3,3-diindolylmethane and related ring substituted analogs inhibit growth of breast cancer cells and induce mitochondrial membrane permeabilization

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1,1-Bis(3-indolyl)methane (DIM) and the 5,5-dibromo ring substituted DIM (5,5-diBrDIM) inhibited growth of MCF-7 and MDA-MB-231 breast cancer cells, and IC₅₀ values were 1-5 and 10-20 μM, respectively, in both cell lines. DIM and 5,5’-diBrDIM did not affect p21 or p27 protein levels or expression of Sp1 or Sp3 proteins in either cell line. In contrast, 10 μM 5,5-diBrDIM downregulated cyclin D1 protein in MCF-7 and MDA-MB-231 cells 12 and 24 hr after treatment. DIM (20 μM) also decreased cyclin D1 in MCF-7 (24 hr) and MDA-MB-231 (12 hr), and the DIM/5,5-diBrDIM-induced degradation of cyclin D1 was blocked by the proteasome inhibitor MG132. Both DIM and 5,5’-diBrDIM induced apoptosis in MCF-7 cells and this was accompanied by decreased Bcl-2, release of mitochondrial cytochrome c, and decreased mitochondrial membrane potential as determined by the red/green fluorescence of JC-1. The mitochondrial membrane potential was decreased by 48% and 19% in MCF-7 cells after 24 hr treatment with 5,5’-diBrDIM and DIM, respectively. DIM and 5,5’-diBrDIM induced extensive necrosis in MDA-MB-231 cells; however, this was accompanied by decreased mitochondrial membrane potential primarily in cells treated with 5,5’-diBrDIM. After 6, 12 and 24 hr treatment of MDA-MB-231 cells with 5,5’-diBrDIM, the mitochondrial membrane potential was decreased 55-71% compared to DMSO (vehicle control). By contrast, DIM only induced a 19% decrease in the mitochondrial membrane potential after 12 hr treatment of MDA-MB-231 cells. Thus, DIM and 5,5’-diBrDIM induce cell death in MCF-7 and MDA-MB-231 cells by overlapping and different pathways, and the ring-substituted DIM represents a novel class of uncharged mitochondrial poisons that inhibit breast cancer cell and tumor growth.