Indole-3-carbinol Inhibits the Expression of Cyclin-dependent Kinase-6 and Induces a G1 Cell Cycle Arrest of Human Breast Cancer Cells Independent of Estrogen Receptor Signaling

Abstract

Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables such as cabbage, broccoli, and Brussels sprouts, has been shown to reduce the incidence of spontaneous and carcinogen-induced mammary tumors. Treatment of cultured human MCF7 breast cancer cells with I3C reversibly suppresses the incorporation of [ 3 H]thymidine without affecting cell viability or estrogen receptor (ER) responsiveness. Flow cytometry of propidium iodide-stained cells revealed that I3C induces a G 1 cell cycle arrest. Concurrent with the I3C-induced growth inhibition, Northern blot and Western blot analyses demonstrated that I3C selectively abolished the expression of cyclin-dependent kinase 6 (CDK6) in a dose-and time-dependent manner. Furthermore, I3C inhibited the endogenous retinoblastoma protein phosphorylation and CDK6 phosphorylation of retinoblastoma in vitro to the same extent. After the MCF7 cells reached their maximal growth arrest, the levels of the p21 and p27 CDK inhibitors increased by 50%. The antiestrogen tamoxifen also suppressed MCF7 cell DNA synthesis but had no effect on CDK6 expression, while a combination of I3C and tamoxifen inhibited MCF7 cell growth more stringently than either agent alone. The I3C-mediated cell cycle arrest and repression of CDK6 production were also observed in estrogen receptor-deficient MDA-MB-231 human breast cancer cells, which demonstrates that this indole can suppress the growth of mammary tumor cells independent of estrogen receptor signaling. Thus, our observations have uncovered a previously undefined antiproliferative pathway for I3C that implicates CDK6 as a target for cell cycle control in human breast cancer cells. Moreover, our results establish for the first time that CDK6 gene expression can be inhibited in response to an extracellular antiproliferative signal.Considerable epidemiological evidence suggests that high vegetable diets correlate with low breast cancer risk (1, 2). This phenomenon is likely due to the diverse spectrum of dietary and environmental compounds that can regulate the function and proliferation of mammalian cells by influencing hormone receptor signal transduction pathways (3, 4). Several classes of these naturally occurring hormone-like chemicals have been implicated in the control of tumor cell growth and as chemopreventative agents. One such substance is the dietary compound indole-3-carbinol (I3C), 1 an autolysis product of a glucosinolate, glucobrassicin, which occurs in Brassica vegetables such as cabbage, broccoli, and Brussels sprouts (5, 6). A recent screen of 90 potential chemopreventative agents in a series of six short term bioassays relevant to carcinogen-induced DNA damage, oxidative stress, and tumor initiation and promotion, revealed I3C to be one of only eight compounds effective in all assays (7). Several studies have shown that exposure to dietary I3C markedly reduces the incidence of spontaneous and carcinogen-induced mammary tumors in rodents (8, 9). For example, I3C administered in the diet or by oral intubation pr...

Indole‐3‐carbinol induces a G1 cell cycle arrest and inhibits prostate‐specific antigen production in human LNCaP prostate carcinoma cells

“…46 It is noteworthy that previous reports have demonstrated that I3C inhibited CDK6 expression in both estrogen-responsive and nonresponsive human breast carcinoma cells. 19,21 Those observations, in combination with our studies involving LNCaP prostate carcinoma cells, suggest that the selective inhibition of CDK6 gene expression may be a hallmark of the cellular mechanism operating in reproductive tumor cells that allows these cell types to respond to I3C. In breast carcinoma cells, we have discovered that I3C treatment alters the function of Sp-1 transcription factors at the specific indoleregulated composite element in the CDK6 promoter, 21 and we currently are attempting to determine whether I3C has a similar response in human prostate carcinoma cells.…”

“…Equal amounts of total cellular protein were mixed with loading buffer (25% glycerol, 0.075% SDS, 1.25 mL 14.4 M 2-mercaptoethanol, 10% bromophenol blue, 3.13% stacking gel buffer), fractionated by electrophoresis on 10% polyacrylamide/0.1% SDS resolving gels, electrophoretically transferred to nitrocellulose membranes, and prepared for Western blotting as described previously. 19,20 The working concentration for all antibodies was 1 g/mL and immunoreactive proteins were detected after 1-3 hours of incubation at room temperature with the appropriate horseradish peroxidaseconjugated secondary antibodies. Blots were treated with ECL reagents (NEN Life Science Products), and all proteins were detected by autoradiography.…”

“…The enzymatic activity of immunoprecipitated CDK2 and CDK4 was determined as previously described. 19,20 …”

“…We have established that the direct treatment of cultured human breast carcinoma cells with I3C potently inhibits the growth of either estrogen-responsive or estrogen-independent human breast carcinoma cells. 19,20 I3C mediates this antiproliferative effect by inducing a G1 cell cycle arrest through disruptions in the expression and activity of specific G1-acting cell cycle components. 19 -21 Although several studies have investigated the apoptotic effects of high concentrations of I3C in prostate carcinoma cells, 22,23 relatively little is known about the cell cycle effects that selectively control the antiproliferative and anticancer response of this indole.…”

BRCA1 and BRCA2 as molecular targets for phytochemicals indole-3-carbinol and genistein in breast and prostate cancer cells

“…It can inhibit cell cycle progression, induce apoptosis, and inhibit tumour invasion and metastasis, even in ER-a-negative cells (Meng et al, 2000a(Meng et al, , 2001Bonnesen et al, 2001;Chen et al, 2001). Indole-3-carbinol downregulates expression of a G1 cyclindependent kinase (CDK6) and upregulates a CDK inhibitor (p21 Cip1 ) (Cover et al, 1998). The major active metabolite of I3C, diindolylmethane (DIM), induces expression of GADD45a, a DNA damage-responsive gene and putative tumour suppressor (Carter et al, 2002).…”