Cytotoxicity of Combretastatin was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, propidium iodide (PI) staining assay and clonogenic survival assay. In vivo microtubule assembly assay, cell cycle analyses, Western blot and cell migration assay were used to study the mechanism of Combretastatin. The effect of intravesical Combretastatin therapy on the development of tumours was studied in the murine orthotopic bladder tumour model.
Bladder cancer is a highly recurrent cancer after intravesical therapy, so new drugs are needed to treat this cancer. Hence, we investigated the anti-cancer activity of Combretastatin an anti-tubulin agent, in human bladder cancer cells and in a murine orthotopic bladder tumour model.
Combretastatin inhibited microtubule polymerization in vivo. Cytotoxic IC(50) values of Combretastatin in human bladder cancer cells were below 4 nM. Analyses of cell-cycle distribution showed Combretastatin obviously induced G(2)-M phase arrest with sub-G(1) formation. The analyses of apoptosis showed that CA-4 induced caspase-3 activation and decreased BubR1 and Bub3 in cancer cells. In addition to apoptosis, Combretastatin was also found to induce the formation of multinucleated cells. CA-4 had a significantly reduced cell migration in vitro. Importantly, the in vivo study revealed that intravesical CA-4 therapy retarded the development of murine bladder tumours.
