Search

Indole-3-carbinol (13C), a compound that occurs naturally in Brassica vegetables such as cabbage and broccoli, can induce a G1 cell-cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (Cdk6) expression and stimulation of [p21.sup.Waf1/Cip1] gene expression. Construction and transfection of a series of promoter-reporter plasmids demonstrate that the indole-regulated changes in Cdk6 and [p21.sup.Waf1/Cip1] levels are due to specific effects on their corresponding promoters. Mutagenic analysis reveals that 13C signaling targets a composite transcriptional element in the Cdk6 promoter that requires both Sp1 and Ets transcription factors for transactivation function. Analysis of protein-DNA complexes formed with nuclear proteins isolated from 13C-treated and -untreated cells demonstrates that the Sp1 DNA element in the Cdk6 promoter interacts with an 13C-inhibited protein-protein complex that contains the Sp1 transcription factor. In indoletreated cells, a fraction of [[.sup.3]H]I3C was converted into its natural diindole product [sup.3]H-labeled 3-3'-diindolylmethane ([[3.sup]]DIM] which accumulates in the nucleus; this suggests that DIM may have a role in the transcriptional activities of I3C. Mutagenic analysis of the [p21.sup.Waf1/Cip1 promoter reveals that in transfected breast cancer cells, DIM (as well as I3C) stimulates [p21.sup.Waf1/Cip p21] transcription through an indole-responsive region of the promoter that contains multiple Sp1 consensus sequences. Furthermore, DIM treatment regulates the presence of a nuclear Sp1 DNA-binding activity. Our results demonstrate that both the Cdk6 and [p21.sup.Waf1/Cip1] promoters are newly defined downstream targets of the indole-signaling pathway, and that the observed transcriptional effects are due to a combination of the cellular activities of 13C and DIM. KEY WORDS: * indoles * 13C * DIM * antiproliferative pathway * regulated promoter * cell-cycle gene * Sp1 transcription factor * reproductive cancer cell

Studies increasingly indicate that dietary indole-3-carbinol (I3C) prevents the development of estrogen-enhanced cancers including breast, endometrial and cervical cancers. Epidemiological, laboratory, animal and translational studies support the efficacy of I3C. Whereas estrogen increases the growth and survival of tumors, I3C causes growth arrest and increased apoptosis and ameliorates the effects of estrogen. Our long-range goal is to best use I3C together with other nutrients to achieve maximum benefits for cancer prevention. This study examines the possibility that induction of growth arrest in response to DNA damage (GADD) in genes by diindolylmethane (DIM), which is the acid-catalyzed condensation product of I3C, promotes metabolically stressed cancer cells to undergo apoptosis. We evaluated whether genistein, which is the major isoflavonoid in soy, would alter the ability of I3C/DIM to cause apoptosis and decrease expression driven by the estrogen receptor (ER)-[alpha]. Expression of GADD was evaluated by real-time reverse transcription-polymerase chain reaction. Proliferation and apoptosis were measured by a mitochondrial function assay and by fluorescence-activated cell sorting analysis. The luciferase reporter assay was used to specifically evaluate expression driven by ER-[alpha]. The estrogen-sensitive MCF-7 breast cancer cell line was used for these studies. We show a synergistic effect of I3C and genistein for induction of GADD expression, thus increasing apoptosis, and for decrease of expression driven by ER-[alpha]. Because of the synergistic effect of I3C and genistein, the potential exists for prophylactic or therapeutic efficacy of lower concentrations of each phytochemical when used in combination. KEY WORDS: * indole-3-carbinol * diindolylmethane * genistein * estradiol