Indole-3-Carbinol (I3C) Inhibits Cyclin-dependent Kinase-2 Function in Human Breast Cancer Cells by Regulating the Size Distribution, Associated Cyclin E Forms, and Subcellular Localization of the CDK2 Protein Complex

Abstract

Indole-3-carbinol (I3C), a dietary compound found in cruciferous vegetables, induces a robust inhibition of CDK2 specific kinase activity as part of a G 1 cell cycle arrest of human breast cancer cells. Treatment with I3C causes a significant shift in the size distribution of the CDK2 protein complex from an enzymatically active 90 kDa complex to a larger 200 kDa complex with significantly reduced kinase activity. Co-immunoprecipitations revealed an increased association of both a 50 kDa cyclin E and a 75 kDa cyclin E immunoreactive protein with the CDK2 protein complex under I3C-treated conditions, whereas the 90 kDa CDK2 protein complexes detected in proliferating control cells contain the lower molecular mass forms of cyclin E. I3C treatment caused no change in the level of CDK2 inhibitors (p21, p27) or in the inhibitory phosphorylation states of CDK2. The effects of I3C are specific for this indole and not a consequence of the cell cycle arrest because treatment of MCF-7 breast cancer cells with either the I3C dimerization product DIM or the anti-estrogen tamoxifen induced a G 1 cell cycle arrest with no changes in the associated cyclin E or subcellular localization of the CDK2 protein complex. Taken together, our results have uncovered a unique effect of I3C on cell cycle control in which the inhibition of CDK2 kinase activity is accompanied by selective alterations in cyclin E composition, size distribution, and subcellular localization of the CDK2 protein complex.Considerable epidemiological evidence show that diets high in vegetable and fiber lead to low cancer risks and confer protection from various forms of cancers, including breast cancer (1, 2). In particular, consumption of vegetables belonging to the Brassica genus, which includes broccoli, cabbage, and cauliflower, have been reported to correlate with a decrease in mammary tumor incidence (3). These epidemiological studies suggest the existence of naturally occurring compounds in dietary sources that represent a largely untapped source of potential chemotherapeutic molecules. One such phytochemical is indole-3-carbinol (I3C), 1 an autolysis product of gluccosinolates present in Brassica vegetables. Dietary exposure to I3C reduced tumor occurrence and decreased the multiplicity of spontaneous as well as carcinogen-induced mammary tumor formation in rodent model systems (4 -8). Furthermore, diets high in cabbage, a good source of I3C, reduced pulmonary metastasis of mammary tumor implants in mice (9). Consistent with these studies, I3C tested positive as a chemopreventative agent in several short term bioassays relevant to carcinogeninduced DNA damage, tumor initiation and promotion, and oxidative stress (10).When ingested, the low pH environment of the stomach converts a large fraction of I3C into a variety of natural indole oligomers (11-15), such as its diindole products 3,3Ј-diindolylmethane (DIM), and indole[3,2-b]carbazole, which account for many of the in vivo anti-estrogenic and anti-tumorigenic biological activities of I3C (14). In contras...

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

“…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]. We have also shown that I3C, but not DIM, down regulates estrogen receptor-alpha expression [32] and acts synergistically with tamoxifen to inhibit the growth of estrogen responsive MCF7 breast cancer cells and suppress CDK2 specific activity [34].…”

“…We previously established that the I3C parent compound induced a G1 cell cycle arrest of human MCF-7 breast cancer cells [27,30]. To quantify the efficacy of the cell cycle arrest of the N-alkoxy I3C derivatives in comparison to I3C, tryptophol, and the DMSO vehicle control, MCF-7 breast cancer cells were treated with varying concentrations of each derivative for 72 hours, and the nuclear DNA content analyzed by flow cytometry analysis of propidium iodide stained nuclei.…”

“…We have established that the inhibition of CDK2 specific enzymatic activity without changes in the level of CDK2 protein is another cellular marker for the cell cycle effects of I3C [10,30,34]. To determine the efficacy of this response for the N-alkoxyl substituents, MCF-7 breast cancer cells were treated with I3C, individual N-alkoxy I3C derivatives, tryptophol, or DMSO vehicle control for 48 hours, and CDK2 immunoprecipitated from isolated nuclear extracts using conditions that maintain the CDK-cyclin interaction [30,34].…”

“…To determine the efficacy of this response for the N-alkoxyl substituents, MCF-7 breast cancer cells were treated with I3C, individual N-alkoxy I3C derivatives, tryptophol, or DMSO vehicle control for 48 hours, and CDK2 immunoprecipitated from isolated nuclear extracts using conditions that maintain the CDK-cyclin interaction [30,34]. One set of control immunoprecipitations utilized non-immune antibodies (No IP).…”

“…In cultured cells a significant portion of I3C is converted into its natural dimerization product DIM [28], and we have shown that the antiproliferative effects of I3C are distinct from and complement the bioactivities of DIM [10,[29][30][31][32]. The I3C mediated G1 cell cycle arrest of human breast cancer cells is mediated in part by an I3C activated transcriptional cascade that leads to the rapid inhibition in expression of the G1-acting cyclin dependent kinase 6 (CDK6) transcripts and protein [27].…”