Quantification and morphologic demonstration of reactive oxygen species produced by Walker 256 tumor cells in vitro and during metastasis in vivo.
BACKGROUND
Pulmonary endothelial damage can be caused by agents that generate oxidants, e.g., bleomycin, hyperoxia, neutrophils or x-irradiation. In animals with intravascular cancer cells, there is increased tumor cell arrest and the subsequent formation of metastatic tumors at the sites of such endothelial injury. We have previously shown that Walker 256 (W256) tumor cells, stimulated with phorbol esters (phorbol 12-myristate 13 acetate) or the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, generate chemiluminescence that is inhibitable by catalase. Such activated cells can injure cultured endothelial monolayers. The purpose of the present study was to quantify and obtain morphological confirmation of the generation of reactive oxygen species by W256 cells in vitro, and to determine if this phenomenon could be morphologically detected in vivo during the metastatic process.


METHODS
The production of oxidants from W256 cells was quantitated in vitro by the scopoletin fluorescence assay, by a ferrithyiocyanate colorimetric assay (Thurman reaction), and confirmed morphologically, in vitro and in vivo, by the formation of cerium perhydroxide (Ce[OH]2OOH) deposits from cerium chloride (CeCl3). To demonstrate generation of reactive oxygen species in vivo, we examined W256 cells collected from the pulmonary circulation and at sites of spontaneous metastasis in the lung after intramuscular tumor transplantation, or cells arrested in the lungs after intravenous injection. The specificity of the CeCl3 reaction was confirmed by blocking in the presence of catalase.


RESULTS
As measured by the loss in scopoletin fluorescence and by generation of ferrithiocyanate 5 x 10(6) activated W256 cells produced an equivalent of 18 nM of H2O2 per hour A. Ce-[OH]2OOH deposits were identified in vitro on the surface of W256 cells, and at points of attachment between W256 cells and cultured endothelial cell monolayers. In vivo, CeCl3-derived deposits were seen on circulating W256 cells and on W256 cells that had arrested in the lungs following the intravenous injection of activated or non-activated W256 cells, or in spontaneous pulmonary metastases which formed after intramuscular tumor inoculation. Pretreatment of tumor-bearing animals with phorbol 12-myristate 13 acetate increased the number of CeCl3-derived deposits more than 2 fold. Catalase inhibited the formation of the electron-dense deposits in vitro and in vivo.


CONCLUSIONS
These data provide morphologic evidence that cancer cells can produce reactive oxygen species in vivo and suggest that free radicals might contribute to endothelial damage during the metastatic process.
Department of Pathology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil.
F A
FA
Soares
S G
SG
Shaughnessy
W R
WR
MacLarkey
F W
FW
Orr
D000818:Animals; D002279:Carcinoma 256, Walker / Q000378:metabolism* / Q000473:pathology*; D002448:Cell Adhesion / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology; D006861:Hydrogen Peroxide / Q000378:metabolism; D008163:Luminescent Measurements; D008297:Male; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009362:Neoplasm Metastasis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D017382:Reactive Oxygen Species; D009240:N-Formylmethionine Leucyl-Phenylalanine; D006861:Hydrogen Peroxide; D013755:Tetradecanoylphorbol Acetate