Indole‐3‐carbinol activates the ATM signaling pathway independent of DNA damage to stabilize p53 and induce G1 arrest of human mammary epithelial cells

Abstract

The phytochemical indole-3-carbinol (I3C), from cruciferous vegetables such as broccoli, has been shown to elicit a potent anti-proliferative response in human breast cancer cell lines. Treatment of the immortalized human mammary epithelial cell line MCF10A with I3C induced a G1 cell cycle arrest, elevated p53 tumor suppressor protein levels and stimulated expression of downstream transcriptional target, p21. I3C treatment also elevated p53 levels in several breast cancer cell lines that express mutant p53. I3C did not arrest MCF10A cells stably transfected with dominant-negative p53, establishing a functional requirement for p53. Cell fractionation and immunolocalization studies revealed a large fraction of stabilized p53 protein in the nucleus of I3C-treated MCF10A cells. With I3C treatment, phosphatidyl-inositol-3-kinase family member ataxia telangiectasia-mutated (ATM) was phosphorylated, as were its substrates p53, CHK2 and BRCA1. Phosphorylation of p53 at the N-terminus has previously been shown to disrupt the interaction between p53 and its ubiquitin ligase, MDM2, and therefore stabilizing p53. Coimmunoprecipitation analysis revealed that I3C reduced by 4-fold the level of MDM2 protein that associated with p53. The p53-MDM2 interaction and absence of p21 production were restored in cells treated with I3C and the ATM inhibitor wortmannin. Significantly, I3C does not increase the number of 53BP1 foci or H2AX phosphorylation, indicating that ATM is activated independent of DNA double-strand breaks. Taken together, our results demonstrate that I3C activates ATM signaling through a novel pathway to stimulate p53 phosphorylation and disruption of the p53-MDM2 interaction, which releases p53 to induce the p21 CDK inhibitor and a G1 cell cycle arrest. ' 2005 Wiley-Liss, Inc.Key words: indole-3-carbinol; breast cancer; p53; MDM2; cell cycle arrest; ATM Indole-3-carbinol (I3C), a phytochemical of Brassica vegetables such as broccoli and cabbage, has been shown to prevent spontaneous and carcinogen-induced mammary tumors.1-3 I3C has a direct anti-proliferative effect in both estrogen-dependent and independent human breast cancer cell lines. 4 In tissue culture, I3C treatment of human adenocarcinoma cells induces a G1 cell cycle arrest that is associated with a decreased expression of cyclindependent kinase 6 (CDK6), and inhibited specific enzymatic activity of CDK2. 4,5 In [ 3 H]I3C-treated MCF-7 cells, the I3C condensation product 3,3 0 -diindolylmethane (DIM) represents 40% of radiolabeled compounds in the nuclear fraction, suggesting that DIM may mediate a subset of the transcriptional responses to I3C. 6 DIM has been shown to decrease CDK2 activity through Sp1-mediated transcriptional upregulation of CDK inhibitor p21. 7In addition to their anti-proliferative effects, treatment with I3C or DIM also causes a p53-independent apoptotic response in cultured breast cancer cells. [8][9][10] I3C was shown to induce apoptosis of the tumorigenic MCF10CA1a cell line, 11 but the mechanism of growth inhibition of the isogen...

The dietary phytochemical indole-3-carbinol is a natural elastase enzymatic inhibitor that disrupts cyclin E protein processing

“…This information is detailed in SI Text. The flow cytometry was performed as we previously described (15), and the Western blots, cyclin E immunoprecipitations, and assays of CDK2 enzymatic activity using histone H1 as a substrate were carried out as described (29).…”

“…In estrogen-responsive breast cancer cells, I3C suppresses estrogen responsiveness (13,14), down-regulates expression of estrogen receptor-␣ (13), and synergizes with the antiproliferative effects of tamoxifen, an anti-estrogen widely used in breast cancer therapies (10). In nontumorigenic human mammary epithelial cells, I3C can induce the ATM signaling pathway independent of DNA damage to stabilize an active p53 tumor suppressor protein (15). I3C can suppress invasion and migration of human breast cancer cells (16) and stimulate IFN-␥ receptor production and IFN-␥ responsiveness (17).…”

Indole-3-carbinol inhibits cell proliferation and induces apoptosis in Hep-2 laryngeal cancer cells

“…A number of studies have shown that I3C induces cell cycle arrest in the G1 phase in cancer cells (8,9), promotes apoptosis (8)(9)(10) and prevents tumor invasion and metastasis (11). It has been found that I3C promotes cell cycle arrest by downregulating cyclin-proteins such as cyclin D1 and cyclin E. Additionally, it is thought that IC3 induces apoptosis by mechanisms that depend on downregulation of the anti-apoptotic genes Bcl-2, Bcl-xL and survivin and by enhancing expression of Bax and by functionally activating caspase-3 and caspase-9 (9)(10)(11)(12).…”

The Anti-Carcinogenic Effect of Indole-3-Carbinol and 3, 3'-Diindolylmethane and their Mechanism of Action

“…Other studies indicated that I3C also induced cell cycle arrest in breast cancer cells and inhibited CdK6, but in high concentration of 200 µM [37][38][39]. In addition, it was demonstrated that I3C activates p53 phosphorylation and disruption of the p53-MDM2 interaction, which releases p53 to induce the p21 CDK inhibition and G1 cell cycle arrest [30,40]. These studies suggested that I3C induced phosphorylation of phosphatidyl-inositol-3-kinase (PI3-K/Akt) family member, ataxia telangiectasia gene that activates p53 through its phosphorelation that induce p21 WAF1 CDK inhibitor and caused a G1 cell cycle arrest.…”