Figure 5

Exogenous supplementation with oleuropein aglycone inhibits HER2 ECD cleavage and HER2 autophosphorylation (activation), down-regulates HER2 expression and synergistically enhances trastuzumab-induced depletion of HER2 in trastuzumab-sensitive SKBR3 cells. A. Overnight serum-starved SKBR3 cells were cultured in DMEM-0.1% FBS in the absence or presence of increasing concentrations of oleuropein aglycone for 24 h. Top. The Oncogene Science HER2 microtiter ELISA was used according to the manufacturer's instructions to compare HER2 ECD concentrations in conditioned media from oleuropein aglycone-treated and untreated control cells. Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. Statistically significant differences (one-factor ANOVA analysis) between experimental conditions and unsupplemented control cells are shown by asterisks (* P < .01, ** P < .001). All statistical tests were two-sided. Bottom. The activation status of HER2 oncoprotein upon treatment with increasing concentrations of oleuropein aglycone was tested using a Phosphor-Tyr1248-HER2 immunoblotting procedure as described in "Material and methods". Figure shows a representative immunoblotting analysis. Equivalent results were obtained in, at least, three independent experiments. B, C. Overnight serum-starved SKBR3 cells were cultured in DMEM-0.1% FBS supplemented with increasing concentrations of oleuropein aglycone in the absence or presence of trastuzumab for 72 h. The Oncogene Science HER2 microtiter ELISA was used according to the manufacturer's instructions to compare HER2 concentrations in cell pellets. Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. Statistically significant differences (one-factor ANOVA analysis) between experimental conditions and unsupplemented control cells are shown by asterisks (* P < .01, ** P < .001). All statistical tests were two-sided.