Figure 3.
RAD001 sensitized breast cancer cells to carboplatin-induced apoptotic cell death through a caspase-independent and apoptosis-inducing factor (AIF)-dependent pathway. A: MCF-7 cells were treated with carboplatin (μg/ml) alone, RAD001 (nM) alone, or the combination of both, either in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK (20 μM) for 7 days. Internucleosomal DNA fragmentation was quantified by assaying for cytoplasmic mononucleosome- and oligonucleosome-associated histone using a cell death detection ELISA kit. The rate of apoptosis is reflected by the enrichment of nucleosomes in the cytoplasm and is expressed as the mean±SD of triplicates. B: MCF-7 cells were treated with carboplatin (10 μg/ml) alone, RAD001 (10 nM) alone, or combination of both for 7 days, and the mitochondrial membrane potential was determined by flow cytometry. Increased permeabilization of the mitochondrial membrane is shown by an increased intensity of green fluorescence (FL1). C: Expression changes of BCL-2, BAX, BAK, p53, and p21 after 7-day treatment with carboplatin (10 μg/ml), RAD001 (10 nM), or both drugs were examined by Western blot. β-Actin expression served as loading control. D: Cytoplasmic and nuclear expression of AIF in MCF-7 and BT-474 cells after treatment with carboplatin (10 μg/ml) alone, RAD001 (10 nM) alone, or their combination.