Figure 4

MM-121 specifically downregulates Survivin and significantly enhances paclitaxel-induced anti-proliferative/anti-survival effects and apoptosis in a trastuzumab-resistant breast cancer cell line. (A) BT474-HR20 cells were untreated, or treated with MM-121 (10 μg/ml) for 24 or 48 h. Cells were collected and subjected to western blot analyses of P-erbB3, erbB3, Survivin, Bcl-xL, Mcl-1, or β-actin. The densitometry analyses of Survivin signals are shown underneath, and the arbitrary numbers indicate the intensities of each sample relative to control, defined as 1.0. (B) BT474-HR20 cells were plated onto 96-well plates and incubated at 37°C with 5% CO₂. After 24 h, the culture medium was replaced with 0.1 ml fresh medium containing 0.5% FBS or the same medium containing the indicated concentrations of paclitaxel in the absence (paclitaxel) or presence (MM-121 + pac) of MM-121 (10 μg/ml) for another 72 h. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS). Bars represent SD. Data are representative of three independent experiments. (C and D) BT474-HR20 cells were untreated, or treated with either MM-121 (10 μg/ml) or paclitaxel (8 nmol/L) alone, or their combinations for 24 h. Cells were collected and subjected to western blot analyses of poly(ADP-ribose) polymerase (PARP), caspase-8, caspase-3, or β-actin (C); or a specific apoptosis ELISA (D). Bars represent SD. ^*P <0.0002 versus single-agent treatment.