3,3′-Diindolylmethane (DIM) induces a G1 cell cycle arrest in human breast cancer cells that is accompanied by Sp1-mediated activation of p21WAF1/CIP1 expression

Abstract

3,3'-Diindolylmethane (DIM) is a promising cancer chemopreventive agent derived from Brassica food plants. To determine whether this natural indole has a direct growth inhibitory effect on human breast cancer cells, we examined the cell cycle regulatory effects of DIM in estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231) human breast cancer cell lines. Results of flow cytometry studies showed that DIM treatment produced a marked increase (from 51 to 79%) in the proportion of cells in the G(1) phase of the cell cycle, regardless of estrogen-receptor status. Analyses of G(1)-acting cell cycle components indicated that the enzymatic activity of cyclin-dependent kinase (CDK) 2 was also strongly reduced. Western blot analyses showed that, concurrent with the DIM-induced cell cycle arrest, DIM stimulated a rapid and pronounced increase in levels of the CDK inhibitor, p21(WAF1/CIP1) (p21). Northern blot analysis demonstrated that DIM increased p21 mRNA expression with a maximal 6-7-fold induction, and exposure to cycloheximide did not block the response. Similar increases in expression of p21 protein and mRNA were observed in both MCF-7 and MDA-MB-231 human breast cancer cells, suggesting that DIM induction of p21 expression is independent of estrogen-receptor signaling and p53. Transient transfection of 5'-deletion constructs of the p21 promoter demonstrated that the first 291 bp segment of the proximal promoter, which contains six promoter specific transcription factor 1 (Sp1) elements, maintained DIM responsiveness. Consistent with a role for Sp1 in this response, a reporter construct driven by three consensus Sp1 binding sites was responsive to DIM. In addition, electrophoretic mobility shift assays showed that DIM induced the binding of Sp1 and Sp3 to the consensus Sp1 responsive element. Thus, our observations have uncovered an antiproliferative pathway for DIM that implicates Sp1/Sp3-induced expression of p21 as a target for cell cycle control in human breast cancer cells.

N-Alkoxy derivatization of indole-3-carbinol increases the efficacy of the G1 cell cycle arrest and of I3C-specific regulation of cell cycle gene transcription and activity in human breast cancer cells

“…Most notably, I3C, but not DIM, inhibits CDK6 gene expression by down-regulating its promoter activity, and inhibits CDK2 enzymatic specific activity by altering the cyclin E products associated with the CDK2 protein complex without any detectable changes in the level of CDK2 protein [10,27,29,30,34]. In contrast, DIM induces expression of p21 CDK inhibitor that leads to an inhibition of CDK2 activity [33]. Thus, the overall profile of the I3C-specific effects on the G1-acting CDKs provides a bioassay for testing the effects of more potent synthetic I3C derivatives.…”

“…For the kinase assay, the remaining half of the immunoprecipitated sample was resuspended in 25 μl kinase buffer containing 20 mM ATP, 5 mM DTT, 0.21 μg Rb carboxy-terminal domain protein substrate (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and 10 μCi [γ 33 P] dATP (3000 Ci/mmol). Reactions were incubated for 15 minutes at 30 °C and stopped by adding an equal volume of 2X loading buffer (10% glycerol, 5% β-mercaptoethanol, 3% SDS, 6.25 mM Tris HCl pH 6.8, and bromophenol blue).…”

“…I3C down regulates CDK6 transcription by inhibiting Sp1 binding and function at a Sp1-Ets composite DNA element in the CDK6 promoter [29]. In contrast, the cell cycle arrest and apoptosis induced by DIM is not accompanied by CDK6 down regulation [23,33]. I3C treatment of breast cancer cells also results in an inhibition of CDK2 specific enzymatic activity due to a disruption in composition and subcellular localization of the CDK2 protein complex [30], whereas, CDK4 enzymatic activity and protein levels remained relatively unaffected [34].…”

Evidence that Sp1 positively and Sp3 negatively regulate and androgen does not directly regulate functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) gene expression in normal human prostate epithelial cells

“…Sp1 and related Sp family members such as Sp3 are the major transcription factors that bind to GC-rich Sp1 sites (Black et al, 2001). Sp1 sites and the Sp family proteins have been implicated in the constitutive expression of many 'housekeeping'genes as well as in tissue and cell specific and highly regulated expression of many other genes (Huang et al, 2000;Hong et al, 2002;Blais et al, 2002). Furthermore, Sp1 sites and the Sp1 protein have been implicated in regulating the basal expression of several other LOXs, including 12-LOX and 15-LOX 1 (Kritzik et al, 1997;Kelavkar et al, 1998;Chen and Chang, 2000).…”

Current principles of effective therapy for ovarian cancer

“…Уста-новлено, что данный метаболит обладает выраженной антипролиферативной активностью [30]. В отсутствие I3C арилкарбоновые рецепторы активируются канце-рогенными углеводородами, что способствует экспрес-сии CYPB1, гидроксилирующей эстрон с образовани-ем 16-альфа-гидроксиэстрона и 4-гидроксиэстрона; 16-альфа-гидроксиэстрон, наоборот, стимулирует кле-точный рост и является агонистом эстрогена [23,31].…”