Disruption of tubulin polymerization and cell proliferation by 1-naphthylarsonic acid

2.1. Chemicals

1-NAA was prepared in the Laboratory of Macromolecules, Institute of Biochemistry and Biophysics, University of Tehran. Human cervical carcinoma cells (HeLa, NCBI C115) and human fetal foreskin fibroblasts (HF2; NCBI C190) were obtained by National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran. Pipes and EDTA were purchased from Merck. GTP, ATP, EGTA, magnesium sulfate, monoclonal anti-α-tubulin antibody, anti-mouse IgG FITC, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], Trypan Blue, trypsin, PBS, Triton X-100, formaldehyde, BSA, DAPI (4′,6-diamidino-2-phenylindole), PBST, uranyl acetate were purchased from Sigma. Phosphocellulose p11 was purchased from Whatman. Formvar carbon-coated copper grids (Pelco), PI (propidium iodide), RPMI 1640 (Gibco) and FBS (fetal bovine serum; Seromed) were also used.

2.2. Equipment

Turbidometry was conducted by UV-Vis spectrophotometer (Varian Cary Bio100). Electron micrographs were taken with a transmission electron microscope (Model 902A, Zeiss). Cell micrographs were prepared by inverted and fluorescence microscopy (Zeiss). MTT colorimetric assay was measured with an ELISA reader (Stat Fax, model 2100, Awareness Technology Inc.). Microtubules were observed after staining by fluorescence microscopy (Axioskop 40, Zeiss).

2.3. Purification of microtubule protein from sheep brain

Sheep brain microtubule protein was purified by a 2 cycle assembly/disassembly process using PEM buffer (containing 100 mM Pipes, pH 6.9, 2 mM MgSO₄ and 1 mM EGTA) and 0.2 mM GTP. The cold homogenized centrifuge pellet was frozen with liquid nitrogen and stored at −70°C. Tubulin concentration was ∼2.5 mg/ml (Mahinpour et al., 2009).

2.4. Tubulin assembly

Tubulin concentration was determined by Bradford method at 595 nm (Ashford et al., 1998) and 280 nm (Marshall and Williams, 1993) with a UV-Vis spectrophotometer. Tubules were assembled in PEM buffer (pH 6.9) and PMG buffers (containing 80 mM Pipes, pH 6.9, 1 mM EGTA, 2 mM MgSO₄ and 3.4 M glycerin). The assembly was initiated by adding GTP at appropriate concentrations at 37°C, and measured by the turbidity at 350 nm after 30 min. A range of concentrations of 1-NAA (0.5–2 mM) was tested on tubulin polymerization at a constant concentration of GTP at 37°C for 10 min. Colchicine was used as a positive control.

To examine whether 1-NAA affected polymer interaction or the activity of MAPs, polymerization was allowed to progress up to the maximum of the elongation phase and small amounts of 1-NAA were added to the solution without a change in the temperature or the concentration of the tubulin solution.

2.5. Electron microscopy

Samples for transmission electron microscopy were prepared by negative staining using uranyl acetate on formvar carbon-coated copper grid, and examined in a TEM (Hitachi Hu-12A) operating at 75 kV (Mahinpour et al., 2009).

2.6. Docking

The 3D tubulin structure was chosen for computing by HEX 4.1 protein docking software (Ritchie, 2003). Blind docking was performed with Tau protein as the first ligand. The whole experiment was performed on the shape and electrostatic relationship. Docking results were generated by HEX, indicating the lowest energy as the best complex of tubulin-ligand. Post-processing refinement was a Newton-like minimization based on an OPLS force-field parameter. The results were saved as a pdb file. As a second run of docking process, 1-NAA was used as a ligand and the same procedure was performed.

2.7. Cell proliferation assay

Proliferation of HeLa and fibroblast cells was measured using the MTT assay. Briefly, the cells were plated into a 96-well microtitre plate (Griener) at 2 × 10⁴ cells/well in RPMI 1640 medium with 10% FBS. The cells were incubated in 1-NAA (0.7–5 mM) at 37°C for 24, 48 and 72 h in a humidified air atmosphere with 5% CO₂. The medium over the cells was removed, 100 μl of MTT (5 mg/ml in PBS) was added to each well, which was incubated for 3 h at 37°C in air with 5% CO₂. The attenuance (D) was recorded on an ELISA reader (Stat Fax, 2100).

2.8. Immunocytochemical assay

Cells were fixed with formaldehyde for 15 min and washed with PBS solution. They were incubated for 10 min in PBS containing 0.25% Triton X-100 and washed for 5 min. Cells were blocked with 1% BSA in PBST for 30 min and incubated in the diluted anti-α-tubulin in PBS (1:100) (Ling et al., 2002) at room temperature for 1 h. After washing thrice with PBS solution, the cells were incubated with FITC-conjugated secondary antibody (1:500) (Ling et al., 2002) in dark room for 1 h. The cells were stained with DAPI in PBS (1 μg/ml) and PI in medium (1 μg/ml). Microtubules were observed by fluorescence microscopy.

2.9. Flow cytometry

To follow apoptosis due to exposure to 1-NAA, an APO-BRDU™ kit with dual colour flow cytometry was used after 24 and 48 h incubation of HeLa cells in the presence of 1-NAA. Positive control cells were incubated with 40 μg/ml etoposide, an anti-tumour drug.

3.1. Tubulin assembly

Administration of 1-NAA (0.5–2 mM) caused a decrease in tubulin polymerization, but an increase in nucleation time at 37°C in the presence of 1 mM GTP (Figure 1). As illustrated in plots 5 and 6, two concentrations of colchicine completely inhibited the polymerization.

Based on the results showed in Figure 1, we investigated in which step of polymerization 1-NAA interacts with microtubule protein. Therefore, 1-NAA was added to the sample 280 s after the initiation of tubulin polymerization under normal conditions (Figure 2, plot 2). Attenuance dropped to a similar level as the microtubule sample incubated with 1-NAA at the inception (Figure 2, plot 1). Electron micrographs of the tubulin polymers produced upon interaction of 1-NAA were in short oligomer lengths, but apparently linked head to tail, forming an apparent long or polymer-like molecule (Figures 3A and 3B).

3.2. Docking

In exploring changes in the microtubule length due to the interaction of 1-NAA, docking was applied by the HEX4 software. 1-NAA binds to the binding site of tau protein on the α-subunit (Figure 4). Deformation in the secondary structure and conformational changes occurred in the α-tubulin subunit, which affected tubulin polymerization.

3.3. Cell proliferation assay

The effect of 0.7–5 mM 1-NAA on proliferation of HeLa cells is shown in Figure 5(A). An increase in toxicity (up to 84%) occurred with increasing concentrations of 1-NAA after 24, 48 and 72 h, although the difference in toxicity at 0.7–5 mM at 48 and 72 h was not significantly different. 1-NAA increased cell death, with cytotoxicity affecting up to 96% of HF2 cells (Figure 5B). Inhibiting tubulin polymerization with 1-NAA results in abnormal microtubules in the human fibroblasts (Kawata et al., 2001).

3.4. Immunocytochemical studies

HeLa cell microtubules labelled with anti-α-tubulin antibody in the presence of 1-NAA (LC50 for 24 h=4.4 mM) showed that microtubule-1-NAA were localized in the central area of the cells (Figure 6a₁), with the cytoskeletal microtubules apparently being pushed towards the cell membrane (Figure 6b₁). Most of the cells were affected (Figures 6a₂ and 6b₂).

3.5. Flow cytometry

The effect of 1-NAA on the HeLa cell apoptosis followed by flowcytometry showed no significant change (Figures 7A–7F) at 24 h but >2-fold increase after 48 h treatment compared with that of 24 h (2.27±1.19 compared with 1.13±0.33). Apoptosis in negative controls was <1% at 24 and 48 h (Figures 7A and 7B). HeLa cells showed evidence of some necrosis after PI staining (Figure 8).