(-)-Oleocanthal treatment induces apoptosis at 25 µM in MDA-MB-231 breast cancer cells.

<p>(<b>A</b>) Flow cytometry analysis. Cells were plated at a density of 5×10⁶ cells/100 mm culture plates, allowed to attach overnight. Afterwards, cells were incubated in the respective control or (-)-oleocanthal-treated RPMI-1640 medium containing 40 ng/ml HGF for 24 h. At the end of the experiment, cells in each treatment group were trypsinized, washed then resuspended in ice-cold 1X Annexin V Binding Buffer. Afterwards, the cells were treated as described in the Materials and Methods. In the dot plot of double variable flow cytometry, LL quadrant (FITC −/PI -) shows living cells; UR quadrant (FITC +/PI +) stands for late apoptotic cells; and LR quadrant (FITC +/PI -) represents early apoptotic cells. (<b>B</b>) Western blot analysis of cleaved caspase 3 and cleaved PARP. (-)-Oleocanthal treatment at 25 µM for 72 h markedly increased levels of cleaved caspase-3 and cleaved PARP. (<b>C</b>) Western blot analysis. (-)-Oleocanthal at 25 µM for 72 h downregulates c-Met levels without affecting EGFR levels, and right panel shows that transfection of MDA-MB-231 cells with c-Met-targeted siRNA totally abolished c-Met protein expression. (<b>D</b>) Western blot analysis of cleaved caspase 8, RIP, cleaved caspase 9 and cytochrome c. Treatment with 25 µM (-)-oleocanthal increased the cleavage of caspase-8 and RIP, but not caspase-9 or cytochrome c. Right panel shows that c-Met-targeted siRNA yielded a pattern of apoptosis that is similar to that following treatment with (-)-oleocanthal at 25 µM for 72 h by causing an increase in caspase-8 and RIP cleavage, with no effect on caspase-9 and cytochrome c levels. (<b>E</b>) Effect of Z-VAD-FMK on (-)-oleocanthal-induced apoptosis. MDA-MB-231 cells were treated with 25 µM (-)-oleocanthal in the presence or absence of caspase inhibitor Z-VAD-FMK (50 µM). After 24-h incubation, cells were analyzed to examine cell death by measuring caspase 3 and caspase 8 cleavage detected by Western blotting. In all the above experiments, whole cell lysates were prepared for subsequent separation by polyacrylamide gel electrophoresis followed by Western blot analysis. Scanning densitometric analysis was performed on all blots done in triplicate and the integrated optical density of each band was normalized with corresponding β-tubulin, as shown in bar graphs beside their respective Western blot images. Vertical bars in the graph indicate the normalized integrated optical density of bands visualized in each lane ± SEM, *<i>P</i><0.05 as compared with vehicle-treated controls.