Effect of GR knockdown and GSH depletion on the in vitro interaction between B16 melanoma cells and the vascular endothelium.

HSE cells (2.5×105cells/well) cultured for 24 h were co-cultured with B16-F10 or iB16-shGCR cells (5.0×105cells/well; pre-cultured for 24 h). Twenty minutes after the addition of tumor cells to the HSE, the plates were washed as described in Materials and Methods. The ratio of tumor cells adhering to the HSE was 1∶1. TNF-α (100 units/ml) and IFN-γ (50 units/ml), which were used as potent activators of NO and H2O2 generation by the HSE, were added to the co-cultures when all tumor cells present were attached to the HSE. In endothelium-induced B16-F10/iB16-shGCR cytotoxicity assays, tumor cytotoxicity (expressed as the % of tumor cells that lost viability within the 3–6-h incubation period) was determined after 6 h of incubation. During the 6-h incubation period, the percentage of HSE cell viability was 98–99% in all cases. When adding cytokines to cultured tumor cells alone, no cytostatic or cytotoxic effects were observed within the next 6 h. During the first 2-hincubation period, both HSE and B16-F10 or iB16-shGCR cells maintained >95% viability (data not shown). Where indicated, B16-F10 or iB16-shGCR cells were incubated for 24 h with BSO (0.5 mM) before co-culturing with endothelial cells. Pretreatment of B16-F10 cells with BSO did not significantly affect control values for tumor cell adhesion. Data are means ± S.D. for 5–6 independent experiments. *pversus B16-F10 + HSE controls in the absence of BSO.