An antitubulin agent BCFMT inhibits proliferation of cancer cells and induces cell death by inhibiting microtubule dynamics

Abstract

Using cell based screening assay, we identified a novel anti-tubulin agent (Z)-5-((5-(4-bromo-3-chlorophenyl)furan-2-yl)methylene)-2-thioxothiazolidin-4-one (BCFMT) that inhibited proliferation of human cervical carcinoma (HeLa) (IC(50), 7.2 ± 1.8 µM), human breast adenocarcinoma (MCF-7) (IC(50), 10.0 ± 0.5 µM), highly metastatic breast adenocarcinoma (MDA-MB-231) (IC(50), 6.0 ± 1 µM), cisplatin-resistant human ovarian carcinoma (A2780-cis) (IC(50), 5.8 ± 0.3 µM) and multi-drug resistant mouse mammary tumor (EMT6/AR1) (IC(50), 6.5 ± 1 µM) cells. Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 µM), BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably) state by 135% and reduced the dynamicity (dimer exchange per unit time) of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3 ± 1.8 µM, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (K(i)) of 5.2 ± 1.5 µM suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2) at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug resistance cells by suppressing microtubule dynamics and indicated that the compound may have chemotherapeutic potential.

Figure 1. Effects of MNFMT, DHBPT and BCFMT on HeLa and MCF-7 cells proliferation.

(A) Structures of MNFMT, DHBPT and BCFMT. (B & C) MNFMT, DHBPT and BCFMT inhibited the proliferation of HeLa (B) and MCF-7 (C) cells in culture. HeLa and MCF-7 cells were incubated with different concentrations of MNFMT (▪), DHBPT (•) and BCFMT (▴) for one cell cycle. The inhibition of cell proliferation was determined by sulforhodamine B assay. Data were an average of three independent experiments. Bars represent ± SD.

Figure 2. BCFMT inhibited tubulin polymerization in vitro.

(A) Tubulin (10 µM) was polymerized in the absence (▪) and presence of 10 (◊), 25 (▴), 50 (×), 75 (○) and 100 (□) µM BCFMT. (B) Electron micrographs of tubulin polymers in the absence and presence of 25 and 50 µM BCFMT. The scale bar is 2000 nm. (C) BCFMT suppressed the GTPase activity of microtubules. Effects of vinblastine on the GTPase activity of microtubules under similar experimental conditions are shown in the inset. Data were an average of three independent experiments. Bars represent ± SD.

Figure 3. BCFMT bound to purified tubulin and inhibited the binding of BODIPY FL-vinblastine to tubulin.

(A) The effects of BCFMT on the tryptophan fluorescence spectra of tubulin are shown. Spectra were monitored in the absence (♦) and presence of 0.25 (▪), 0.5 (▴), 1 (×), 2 (−), 5 (○), 7 (l) and 10 (•) µM BCFMT. (B) The change in the fluorescence intensity of tubulin (ΔF) was plotted against concentration of BCFMT. The dissociation constant (K_d) for BCFMT binding to tubulin was estimated using an equation described in the methods. Data were the average of four independent experiments. (C) Reduction in the fluorescence intensity of tubulin- BODIPY FL-vinblastine complex in the absence (♦) and presence of 10 (▪), 25 (▴) and 50 (•) µM BCFMT. (D) Tubulin (2 µM) in 25 mM PIPES buffer (pH 6.8) was incubated without and with different concentrations (5, 10, 15, 20, 25 µM) of BCFMT at 25°C for 20 min. Three such different sets were prepared. After 20 min incubation, in one set 2 µM (♦), in the second set 4 µM (▴) and in the third set 6 µM (▪) BODIPY FL-vinblastine was added. Fluorescence of tubulin-BODIPY FL-vinblastine complex was measured and the inhibitory concentration (Ki) was calculated from the modified Dixon plot.

Figure 4. BCFMT depolymerized microtubules of MCF-7 cells.

(A) Cells were treated without and with 40 µM BCFMT for 3 h and microtubules were stained using antibody against α-tubulin (red). (B) BCFMT perturbed interphase microtubule organization of MCF-7 cells. MCF-7 cells were incubated in the absence and presence of different concentrations of BCFMT for 48 h. Cells were fixed and stained using antibody against α-tubulin (red). (C) BCFMT decreased the ratio of polymeric/soluble tubulin in MCF-7 cells determined by western blot. MCF-7 cells were treated without (lane 1) or with 20 µM (lane 4) and 40 µM (lane 5) of BCFMT for 36 h. 20 nM taxol (lane 2) and 200 nM nocodazole (lane 3) were also used under similar experimental conditions. Polymeric and soluble tubulin fractions were isolated and equal amounts of proteins were loaded on SDS-PAGE. Immunoblotting was done with α-tubulin antibody. Experiment was performed independently three times. Shown is the representative blot. (D) BCFMT depolymerized spindle microtubules in MCF-7 cells. DNA stained in blue. Scale bar is 10 µm.

Figure 5. BCFMT-treatment dampened dynamics of individual microtubules and accumulated spindle assembly checkpoint proteins at the kinetochores in MCF-7 cells.

(A & B) Life history traces shows microtubule length changes with time in the absence (A) and presence of 10 µM BCFMT (B). The initial length represents a length from an arbitrary fixed point of a microtubule to the plus end of the microtubule. (C & D) BCFMT treatment accumulated checkpoint proteins Mad2 (green) (C) and BubR1 (red) (D) at the kinetochores. The position of kinetochores was visualized using antibody against Hec1 (red) (C). DNA stained in blue. Scale bar is 10 µm.

Figure 6. BCFMT activated p53 dependent apoptotic pathway in MCF-7 cells.

MCF-7 cells were incubated without and with different concentrations of BCFMT for 48 h. (A) Cells were processed for Annexin V/PI staining. Annexin V stained cells are in green and PI stained cells are in red. (B & C) MCF-7 cells treated without or with BCFMT were fixed and stained with antibody specific for p53 (red) (B) and p21 (red) (C). DNA was stained in blue. Scale bar is 10 µm.