Induction of growth arrest and apoptosis in human breast cancer cells by 3,3-diindolylmethane is associated with induction and nuclear localization of p27

3,3¶-Diindolylmethane (DIM) is a stable condensation product of indole-3-carbanol, a potential breast cancer chemoprevention agent. Human breast cancer cell lines were studied to better understand its mechanisms. In vitro experiments were done in MCF-7, T47D, BT-20 and BT-474 cells using MTT, ELISA, immunoblotting assays, reverse transcription-PCR, protein half-life, confocal microscopy, cell fractionation, and immunoprecipitation assays. We found that DIM inhibited the growth of all four breast cancer cell lines (IC50s, 25-56 Mmol/L). Because BT-20 and BT-474 overexpressed Her-2 and activated Akt, and BT-20 lacks estrogen receptor, these were studied further. In both cell lines, DIM appeared to induce expression of p27 protein before the loss of cell viability and apoptosis. In BT-20 cells, DIM also inhibited expression of activated Akt, but this appeared after p27 induction. In both cell lines, DIM induced p27 transcript expression within 6 h. DIM prolonged the p27 protein half-life in BT-20 but not BT-474 cells. We also showed, for the first time, that DIM induced nuclear localization of p27 in both cell lines. Moreover, in BT-20 cells, DIM induced a decrease in p27 phosphorylation at Thr, and its association with the 14-3-3 protein, which helped to explain the protein half-life increase and nuclear localization, respectively. DIM modulates p27 through transcription, prolongation of protein half-life, and nuclear localization. These effects appear to be independent of Her-2, Akt, or estrogen receptor status and should support further study for its chemoprevention potential in breast cancer. [Mol Cancer Ther 2008;7(2):341–9]