Cucurbitacin B inhibits microtubule polymerization. A. Effect of cucurbitacin B on the organizations of cellular microtubule network. Immunofluorescent staining for α-tubulin was inspected in MCF-7 and MDA-MB-231 cells treated with cucurbitacin B. Cells were fixed, permeabilized and stained with anti-α-tubulin monoclonal antibody. The cells were analyzed using confocal microscopy. DAPI was used for nuclear localization. Change of tubulin organization within the cells was observed after cucurbitacin B treatment. B. Effect of cucurbitacin B on microtubule polymerization in a cell-free system was determined in vitro. Bovine brain tubulin protein (3 mg/mL) was mixed with special reaction buffer as described in materials and method, and incubated with 2.5, 5, 10 μM cucurbitacin B, 10 μM vincristine or 10 μM paclitaxel. The polymerization of tubulin was determined on the basis of the increase in optical density. The O.D. (340 nm) was measured each minute for up to 40 minutes at 37°C using microplate reader. Unlike paclitaxel and vincristine, treatment with cucurbitacin B in cell-free system has no effect on tubulin density.