RESUMO
The current study was focused on the induction of apoptotic effects of resveratrol along with the combination treatments of nutlin-3 and transforming growth factor-ß (TGF-ß) against the human ovarian cancer cell line A2780/CP70. To determine the extent of apoptosis following the above-mentioned treatments, we assessed the execution of apoptotic events that proceed via caspase activation and cytochrome c release. We estimated the caspase-3 and -9 activities using a direct enzymatic assay that measures the cleavage of synthetic peptide substrate (N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide). Our experiments showed an increase in caspase-3 and -9 activities in the cells that were treated with the combination of resveratrol (5 µM) with nutlin-3 (5 µM) or TGF-ß (1 µg/mL). Since activation of procaspase-3 by caspase-9 requires the release of cytochrome c into the cytoplasm, we measured the levels of cytochrome c in the cytoplasm by western blot experiments. The data indicated a considerable increase in caspase-3 and cytochrome c levels when cells were treated with drugs for 24 hours. Experiments with 4,6'-diamino-2-phenylindole dihydrochloride (DAPI) staining also confirmed the induction of apoptosis in all the above-mentioned treatments done at 24 and 48 hours. These results support our hypothesis that resveratrol combination can induce programmed cell death at doses that are less than half of what is typically needed for nutlin-3 and TGF-ß to induce apoptosis.
