Tubulin Polymerization Assay

In order to determine the direct action of the test compounds on the microtubular system, an in vitro tubulin polymerization assay was performed using a commercially available kit (Cytoskeleton Inc., Denver, CO, United States) in accordance with the provider’s instructions. The assay reactions were performed on a pre-warmed (37°C), UV-transparent 96-well microplate. Ten microliters of the test solutions were placed on the wells supplemented with 2 mM MgCl₂, 0.5 mM ethylene glycol tetraacetic acid (EGTA), 1 mM guanosine triphosphate (GTP) and 10.2% glycerol. Ten microliters of general tubulin buffer was used as untreated control, and paclitaxel (PAC) served as the reference compound. The polymerization reaction was initiated by adding 100 μL of 3.0 mg/mL tubulin in 80 mM PIPES, pH 6.9, to each sample. Absorbance of the samples was measured per minute, at 340 nm, using a 60-min kinetic measurement protocol. Each sample was prepared in two parallels. To characterize the process, polymerization curves were fitted to the measured data. The highest difference between the absorbances measured at two consecutive time points was regarded as V_max (Δabsorbance/min) for the tested compound. A clinically applied reference agent, PAC was used at a relatively high concentration (10 μM) as recommended by the manufacturer. This concentration is approximately 1,000-fold higher than the IC₅₀ value of PAC on HeLa cells (Jordan et al., 1996). Since similarly high concentrations of the tested compounds were not possible to be applied because of the limited solubility of the substances in the recommended buffer, we used the highest concentrations reflecting the differences in the efficacies of the tested compounds.