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, 3 (7), 818-28

A Novel Mechanism of indole-3-carbinol Effects on Breast Carcinogenesis Involves Induction of Cdc25A Degradation

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A Novel Mechanism of indole-3-carbinol Effects on Breast Carcinogenesis Involves Induction of Cdc25A Degradation

Yongsheng Wu et al. Cancer Prev Res (Phila).

Abstract

The natural compound indole-3-carbinol (I3C; found in vegetables of the genus Brassica) is a promising cancer prevention or therapy agent. The cell division cycle 25A (Cdc25A) phosphatase is overexpressed in a variety of human cancers and other diseases. In the present study, I3C induced degradation of Cdc25A, arrest of the G(1) cell cycle, and inhibition of the growth of breast cancer cells. We also showed that the Ser124 site of Cdc25A, which is related to cyclin-dependent kinase 2, is required for I3C-induced degradation of Cdc25A in breast cancer cells, and that interruption of the ATM-Chk2 pathway suppressed I3C-induced destruction of Cdc25A. Our in vivo studies of different mutated forms of Cdc25A found that the mutation Cdc25A(S124A) (Ser124 to Ala124), which confers resistance to I3C-induced degradation of Cdc25A, attenuated I3C inhibition of breast tumorigenesis in a mouse xenograft model. The present in vitro and in vivo studies together show that I3C-induced activation of the ATM-Chk2 pathway and degradation of Cdc25A represent a novel molecular mechanism of I3C in arresting the G(1) cell cycle and inhibiting the growth of breast cancer cells. The finding that I3C induces Cdc25A degradation underscores the potential use of this agent for preventing and treating cancers and other human diseases with Cdc25A overexpression.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Fig. 1
Fig. 1
Analysis of I3C-induced profile of Cdks in different breast cancer cells, G1 cell cycle arrest, and cell growth inhibition. A to C, profiles of the I3C-induced G1 Cdks. MCF7, MDA-MB-231, and MDA-MB-468 breast cancer cells were treated with 0.1% DMSO alone or with 100 to 200 μmol/L I3C for 24, 48, and 72 h. Cell lysates were prepared and Western blot analysis was done with the indicated antibodies. Representative data from one experiment are shown (n = 5). D to F, analysis of cell cycle of the breast cancer cells. The cells, treated with either 0.1% DMSO alone or with I3C, were harvested in P3S and stained with a hypotonic solution containing propidium iodide. Stained nuclei were subjected to flow cytometry analysis. G to I, cell growth inhibition by I3C. The breast cancer cells were treated with 100 to 200 μmol/L I3C and control cells with 0.1% DMSO and then harvested by trypsinization; cell numbers were determined. The number of living cells was plotted versus the days of I3C exposure. Points, mean (n = 5); bars, SEM. Differences between solvent control and reatment are sgnificant (P < C.05).
Fig. 2
Fig. 2
I3C reduces the level of Cdc25A protein in breast cancer cells. MDA-MB-231 (A), MDA-MB-468 (B), and MCF7 (C) breast cancer cells were treated with 0.1% DMSO alone or with 100 to 200 μmol/L I3C for 24, 48, and 72 h. Cell lysates were prepared and Western blot analysis was done with the indicated antibodies. Cdk6 was reduced only in the I3C-treated MCF7 cells, whereas reduced levels of Cdc25A were observed in all cell types with I3C treatment, which attenuated the dephosphorylation of its downstream Cdk2. Representative data from one experiment are shown (n = 3).
Fig. 3
Fig. 3
The Ser124 site of Cdc25A is required for Cdc25A degradation in response to I3C induction. A, a sketch for multiple pathways related to Chk1, Smad3, and Chk2 regulating Cdc25A degradation (–25), B, Dox-induced expression of Cdc25A and its derivatives. The wild-type Cdc25A cDNA from MCF10A cells was cloned into the pCDNA4/TO vector with Tet-on system, and each mutated derivative, Cdc25AS76A, Cdc25AS82A, and Cdc25As124A, in the vectors was constructed according to the procedures described in Materials and Methods. In cultured medium with 10 ng/mL Dox, Cdc25A and its derivatives were effectively induced. C and D, response of the mutant Cdc25A derivatives to I3C treatment in MDA-MB-231 breast cancer cells (C) and MDA-MB-468 breast cancer cells (D). Cell lysates were prepared and Western blot analysis was done with the indicated Cdc25A and control antibodies. Representative data from one experiment are shown (n = 3).
Fig. 4
Fig. 4
Detection of protein levels of Cdc25AWT and its mutation derivatives in breast cancer cells with I3C treatment. Cdc25AWT and its derivatives continued to be induced in MDA-MD-231 breast cancer cells with 10 ng/mL Dox while the cells were treated with either 0.1% DMSO alone or with 200 μmol/L I3C for 48 h. The cells were then stained with anti-Cdc25A mAb. Signals were visualized by incubation with fluorescein-conjugated anti-mouse immunoglobulin G antibody, followed by analysis with a fluorescence microscope. The cells maintain normal cell shape (phase-contrast images) and 4′,6-diamidino-2-phenylindole staining also shows normal nuclear shape (data not shown). Results are representative of three independent sets of experiments. A, Cdc25AWT; B, Cdc25AS76A; C, Cdc25AS82A; D, Cdc25AS124A.
Fig. 5
Fig. 5
I3C activates the ATM-Chk2-Cdc25A pathway in human breast cancer cells. Breast cancer MAD-MB-231 cells were treated with 0.1% DMSO alone, with 200 μmol/L I3C, or with 200 μmol/L I3C and 10 μmol/L of ATM inhibitor KU55933 (KU) for 36 and 60 h, respectively. Cell lysates were then prepared and Western blot analysis was done with the indicated antibodies. The inhibitor KU55933 can effectively block the I3C-induced phosphorylation of ATM at residue 1981 the activity of downstream Chk2 by phosphorylation at Tyr68, and the degradation of downstream Cdc25A. Representative data from one experiment are shown (n = 3).
Fig. 6
Fig. 6
The mutant Cdc25AS124A is resistant to the effect of I3C on breast cancer in a mouse xenograft model. A, representative images of mice from each cell line and group, photographed at time of sacrifice. B, statistical analysis for tumor volume from each cell line and treatment group at different time points. The mice were inoculated s.c. in the lateral flanks with 0.1 mL of PBS solution containing 1 × 106 MDA-MB-231 human breast cancer cells. The mice in the intervention group were given I3C (1 mg/d per mouse) by oral gavage everyday for 6 wk as described in Materials and Methods. The control mice received only sesame seed oil without I3C. For Dox induction, Dox (2 mg/mL) was added into daily feeder water as soon as the cells were inoculated in the flanks of mice. Fresh water was replaced twice weekly. The mice were divided into four groups for each of the tested cell lines: without Dox induction or I3C treatment, group 1 (DOXI3C); group 2 (DOX+I3C); group 3 (DoxI3C+); and group 4 (Dox+I3C+). Each group contained 10 mice and 3 repeats. The cells and treatment methods used in each case are indicated in the figure. The palpable tumor diameters were measured and volumes were calculated twice per week. Under the experimental conditions, the mutation Cdc25AS124A significantly inhibited the effect of I3C on breast cancer cell tumorigenesis in nude mice (P < 0.05).

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