Fig. 6.

A, Cytotoxic effects of betulinic acid on MDA-MB-231, MCF-7, U266, MM.1S and Du-145, PC-3 cells. Cells were plated in triplicate, treated with indicated concentrations of betulinic acid for 72 h, and then subjected to MTT assay to analyze the viability of cells. B, Over expression of constitutive STAT3 rescues A293 cells from betulinic acid -induced cytotoxicity. First, A293 cells were transfected with constitutive STAT3 plasmid. After 24 h of transfection, the cells were treated with indicated concentration of betulinic acid for 24 h, and then the cytotoxicity was determined by Live/Dead assay and 20 random fields were counted. C, Betulinic acid potentiates the apoptotic effect of bortezomib and thalidomide. U266 cells (5000 cells/well) were treated with indicated amounts of betulinic acid for 4h followed by indicated amounts of thalidomide or bortezomib for 24 h at 37°C. Cell viability was assessed by MTT uptake method. For live and dead assay, U266 cells (1 × 10⁶/mL) were treated with 25 μM betulinic acid and 10 μg/mL thalidomide or 20 nM bortezomib alone or in combination for 24 h at 37°C. Cells were stained with a Live/Dead assay reagent for 30 min and then analyzed under a fluorescence microscope and 20 random fields were counted.