewer cytochalasin B-treated W256 cells were attached to the endothelial cell monolayer than in untreated controls. It is concluded that the W256 tumor cells can damage endothelial cells directly via a mechanism involving production of reactive oxygen species.
authors.affiliation: Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
authors.forename: S G
authors.initials: SG
authors.lastname: Shaughnessy
authors.forename: M R
authors.initials: MR
authors.lastname: Buchanan
authors.forename: S
authors.initials: S
authors.lastname: Turple
authors.forename: M
authors.initials: M
authors.lastname: Richardson
authors.forename: F W
authors.initials: FW
authors.lastname: Orr
mesh_terms: D000818:Animals; D002279:Carcinoma 256, Walker / Q000378:metabolism / Q000473:pathology*; D002374:Catalase / Q000494:pharmacology; D002450:Cell Communication; D002460:Cell Line; D003847:Deoxyglucose / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism / Q000473:pathology*; D005609:Free Radicals; D008163:Luminescent Measurements; D008297:Male; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013482:Superoxide Dismutase / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000494:pharmacology
chemical_list: D005609:Free Radicals; D003847:Deoxyglucose; D002374:Catalase; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D010100:Oxygen