Antiproliferative Activity of Crocin Involves Targeting of Microtubules in Breast Cancer Cells

Abstract

Crocin, a component of saffron spice, is known to have an anticancer activity. However, the targets of crocin are not known. In this study, crocin was found to inhibit the proliferation of HCC70, HCC1806, HeLa and CCD1059sk cells by targeting microtubules. Crocin depolymerized both the interphase and mitotic microtubules of different cancer cells, inhibited mitosis and induced multipolar spindle formation in these cells. In vitro, crocin inhibited the assembly of pure tubulin as well as the assembly of microtubule-associated protein rich tubulin. Electron microscopic analysis showed that crocin inhibited microtubule assembly while it induced aggregation of tubulin at higher concentrations. Crocin co-eluted with tubulin suggesting that it binds to tubulin. Vinblastine inhibited the binding of crocin to tubulin while podophyllotoxin did not inhibit the crocin binding indicating that crocin binds at the vinblastine site on tubulin. The results suggested that crocin inhibited cell proliferation mainly by disrupting the microtubule network.

Figure 1

Crocin inhibited the proliferation of (a) cancer and (b) normal fibroblasts cells. Cancer cell types HeLa (○), HCC1806 (□), HCC70 (Δ) and normal fibroblasts CCD1059sk (●) were incubated with different concentrations of crocin for 24 h. The cell proliferation was determined by counting dead and live cells with trypan blue. Three sets of experiments were performed and the error bars indicate the standard deviation. (c) The cell cycle analysis of HeLa cells treated without and with 1 and 2 μM crocin for 24 h. DNA was stained with PI and analyzed by FACS. The experiment was performed twice.

Figure 2. Crocin induced mitotic block and produced multipolar spindles in cancer cells.

(a) HCC1806 (■), HCC70 (♦) HeLa (Δ) and normal fibroblast CCD1059sk (○) were treated with increasing concentration of crocin and the percentage of cells in mitosis were determined. In each case 300 cells were counted. Error bars indicate the SD. (b) Crocin increased the number of multipolar mitotic cells in cancer cell lines. The percentage of normal (grey bars) and multipolar (black bars) mitotic cells are shown when treated with different concentrations of crocin.

Figure 3. Effects of crocin on the interphase microtubules.

The cancer cells and normal fibroblast cells were incubated in the absence (control) or the presence (treated) of 1 μM crocin for 24 h. Microtubules are shown in red and the nuclear stain is shown in blue.

Figure 4. Crocin treatment induced multipolar spindle formation and caused chromosomal misalignment in the mitotic cells.

(a) Mitotic spindles of HeLa, MCF-7, HCC70, HCC1806 cells in the absence (control) and presence of 1.0 μM crocin for 24 h are shown. Microtubules are shown in green and DAPI stained nuclei or chromosomes are shown in red. (b) HeLa cells were treated without and with crocin (1 μM) for 24 h and immunostained with γ-tubulin IgG and Hoechst (DNA). The scale bar indicates a length of 10 μm.

Figure 5. Crocin inhibited the assembly of tubulin in vitro.

Tubulin (10 μM) was incubated in the absence (■) and presence of different concentrations (10 (●), 20 (▲), 30 (▼), 40 (♦) and 50 (□) μM of crocin for 5 min on ice in PEM buffer containing either 1 M monosodium glutamate (a) or 10% DMSO (b). Further, GTP (1 mM) was added to the reaction mixtures. The assembly of microtubules was monitored by light scattering at 350 nm. (c) MAP-rich (2 mg/mL) tubulin was incubated in the absence (■) and presence of different concentrations (10 (●), 20 (▲), 30 (▼), 40 (♦) and 50 (□) μM) of crocin for 10 min on ice. Subsequently, 1 mM GTP was added to the reaction mixtures and the assembly of MAP-rich tubulin was monitored at 37 °C. The experiment was performed three times. (d) Tubulin (10 μM) was polymerized in the absence and presence of 20 and 50 μM crocin for 10 min with 1 M monosodium glutamate and 1 mM GTP. Electron micrographs of polymers in the absence and presence of crocin are shown. The scale bars are shown in the figures.

Figure 6. Crocin bound to purified tubulin in vitro.

The elution profiles of free 20 μM tubulin (■) and 120 μM crocin (Δ) are shown. Tubulin (20 μM) was incubated with 120 μM crocin for 30 min at 25 °C and then, eluted through the same column. The elution of tubulin (●) and crocin (○) are shown from combination of tubulin-crocin. The experiment was performed three times.

Figure 7. Effects of vinblastine and podophyllotoxin on the binding of crocin to tubulin.

(a) Tubulin (10 μM) was first incubated without or with different concentrations of vinblastine and then, 10 μM crocin was added to the reaction mixtures. The absorption spectra of crocin (10 μM) in the absence (■) and presence of tubulin (●) are shown. The absorption spectra of tubulin-crocin complex in the presence of increasing concentrations of vinblastine 3 (▲), 5 (▼), 10 (◄) and 15 (►) μM are also shown. The experiment was done 3 times. (b) The change in the tryptophan fluorescence intensity was plotted against different concentrations of vinblastine in the absence (●) and presence of 25 μM crocin (■). The data were fitted in a binding equation as mentioned in the method section. The experiment was done 4 times. (c) Tubulin (10 μM) was first incubated without or with 15 μM podophyllotoxin and then, 10 μM crocin was added to the reaction mixtures. The absorption spectra of free crocin (10 μM) (■), crocin-tubulin (●) complex without podophyllotoxin and crocin-tubulin complex (▲) in the presence of 15 μM podophyllotoxin are shown. The experiment was performed 3 times.

Figure 8. A model showing the effect of crocin on tubulin polymerization.

Crocin can bind to a tubulin dimer. Alternatively, a long and flexible crocin molecule may bind to two tubulin dimers as shown. Crocin can inhibit microtubule polymerization either (a) by inducing the formation of tubulin oligomers and aggregates or (b) by producing defective microtubules. Crocin can inhibit the addition of tubulin dimers at the growing end of a microtubule by causing steric hindrance. In addition, the presence of several crocin molecules into a microtubule could produce significant distortion in the microtubule lattice leading to its disassembly.