Cucurbitacin B inhibits human breast cancer cell proliferation through disruption of microtubule polymerization and nucleophosmin/B23 translocation

Abstract

Background: Cucurbitacin B, an oxygenated tetracyclic triterpenoid compound extracted from the Thai medicinal plant Trichosanthes cucumerina L., has been reported to have several biological activities including anti-inflammatory, antimicrobial and anticancer. Cucurbitacin B is great of interest because of its biological activity. This agent inhibits growth of various types of human cancer cells lines.

Methods: In this study, we explored the novel molecular response of cucurbitacin B in human breast cancer cells, MCF-7 and MDA-MB-231. The growth inhibitory effect of cucurbitacin B on breast cancer cells was assessed by MTT assay. The effects of cucurbitacin B on microtubules morphological structure and tubulin polymerization were analyzed using immunofluorescence technique and tubulin polymerization assay kit, respectively. Proteomic analysis was used to identify the target-specific proteins that involved in cucurbitacin B treatment. Some of the differentially expressed genes and protein products were validated by real-time RT-PCR and western blot analysis. Cell cycle distributions and apoptosis were investigated using flow cytometry.

Results: Cucurbitacin B exhibited strong antiproliferative effects against breast cancer cells in a dose-dependent manner. We show that cucurbitacin B prominently alters the cytoskeletal network of breast cancer cells, inducing rapid morphologic changes and improper polymerization of the microtubule network. Moreover, the results of 2D-PAGE, real-time RT-PCR, and western blot analysis revealed that the expression of nucleophosmin/B23 and c-Myc decreased markedly after cucurbitacin B treatment. Immunofluorescence microscopy showed that cucurbitacin B induced translocation of nucleophosmin/B23 from the nucleolus to nucleoplasm. Treatment with cucurbitacin B resulted in cell cycle arrest at G2/M phase and the enhancement of apoptosis.

Conclusions: Our findings suggest that cucurbitacin B may inhibit the proliferation of human breast cancer cells through disruption of the microtubule network and down-regulation of c-Myc and nucleophosmin/B23 as well as the perturbation in nucleophosmin/B23 trafficking from the nucleolus to nucleoplasm, resulting in G2/M arrest.

Figure 1

Chemical structure of cucurbitacin B (25-acetoxy-2β,16α,20β-trihydroxy-9β-methyl-19nor-10α-lanosta-5,23-diene-3,11,22-trione).

Figure 2

Cucurbitacin B inhibits growth of human breast cancer cells. MCF-7 and MDA-MB-231 were treated with cucurbitacin B at a final concentration, ranging from 0 to 100 μM for 48 hr. Growth inhibition was determined by the MTT assay. The percentage of cell survival was calculated by defining the absorption of cells without cucurbitacin B treatment as 100%. Results are the average of three independent experiments *, p<0.05 (treated vs untreated control).

Figure 3

Effect of cucurbitacin B on cell-cycle distribution. MCF-7 and MDA-MB-231 were treated with 0, 2.5, and 5 μM cucurbitacin B for 24 hr, and then stained with propidium iodide (PI) before subjected to flow cytometric analysis. A. The cell cycle/DNA content histograms represent the cell population at each cell cycle phase as determined by the level of DNA content in each cucurbitacin B treatmented group. Blockage at G₂/M and apoptotic induction was observed. B. The values indicate percentage of cells in each phases of the cell cycle. *, p<0.05 (treated vs untreated control).

Figure 4

Apoptotic induction by cucurbitacin B. MCF-7 and MDA-MB-231 were incubated with cucurbitacin B for 24 hr and apoptosis was analyzed by Annexin V-FITC. A. Annexin V-FITC staining is represented on the X-axis and PI staining is represented on the Y-axis. B. The values indicate the percentage of apoptotic cells in each concentration. Results shown are the average of three independent experiments. *, p<0.05 (treated vs untreated control).

Figure 5

Effect of cucurbitacin B on the level of nucleophosmin/B23, STAT3, tubulin, and c-Myc expression. MCF-7 and MDA-MB-231 were treated with 0, 2.5, and 5 μM cucurbitacin B for 48 hr. A. RNA was extracted for real-time RT-PCR to quantitate the gene expression levels of nucleophosmin/B23, STAT3, tubulin, and c-Myc after cucurbitacin B treatment. Results shown are the average of three independent experiments. *, P < 0.05 (treated vs untreated control) B. Western blot was analyzed for protein expression. GAPDH was used as loading control.

Figure 6

Cucurbitacin B changes nucleophosmin/B23 localization in human breast cancer cells. MCF-7 and MDA-MB-231 cells were stained with anti-nucleophosmin/B23 primary antibody. Alexa 568-conjugated anti-mouse IgG rabbit antibody was used as secondary antibody. DAPI was used for nuclear localization. Stained cells were analyzed by using confocal microscope. After exposure to 2.5 μM cucurbitacin B for 20 min. Translocation of nucleophosmin/B23 from nucleolus (red) to nucleoplasm (green) was observed.

Figure 7

Morphological changes by cucurbitacin B treatment of breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with 2.5 μM cucurbitacin B for 24 hr, and the cells were visualized under phase-contrast microscopy to investigate the morphological alteration. Shrinkage and rounding of the cell shape were observed in both cell types.

Figure 8

Cucurbitacin B inhibits microtubule polymerization. A. Effect of cucurbitacin B on the organizations of cellular microtubule network. Immunofluorescent staining for α-tubulin was inspected in MCF-7 and MDA-MB-231 cells treated with cucurbitacin B. Cells were fixed, permeabilized and stained with anti-α-tubulin monoclonal antibody. The cells were analyzed using confocal microscopy. DAPI was used for nuclear localization. Change of tubulin organization within the cells was observed after cucurbitacin B treatment. B. Effect of cucurbitacin B on microtubule polymerization in a cell-free system was determined in vitro. Bovine brain tubulin protein (3 mg/mL) was mixed with special reaction buffer as described in materials and method, and incubated with 2.5, 5, 10 μM cucurbitacin B, 10 μM vincristine or 10 μM paclitaxel. The polymerization of tubulin was determined on the basis of the increase in optical density. The O.D. (340 nm) was measured each minute for up to 40 minutes at 37°C using microplate reader. Unlike paclitaxel and vincristine, treatment with cucurbitacin B in cell-free system has no effect on tubulin density.