Materials and methods Chemicals and cell cultures The PEITC was purchased from LKT Labs with 98% purity. The PEITC was in Paclitaxel powder was dissolved in DMSO to a MG132 CAS stock concentration of 200 nM. The MCF7 and MDA MB 231 cell lines were obtained from American Type Cell Cultures. The cells were seeded at 0. 4 106 per ml and 0. 2 106 per ml, respectively, of PRMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum and maintained at 37 C in a humidified atmosphere containing 5% CO2. The cells in exponential growth were exposed to PEITC and taxol at various concentrations. The control cultures were supple mented with DMSO as the vehicle control. At the specified time points, the cells were harvested. Cell num ber and viability were determined from at least triplicate cultures by the trypan blue exclusion method.

Cell cycle analysis The analysis of cell cycle phases was performed using a Becton Dickinson FACScan flow cytometer according to the methods described previously. The cells were stained with propidium iodide solution on ice, and at least 10,000 cells were analyzed. Apoptosis analysis Apoptotic cells were determined by the terminal deoxynu cleotidyl transferase mediated biotinylated UTP nick end labeling assay. The TUNEL assay, according to the methods described previously, was performed in situ with a cell death detection kit. To enumerate the apoptotic cells, six different fields on each section were examined. At least 100 cells from each field were counted. The mean populations of apoptotic cells per section from the control group and experimental group were reported.

Statistical analysis Results from 3 of more experiments were analyzed and expressed as the mean SD. Results were evaluated by a two sided paired Students t test for statistical difference between treatments. P 0. 05 was considered to be statistically significant. IC50, the concentration at which 50% of cell growth is inhib ited, was calculated using the Calcusyn software. Synergism was assessed by the dose effect curves of single versus combined drug treatment using the Calcusyn software. Results Effect of PEITC and taxol on breast cancer cells To test the effect of PEITC and taxol on breast can cer cells, the agents were added to the MCF7 and MDA MB 231 cell cultures at serial dilu tions for 24 and 48 hours, respectively.

The PEITC concentration ranged from 1 to 40 uM, and taxol concentration ranged from 0. 1 to 10,000 nM. PEITC suppressed cell growth in a time and concentration dependent manner. The IC50 of PEITC for MCF cells at 48 hours is 5. 6 uM, the IC50 of Brefeldin_A PEITC for MB cells at 48 hours is 15. 6 uM. It appears that 5 uM and 10 uM are the concentrations that can cause growth suppression in a linear fashion for MCF and MB cells, respectively. These concentrations were therefore chosen for fur ther combination studies. The IC50 of taxol for MCF and MB cells at 48 hours is 111 nM and 410 nM, re spectively.