Involvement of a transforming‐growth‐factor‐β‐like molecule in tumor‐cell‐derived inhibition of nitric‐oxide synthesis in cerebral endothelial cells

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Abstract
Nitric oxide (NO) has been shown to exert cytotoxic effects on tumor cells. We have reported that EC219 cells, a rat‐brain‐microvessel‐derived endothelial cell line, produced NO through cytokine‐inducible NO synthase (iNOS), the induction of which was significantly decreased by (a) soluble factor(s) secreted by DHD/PROb, an invasive sub‐clone of a rat colon‐carcinoma cell line. In this study, the DHD/PROb cell‐derived NO‐inhibitory factor was characterized. Northern‐blot analysis demonstrated that the induction of iNOS mRNA in cytokine‐activated EC219 cells was decreased by PROb‐cell‐conditioned medium. When DHD/PROb cell supernatant was fractionated by affinity chromatography using Con A‐Sepharose or heparin‐Sepharose, the NO‐inhibitory activity was found only in Con A‐unbound or heparin‐unbound fractions, respectively, indicating that the PROb‐derived inhibitory factor was likely to be a non‐glycosylated and non‐heparin‐binding molecule. Pre‐incubation of DHD/PROb‐cell supernatant with anti‐TGF‐β neutralizing antibody completely blocked the DHD/PROb‐derived inhibition of NO production by EC219 cells. Addition of exogenous TGF‐β, dose‐dependently inhibited NO release by EC219 cells. The presence of active TGF‐β in the DHD/PROb cell supernatant was demonstrated using a growth‐inhibition assay. Moreover, heat treatment of medium conditioned by the less invasive DHD/REGb cells, which constitutively secreted very low levels of active TGF‐β, increased both TGF‐β activity and the ability to inhibit NO production in EC219 cells. Thus, DHD/PROb colon‐carcinoma cells inhibited NO production in EC219 cells by secreting a factor identical or very similar to TGF‐β.

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