Mechanism of interaction between ABZ and 2ME. A, Effect of combination therapy on tubulin polymerization. Cells were treated with the indicated concentrations of ABZ, 2ME, and their combination for 24 hours The percentage of polymerized tubulin was determined by dividing the densitometric value of polymerized tubulin by the total tubulin content. The blot is a representative blot from four experiments. B, Effect of ABZ and 2ME on caspase activation. HCT-116 cells were incubated with vehicle, ABZ, 2ME, and their combination at indicated concentrations for 4, 8, 16, 24 hours. Caspase activity was determined by caspase substrates labeled with p-nitroaniline (pNA) using colorimetic assay. The data represent the mean values for duplicate measurements from three experiments. C, Effect of ABZ, 2ME and their combination on DR5 and P53 protein expression. HCT-116 cells were treated with vehicle, ABZ, 2ME, and ABZ plus 2ME at indicated concentrations for 24 hours. Cells were then lysed and subjected to western blot analysis. β-actin was used as a control for equal protein loading. The bar graphs represent the mean protein expression of three individual experiments, with error bars showing SEM. (*P < 0.05 compared with single agents).