Carbendazim inhibits cancer cell proliferation by suppressing microtubule dynamics

Abstract

Carbendazim (methyl 2-benzimidazolecarbamate) is widely used as a systemic fungicide in human food production and appears to act on fungal tubulin. However, it also inhibits proliferation of human cancer cells, including drug- and multidrug-resistant and p53-deficient cell lines. Because of its promising preclinical anti-tumor activity, it has undergone phase I clinical trials and is under further clinical development. Although it weakly inhibits polymerization of brain microtubules and induces G(2)/M arrest in tumor cells, its mechanism of action in human cells has not been fully elucidated. We examined its mechanism of action in MCF7 human breast cancer cells and found that it inhibits proliferation (IC(50), 10 microM) and half-maximally arrests mitosis at a similar concentration (8 microM), in concert with suppression of microtubule dynamic instability without appreciable microtubule depolymerization. It induces mitotic spindle abnormalities and reduces the metaphase intercentromere distance of sister chromatids, indicating reduction of tension on kinetochores, thus leading to metaphase arrest. With microtubules assembled in vitro from pure tubulin, carbendazim also suppresses dynamic instability, reducing the dynamicity by 50% at 10 microM, with only minimal (21%) reduction of polymer mass. Carbendazim binds to mammalian tubulin (K(d), 42.8 +/- 4.0 microM). Unlike some benzimidazoles that bind to the colchicine site in tubulin, carbendazim neither competes with colchicine nor competes with vinblastine for binding to brain tubulin. Thus, carbendazim binds to an as yet unidentified site in tubulin and inhibits tumor cell proliferation by suppressing the growing and shortening phases of microtubule dynamic instability, thus inducing mitotic arrest.

Fig. 1.

Inhibition of MCF7/GFP cell proliferation (•) and induction of mitotic index (bars) by carbendazim. Cells were seeded in six-well plates for 24 h, followed by incubation with 0.5 to 50 μM carbendazim for 48 h. Live cells were counted by hemacytometer after staining with trypan blue for estimating cell proliferation. Mitotic cells were determined after fixation and staining with DAPI and phosphohistone H3 antibody. Results are mean and S.E.M. of three experiments. Inset, structure of carbendazim.

Fig. 2.

Effects of carbendazim on microtubule spindle organization in MCF7/GFP cells. Cells were fixed and stained with anti-α-tubulin to stain microtubules and DAPI to stain chromosomes and chromatin (Materials and Methods). Control interphase cell with normal dense network of microtubules (A), control mitotic cell with well defined compact metaphase plate (B), 5 μM carbendazim induced asymmetric mitosis (C) and uncongressed chromosomes in mitosis (D), 15 μM carbendazim induced depolymerization of microtubules in interphase cells (E) and tripolar spindles in mitotic cells (F), 30 μM carbendazim depolymerized many microtubules in interphase cells (G) and induced multipolar spindles (H), increased depolymerization of microtubule network at 50 μM carbendazim in interphase cells (I), and highly multipolar spindle (J).

Fig. 3.

Concentration-dependent suppression of dynamic instability parameters in living MCF7/GFP cells by carbendazim. Effect of increasing concentrations of carbendazim on the growth rate (⋄), growth length (□), shortening rate (▵), shortening length (▾), and dynamicity (•). Data are from Table 1.

Fig. 4.

Concentration-dependent suppression of in vitro dynamic instability of microtubule plus ends by carbendazim. Life history traces of length changes of individual control microtubules (A) and microtubules assembled in the presence of 30 μM carbendazim (B) show carbendazim's suppressive effect on dynamic instability. C, effect of increasing concentrations of carbendazim on the growth rate (⋄), growth length (□), shortening rate (▵), and dynamicity (•) of reassembled bovine brain microtubules in vitro. Data are from Table 2.

Fig. 5.

Determination of binding affinity and binding site for carbendazim in tubulin. A, effects of carbendazim on tubulin-bound ANS fluorescence. Tubulin (2 μM) was incubated with carbendazim for 30 min, followed by incubation with ANS (40 μM) for 15 min (Materials and Methods). Inset, double-reciprocal plot of binding of carbendazim to tubulin. X and L_f represent the fraction of the binding sites and the free carbendazim concentration, respectively. The dissociation constant (K_d) was determined using the relationship, 1/X = 1 + K_d/L_f. Results are expressed as mean and S.E.M. for four experiments. B, inhibition of [³H]colchicine binding by carbendazim. Tubulin (9 μM) was incubated with [³H]colchicine (20 μM) and increasing concentrations of carbendazim simultaneously for 16 h (•) or preincubated with a range of concentrations of carbendazim for 2 h followed by incubation with 5 μM colchicine for 1 h (□). The tubulin-colchicine complex was separated from free colchicine by gel filtration, and radioactivity of bound colchicine was determined. Data for preincubation experiment are mean and S.E.M. of two independent experiments; S.E.M. values are smaller than the dimensions of the symbols. C, inhibition of [³H]vinblastine binding by carbendazim. Tubulin (5 μM) was incubated with increasing concentrations of carbendazim for 30 min, followed by incubation with [³H]vinblastine (5 μM) for 30 min. The unbound vinblastine was separated by centrifugal filtration, and radioactivity was determined. The concentration of bound vinblastine was calculated from the difference between total and unbound vinblastine. Results are expressed as mean and S.E.M. for two experiments.