Figure 1.
Effects of erucin on MCF7 cell proliferation, mitotic index, cell cycle progression and apoptosis.
(A) Erucin (inset) inhibits proliferation along with mitotic arrest. Cells were incubated with a range of erucin concentrations for 72 hours and SRB cell proliferation assays were performed to assess cell proliferation (IC50 = 28 µM, -•-). To determine the mitotic index (IC50 = 13 µM, -▪-), cells were incubated with erucin for 24 hours, fixed, and stained with DAPI to visualize DNA (Materials and Methods). Data are the mean of three to four independent experiments; bars, ± SEM. (B) Erucin arrests cells at G2/M. Non-synchronized cells were treated with a range of erucin concentrations for 24 hours and analyzed by flow cytometry (Materials and Methods). White bars represent G1 phase, gray bars, S phase, and black bars, G2/M phase of the cell cycle. (C) Erucin induces time-, and concentration-dependent apoptosis. Cells were incubated with a range of erucin concentrations for 24 hours (-○-) and 48 hours (-□-) and the total number of apoptotic cells (early and late apoptotic) for each condition was determined by flow cytometry (Materials and Methods). Results are the mean ± SEM of at least three independent experiments performed in duplicate.
Figure 2.
Concentration-dependence for the effects of erucin on cellular microtubule morphology, spindle morphology and microtubule acetylation in MCF7 cells.
(A) Interphase control cells, (D) 15 µM, (G) 30 µM, (J) 50 µM, and (K) 25 µM erucin (a multinucleated cell). Control cells and cells incubated with 15 µM erucin show intact microtubule networks with similar overall morphologies. At 30 µM erucin, microtubules were somewhat shorter, reduced in number, and very curvy. At 50 µM erucin, the majority of microtubules were depolymerized with the remaining microtubules clustered around nucleus. Erucin also induced giant multinucleated cells (K, 25 µM erucin) at low and especially at high concentrations (data not shown). Metaphase spindles in control cells (B) were bipolar with all chromosomes congressed to the metaphase plate. The majority of cells arrested in mitosis by erucin displayed some abnormalities in metaphase: poorly defined bipolar spindles with uncongressed chromosomes ((E) 15 µM erucin, arrowhead) and an increased number of astral microtubules ((E) 15 µM erucin, arrows), or monopolar spindles that contained many uncongressed chromosomes ((H) 25 µM erucin, arrowhead). A, B, D, E, G, H, J, and K: chromosomes are blue, microtubules are green. C, F, I, L: acetylated microtubules in the presence of 0–100 µM erucin. Erucin promotes acetylation of microtubules in MCF7 cells in a concentration-dependent manner. (C) In control cells the majority of microtubules are dynamic (red color) with a very few acetylated microtubules (green microtubules). (F) At 15 µM erucin, there were more stabilized (acetylated) microtubules compared with those in controls. (I) At 30 µM erucin (approximately 2×mitotic IC50), there were fewer microtubules, and the majority of remaining microtubules were curved and acetylated (green). (L) In the presence of 50 µM erucin the majority of microtubules were depolymerized and strongly acetylated. Overall, the dynamics of microtubules was significantly stabilized by erucin, reducing the microtubule turnover. C, F, I, L: chromosomes are blue, microtubules are red, acetylated microtubules are green. Scale bar is 10 µm.
Figure 3.
Dynamic instability of microtubules in MCF7 cells incubated with erucin.
Time-lapse sequences of fluorescent microtubules at their plus ends: the positions of microtubule plus ends were tracked over time to generate life history plots in the absence (A) or presence (B) of 15 µM erucin, from which the dynamic instability parameters were calculated. Most microtubules in control cells grew (A, arrows) and shortened (A, arrowheads) within the course of the 2-min observation period. The dynamics were significantly suppressed by 15 µM erucin (B). Bar = 5 µm. (C) Concentration-dependent suppression of microtubule dynamics parameters in MCF7 cells by erucin: the growth rate (♦), growth length (◊), shortening rate (▪), shortening length (□), and dynamicity (×). Data are from Table 1.
Table 1.
Effects of Erucin on Dynamic Instability of Microtubules in MCF7-EGFP-α-Tubulin Breast Cancer Cells.
Figure 4.
Erucin inhibits tubulin polymerization in vitro.
(A) Polymerization of purified tubulin (2.75 mg/mL), pre-incubated with or without the drug for 35 minutes, was carried out at 35°C by initiation with nucleating microtubule seeds (Materials and Methods) in the absence (□) control, or presence of (◊) 15, (○) 25, (•) 50 µM, and (Δ) 100 µM erucin for 60 minutes. (B) Total microtubule polymer mass was determined by centrifugation 60 minutes after the initiation of assembly. Bars are ± SEM. Values with *are significantly different from control at ≥95% confidence interval by Student’s t-test.
Figure 5.
Concentration-dependent suppression of dynamic instability at microtubule plus-ends in vitro by erucin.
Microtubule ends were tracked over time to produce life history plots of individual microtubules assembled in vitro from purified bovine brain tubulin. Life history plots are the length changes of individual microtubules in controls (A), and in the presence of 15 µM erucin (B). (C) Concentration-dependent suppression of microtubule dynamics parameters in vitro by erucin: the growth rate (♦), growth length (◊), shortening rate (▪), shortening length (□), and dynamicity (×). Data are from Table 2.
Table 2.
Effects of Erucin on Dynamic Instability of Steady-State Purified Microtubules in Vitro.