Figure 6

Degradation of biotinylated HS by heparanase in SKBR3, MCF7 and MCF7-HPSE1 in the presence or absence of trastuzumab. HPSE1 action was measured by an ELISA-like method using HS biotinylated. Briefly, the cells were cultured in the absence or presence of trastuzumab (25 μg/ml) for 3 days on 60 mm plates. The cells were scraped with sodium acetate 25 mM, pH 5.0, containing protease inhibitors. The cell extract was incubated with the biotinylated HS pre-coated on the plate, revealed with europium-labeled streptavidin, washed and submitted to the enhancement solution. Free europium was measured and the data analyzed using MultiCalc software (PerkinElmer Life Sciences-Wallac Oy). *P < 0.001, compared to the non-degraded biotinylated HS (Control), **P < 0.05, treated versus non-treated cells, ***P < 0.05 MCF7-HPSE1 versus MCF7. Each bar indicates the mean ± SD. The assays were performed in triplicate.