Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine

Abstract

Background: Griseofulvin, an antifungal drug, has recently been shown to inhibit proliferation of various types of cancer cells and to inhibit tumor growth in athymic mice. Due to its low toxicity, griseofulvin has drawn considerable attention for its potential use in cancer chemotherapy. This work aims to understand how griseofulvin suppresses microtubule dynamics in living cells and sought to elucidate the antimitotic and antiproliferative action of the drug.

Methods: The effects of griseofulvin on the dynamics of individual microtubules in live MCF-7 cells were measured by confocal microscopy. Immunofluorescence microscopy, western blotting and flow cytometry were used to analyze the effects of griseofulvin on spindle microtubule organization, cell cycle progression and apoptosis. Further, interactions of purified tubulin with griseofulvin were studied in vitro by spectrophotometry and spectrofluorimetry. Docking analysis was performed using autodock4 and LigandFit module of Discovery Studio 2.1.

Results: Griseofulvin strongly suppressed the dynamic instability of individual microtubules in live MCF-7 cells by reducing the rate and extent of the growing and shortening phases. At or near half-maximal proliferation inhibitory concentration, griseofulvin dampened the dynamicity of microtubules in MCF-7 cells without significantly disrupting the microtubule network. Griseofulvin-induced mitotic arrest was associated with several mitotic abnormalities like misaligned chromosomes, multipolar spindles, misegregated chromosomes resulting in cells containing fragmented nuclei. These fragmented nuclei were found to contain increased concentration of p53. Using both computational and experimental approaches, we provided evidence suggesting that griseofulvin binds to tubulin in two different sites; one site overlaps with the paclitaxel binding site while the second site is located at the alphabeta intra-dimer interface. In combination studies, griseofulvin and vinblastine were found to exert synergistic effects against MCF-7 cell proliferation.

Conclusions: The study provided evidence suggesting that griseofulvin shares its binding site in tubulin with paclitaxel and kinetically suppresses microtubule dynamics in a similar manner. The results revealed the antimitotic mechanism of action of griseofulvin and provided evidence suggesting that griseofulvin alone and/or in combination with vinblastine may have promising role in breast cancer chemotherapy.

Figure 1

GF inhibited MCF-7 cell proliferation, arrested cell cycle progression at G2/M phase, increased the accumulation of Mad2 at the kinetochores and induced apoptosis. (A) The effect of GF on cell proliferation (closed circle) was determined by counting the cells after 48 h of incubation. The mitotic index (closed triangle) was calculated by Hoechst staining method after incubating the cells without or with GF for 24 h. Each experiment was performed four times. Data represent mean ± SD. (B) Flow cytometric analysis showing the effects of GF on the MCF-7 cell cycle (C) Effect of GF on Mad2 (green) localization. Staining was also done for a kinetochore protein Hec1 (red) (D) GF induced apoptosis in MCF-7 cells. Bars equal to 10 μm

Figure 2

GF treatment increased the nuclear accumulation of p53 and p21. MCF-7 cells were incubated with different concentrations of GF (15-90 μM) for 48 hours, fixed and processed to visualize either (A) p53 (red) and DNA (blue) or (B) p21 (red) and DNA (blue). Bar equals to 20 μm.

Figure 3

Effects of GF on the microtubules of MCF-7 cells. MCF-7 cells were incubated without or with different concentrations of GF for 48 h. (A) Microtubules (red) and chromosomes (blue) of interphase cells or (B) Microtubules (red) of metaphase cells are shown. Arrows point towards the poles. Bar equals to 20 and 10 μm for an interphase and a metaphase cell, respectively. (C) Effects of GF on the polymer mass of tubulin in MCF-7 cells. (D) Band intensities of the polymeric and soluble tubulin fractions in MCF-7 cells treated with different GF concentrations relative to the vehicle-treated cells are shown. The experiment was performed three times. Data represent mean ± SD.

Figure 4

Life history traces of individual microtubules in the absence or presence of 5 and 15 μM of GF are shown.

Figure 5

Mode of interaction of GF with tubulin. (A) Structure of GF. The conformation of the O-Me groups are chosen arbitrarily. Color code: carbon, blue; oxygen, red; hydrogen, grey; chlorine, green. (B) Cartoon rendering of the αβ tubulin heterodimer (PDB id 1TVK) showing the location of the binding sites for colchicine, paclitaxel and vinblastine (purple), GTP and GDP (black) and GF (blue). The "upper" domain is α tubulin and the "lower" domain is β tubulin. In this view, GTP and GDP are partly hidden. Vinblastine binds at the inter-dimer (αβ)-interface and hence, is shown both at the top and bottom. Colchicine binds at the intra-dimer interface (αβ) and paclitaxel binds to the β-subunit. The binding sites for GF, one at the interface (site A) and the other overlapping with that of paclitaxel (site B), are predicted by docking; all others are based on X-ray crystallographic studies. (C) and (D) Representative binding modes of GF in site A (C) and site B (D) obtained by docking. Atoms are colored by element type except for carbon (cyan for GF and grey for binding site residues). Hydrophobic residues constituting the binding pocket and polar residues within 3 Å of GF are shown.

Figure 6

Combination of GF and vinblastine exerted synergistic effects in inhibiting MCF-7 cell proliferation. The median effect plots for the inhibition of cell proliferation in the presence of GF (A) and vinblastine (B) are shown. (C) The effects of the combination of GF and vinblastine (Vinb) on MCF-7 cell proliferation after 48 h treatment are shown as combination index. Data are average of four experiments and error bars represent SD.