3,3'-Diindolylmethane enhances taxotere-induced growth inhibition of breast cancer cells through downregulation of FoxM1

Abstract

Emerging evidence suggests that the transcription factor Forkhead Box M1 (FoxM1) is associated with aggressive human carcinomas, including breast cancer. Because elevated expression of FoxM1 has been observed in human breast cancers, FoxM1 has attracted much attention in recent years as a potential target for the prevention and/or therapeutic intervention in breast cancer. However, no information is currently available regarding how downregulation of FoxM1 could be achieved for breast cancer prevention and therapy. Here, we report for the first time that 3,3'-diindolylmethane (DIM), a nontoxic dietary chemopreventive agent could effectively downregulate FoxM1 in various breast cancer cell lines. Using gene transfection, real-time reverse transcription-PCR, Western blotting, invasion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, we found that DIM could enhance Taxotere-induced growth inhibition of breast cancer cells, and decreased invasive capacity of breast cancer cells was observed after either treatment alone or the combination. These effects were associated with downregulation of FoxM1. We also found that knock down of FoxM1 expression by small interfering RNA (siRNA) transfection increased DIM-induced cell growth inhibition, whereas over-expression of FoxM1 by cDNA transfection attenuated DIM-induced cell growth inhibition, suggesting the mechanistic role of FoxM1. Most importantly, the combination treatment significantly inhibited tumor growth in severe combined immunodeficiency (SCID) mice, and the results were correlated with the downregulation of FoxM1 in tumor remnants. We conclude that inactivation of FoxM1 and its target genes by DIM could enhance the therapeutic efficacy of Taxotere in breast cancer, which could be a useful strategy for the prevention and/or treatment of breast cancer.

Figure 1

FoxM1 expression and its effects on cell growth after DIM and Taxotere treatment. For single-agent treatment, breast cancer cells were treated with DIM (5, 10, 25, or 40 μM), Taxotere (0.5, 1.0, or 1.5 nM) alone for 72 hr (a and b). For combination, breast cancer cells were treated with 25 or 40 (MDA-MB-231 only) μM of DIM and then exposed to 1.0 nM of Taxotere for 72 hr (c). DIM in combination with Taxotere significantly inhibited cell proliferation in all breast cancer cells (c) as measured by MTT assay. FoxM1 expression was evaluated by real-time RT-PCR and Western blotting (d). FoxM1 mRNA levels were downregulated by DIM, followed by Taxotere treatment as measured by real-time RT-PCR (e–h). Tax, Taxotere. β-actin protein was used as protein loading control for Western blot, whereas GAPDH was used as internal control for the real-time RT-PCR analysis. The data was obtained from three individual experiments. *, p < 0.05; **, p < 0.01 relatively control.

Figure 2

Regulation of FoxM1 expression by siRNA or cDNA followed by DIM and Taxotere treatment. Downregulation of FoxM1 expression by siRNA promotes DIM and Taxotere-induced cell growth inhibition in breast cancer cells (a and b). NS, Nonspecific. Overexpression of FoxM1 by FoxM1 cDNA transfection abrogated DIM and Taxotere-induced cell growth inhibition in breast cancer cells (c and d). The experiments were repeated thrice. *, p < 0.05; **, p < 0.01 relatively control.

Figure 3

Effect of FoxM1 on cell cycle regulatory factors. The expression level of several known cell cycle regulatory factors, as measured by real-time RT-PCR (a) and Western blotting (b) in all four breast cancer cells. C, Control; D, DIM [25 or 40 (MDA-MB-231) μM]; T, Taxotere (1.0 nM); D+T, DIM + Taxotere. The experiments were repeated thrice. *, p < 0.05; **, p < 0.01 relatively control.

Figure 4

Downregulation of FoxM1 decreased MMP-9 and VEGF activities. Effects of FoxM1 downregulation on the activities of MMP-9 and VEGF, as measured by respective ELISA analysis conducted using conditioned media. C, Control; D, DIM [25 or 40 (MDA-MB-231) μM]; T, Taxotere (1.0 nM); D+T, DIM + Taxotere. All the analyses were done at least thrice. *, p < 0.05; **, p < 0.01 relatively control.

Figure 5

DIM decreased breast cancer cell invasion. Invasion assay showing that DIM-treated breast cancer cells resulted in low penetration through the Matrigel-coated membrane, compared with control cells. MDA-MB-231 and MDA-MB-468 cell were with 25 and 40 μM of DIM, respectively. The bar graphs show the fluorescence values from the invasive cells. The value indicated the comparative amount of invaded cells. C, Control; D, DIM 25 or 40 μM; T, Taxotere (1.0 nM); D+T, DIM + Taxotere; Tax, Taxotere; DIM+Tax, DIM + Taxotere. The experiments were repeated thrice. *, p < 0.05; **, p < 0.01 relatively control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 6

Inhibition of tumor growth in vivo by DIM alone or in combination with Taxotere. DIM or Taxotere alone significantly inhibited MDA-MB-231 tumor growth (40% and 65%, respectively). Treatment of animals with DIM in combination with Taxotere caused 80% reduction in tumor volume compared with control group (a). DIM+Tax, DIM + Taxotere. *, p < 0.05 relatively control. Immunohistochemical staining for FoxM1 in DIM plus Taxotere-treated and -untreated animal tumors done on randomly selected tumor tissues (b). Tumor cells in untreated control group show intensive staining of FoxM1 (b). DIM+Tax, DIM + Taxotere. In contrast, tumor cells in DIM plus Taxotere-treated group show weaker staining of FoxM1 (b). The total proteins were extracted from randomly selected frozen tumor tissues obtained from each treatment group of animals and subjected to Western blot analysis (c). Results showed that the combination of DIM and Taxotere was effective in downregulating FoxM1, E2F1, CDK2, p27 and survivin in treated animals relative to the control tumors (c). Tax, Taxotere; DIM+Tax, DIM + Taxotere. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]