Anticancer activity of a novel small molecule tubulin inhibitor STK899704
Fig 3
STK899704 inhibited tubulin polymerization and mitotic spindle organization.
(A) Tubulin polymerization assay. The effect of STK899704 (5 μM) on polymerization of purified tubulin in vitro was examined in a GTP-containing buffer. DMSO was used as a negative control. Tubulin-targeting agents Taxol (5 μM) and vinblastine (5 μM) were also used as controls for tubulin-stabilizing and tubulin-destabilizing agents, respectively. Assembly of tubulin into microtubules was determined by the degree of turbidity at 340 nm. (B) Immunofluorescence staining of microtubules in HeLa cells. Cells were treated with DMSO, Taxol (100 nM), nocodazole (200 ng/ml), or indicated concentrations of STK899704 for 17 h. Cells were then fixed and stained with Alexa Fluor 488-conjugated anti-tubulin antibody and Hoechst 33342 to visualize α-tubulin and DNA, respectively. Scale bar, 10 μm. (C) Fraction of mitotic cells. At least 100 cells from (B) were counted from the different regions. Percentages of normal metaphase, misaligned, multipolar, and tubulin aggregate phenotypes were shown. (D) Proposed binding model of STK899704 on tubulin. The αβ-tubulin heterodimer from PDB entry 1SA0 is shown as ribbon (gray, α-tubulin; cyan, β-tubulin). STK899704 is presented in red stick while colchicine is shown in green. (E) Effect of STK899704 on tubulin binding. Tubulin binding was tested with a SPA-based competition assay. Error bars represent mean ± SDs from three independent experiments.