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* Department of Molecular and Cell Biology and Cancer Research Laboratory and Department of Nutritional Sciences and Toxicology, University of California at Berkeley, Berkeley, CA 94720-3200
3 To whom correspondence should be addressed. E-mail: glfire{at}uclink4.berkeley.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • indoles • I3C • DIM • antiproliferative pathway • regulated promoter • cell-cycle gene • Sp1 transcription factor • reproductive cancer cell
One of the complexities of reproductive cell cancers such as breast cancer is that several distinct classes of tumors can be produced that differ in their proliferative responses to hormonal and other environmental cues (
1). Only 
35% of breast cancers are estrogen responsive and can be initially treated with the nonsteroidal antiestrogen tamoxifen (
2–
4), although these cancers eventually progress into a steroid-independent state (
1). A critical problem in the clinical management of human breast cancer is the need to develop new therapeutic strategies, because the current treatment options for estrogen-independent mammary tumors are surgical removal of the tumors, general chemotherapy and/or radiation therapy. A potential source of new classes of chemotherapeutic and chemopreventative agents to control reproductive cancers with reduced side effects are compounds found in the diet. Epidemiological findings show that dietary factors play a major role in the pathogenesis of several types of cancers (
1,
5–
7), and increased consumption of phytochemicals from whole grains, vegetables and fruits is directly associated with decreased risk for breast cancer (
1,
3,
8–
12). These studies suggest that dietary plants produce unique compounds that represent a largely untapped source of potentially potent chemotherapeutic molecules. One such phytochemical is indole-3-carbinol (I3C),4 a naturally occurring component of Brassica vegetables such as cabbage, broccoli and brussels sprouts (
10,
13,
14).
Early studies established that high doses of I3C fed in the diet or administered by oral intubation to rodents greatly reduces the incidence of spontaneous and carcinogen-induced tumors of the mammary gland and endometrium as well as other cancer types such as colon, lung, skin, liver and cervix with negligible levels of toxicity ( 12, 14– 23). For example, I3C treatment prevents the formation of 7,12-dimethyl-benz(a)anthracene (DMBA)-induced mammary tumors in rats ( 14, 18, 24) and benzo(a)pyrene-induced tumors of the forestomach and pulmonary adenomas in mice ( 12, 20). Dietary supplementation with cabbage or broccoli, both of which are good sources of I3C, also results in decreased mammary tumor formation in DMBA-treated rats ( 16) as well as a 50% lower spontaneous mammary tumor incidence and multiplicity in female mice ( 14, 18, 19, 25). Consistent with these studies, I3C tested positive as a chemopreventative agent in several short-term bioassays that are relevant to carcinogen-induced DNA damage, tumor initiation and promotion and oxidative stress ( 24).
Recently emerging evidence documents that I3C has potent antiproliferative effects in certain cancer cells. For example, ectopic application of I3C directly inhibits skin tumor formation in mouse models ( 21). Studies that we have conducted ( 26– 28) and studies by others ( 29– 33) show that I3C has both antiproliferative and apoptotic effects on cultured human breast cancer cells that generally depend on the concentration of this dietary indole used in the assays. In one study, I3C was shown to alter the level of BRCA1 gene expression, although the cellular significance of this observation remains unknown ( 32). Several studies show that 3-3'-diindolylmethane (DIM), which is the predominate natural diindole product of I3C, can reduce the incidence of different classes of reproductive tissue tumors ( 17, 34– 36). Furthermore, we showed that DIM inhibits the growth ( 37) and induces programmed cell death ( 38) of human breast cancer cells as well as endometrial tumor cells ( 36). I3C or DIM has little affinity for the aryl hydrocarbon, estrogen or androgen receptors ( 34, 39– 41), which suggests that these indoles act through an unknown target protein.
Before our studies, little was known about the signal transduction pathway or the nature of the regulated gene targets through which natural indoles exert their growth-inhibitory effect on reproductive cancer cells. We discovered that the direct exposure of human breast cancer cells to I3C activates a novel antiproliferative pathway that induces a G1 cell-cycle arrest accompanied by the selective and rapid downregulation of cyclin-dependent kinase (Cdk)6 gene expression and strong stimulation of p21Waf1/Cip1 gene expression ( 26– 28). The G1 cell-cycle arrest occurs through an estrogen-receptor–independent pathway ( 26, 27). I3C treatment also inhibits Cdk2-specific enzymatic activity without having any effects on Cdk4 activity or protein expression ( 26, 27). As discussed below, in indole-treated breast cancer cells, a fraction of I3C is converted into its natural diindole product DIM, and the newly formed DIM accumulates in the nucleus, which suggests that this product may have a role in the cellular biological activities of I3C ( 42). Consistent with this notion, DIM mimics some of the I3C effects in I3C-responsive human breast cancer cells including induction of a G1 cell-cycle arrest, strong stimulation of p21Waf1/Cip1 gene expression and apoptotic response ( 37, 38). We also document that the cell-cycle arrest induced by I3C or DIM occurs with indole-specific changes in the transcription of cell-cycle genes. Treatment with I3C but not with DIM downregulates Cdk6 promoter activity and disrupts the interaction of the Sp1 transcription factor at a composite transcriptional element in the Cdk6 promoter. In contrast, treatment with either I3C or DIM promotes the interaction of Sp1 with the p21Waf1/Cip1 promoter. Our results demonstrate that both the Cdk6 and p21Waf1/Cip1 promoters are newly defined downstream targets of the indole-signaling pathway in breast cancer cells, and that the observed transcriptional and cell-cycle effects are due to a combination of the cellular activities of I3C and DIM.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Our initial efforts tested whether I3C treatment can inhibit the proliferation of two widely used human breast cancer cell lines: estrogen-responsive MCF-7 cells (see
Table 1) and estrogen-receptor-
–deficient MDA-MB-231 cells (
26). Both breast cancer cell lines were originally developed from pleural effusions of primary metastatic tumors. Cell proliferation was analyzed by cell number, incorporation of [3H]thymidine (2-h pulse) and flow cytometry for DNA content. Treatment with 100 µmol I3C/L maximally inhibited the cell proliferation of both breast cancer cell types with no obvious morphology changes or effects on cell viability (higher concentrations caused apoptosis); this demonstrates that I3C mediates its antiproliferative effects independent of estrogen. The growth-inhibitory effect is rapid and completely reversible. In the case of estrogen-nonresponsive MDA-MB-231 cells, indole treatment inhibited proliferation under conditions in which the antiestrogen tamoxifen had no effect (
27), which suggests that a wider range of breast cancer cells responds to indoles than estrogen antagonists.
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13% of the cells remain with a G2/M DNA content. Other breast cancer cell lines such as the highly tumorigenic and metastatic MDA-MB-435 cells, T-47D cells and the nontumorigenic MCF-10A cells also display this indole-responsive cell-cycle response. Selective control of cell-cycle gene expression and activity by I3C
Regulation of the cyclin-Cdk protein complexes plays a critical role in cell-cycle progression and is disrupted during the development of many human cancers ( 43– 45). Therefore, we tested whether the I3C growth-suppression pathway targets specific components of the G1 phase of the cell cycle in human breast cancer cells. Expression of the major G1-acting cell-cycle components was screened in cultured reproductive cancer cells over 96-h time courses of indole treatment (see Table 1). Western blot, Northern blot and semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analyses revealed that I3C selectively and rapidly downregulates the expression of Cdk6 and upregulates the expression of the Cdk inhibitor p21Waf1/Cip1 within 24 h of indole treatment ( 26, 27). I3C also stimulates a small but reproducible increase in levels of p27 (another Cdk inhibitor), which coincides with the maximal cell-cycle arrest. As summarized in Table 1, I3C has no other significant effects on the production of other key G1-acting cell-cycle components including Cdk2 or Cdk4, cyclin D (all three isoforms), cyclin E or the other small-molecule Cdk inhibitors ( 26, 27); this demonstrates the specificity of the indole response. Because Cdk levels generally remain constant throughout the cell cycle ( 46), a unique feature of the I3C antiproliferative pathway is rapid inhibition of expression of Cdk6 transcripts and protein. Our studies uncovered the first evidence for selective targeting of cell-cycle components by the direct treatment of cancer cells with natural indoles, and the Cdk6 observation in particular suggests that indoles activate a previously unknown pathway.
It is well established that p21Waf1/Cip1 binds and inhibits the activity of a variety of Cdk such as Cdk2, Cdk4 and Cdk6, which in some systems is sufficient to suppress the progression of the cell cycle from G1 to S phase (
45,
46). To determine whether the indole downregulation of Cdk6 and stimulation of p21Waf1/Cip1 levels alters total cellular Cdk activities, the three G1-acting Cdk were immunoprecipitated from I3C-treated and -untreated cells and their respective kinase activities were assayed by their ability to phosphorylate the retinoblastoma (Rb) protein in vitro in the presence of [
-32P]ATP (
26,
27). I3C strongly inhibits Cdk2-specific activity (loss of enzymatic activity without any effects on Cdk2 protein levels) without any effects on Cdk4 specific activity (or total protein levels). I3C treatment causes the loss of total cellular Cdk6 kinase activity, which is accounted for by the downregulation of Cdk6 transcripts and protein (see
Table 1). Immunoprecipitation of I3C-treated and -untreated cells reveals that a key ramification of the loss of total cellular Cdk2 and Cdk6 functions results in a significant reduction in the endogenous phosphorylated form of Rb, which causes the block in cell-cycle progression (summarized in
Table 1). Thus, the G1 cell-cycle arrest of human breast cancer cells results from the selective inhibition of Cdk6 expression and inhibition of Cdk2 specific activity.
Synergistic effects of I3C and tamoxifen on cell-cycle control
A key clinical issue is that one-third of breast cancers are estrogen responsive for cell growth, and for these patients, the estrogen antagonist tamoxifen can be used as an initial treatment therapy. I3C but not tamoxifen reduces the level of Cdk6 expression ( 27). The unique pattern of I3C-regulated gene expression and activity of cell-cycle components suggest that this indole functions through an antiproliferative pathway that is distinct from tamoxifen; therefore, I3C may synergize with this estrogen anatagonist in controlling cell growth. Flow cytometry profiles and incorporation of [3H]thymidine demonstrate that a combination of tamoxifen and I3C more effectively induce G1 cell-cycle arrest in the estrogen-responsive MCF-7 breast cancer cells than in cells treated with either agent alone ( 27). Moreover, a combination of both agents also causes a modest increase in production of p21Waf1/Cip1 as compared with the effects of the individual treatments and completely ablates the production of phosphorylated Rb, a form that is critical for cell-cycle progression ( 27).
I3C transcriptional mechanism of action: control of Cdk6 promoter activity through a composite Sp1-containing DNA element
Northern blot and RT-PCR analyses of Cdk6 mRNA decay rates in breast cancer cells reveal that I3C rapidly downregulates Cdk6 transcript levels without any effect on Cdk6 transcript stability (
28). These results imply that I3C treatment regulates transcription of the Cdk6 gene as an integral feature of the indole-mediated G1 cell-cycle arrest. To further elucidate the mechanism by which I3C downregulates Cdk6 transcription, we conducted the initial cloning and functional characterization of the human Cdk6 gene promoter (
28), which surprisingly had not been identified before our studies (GenBank database, accession no. AF332591). The Cdk6 gene is located in chromosome 7q21 and comprises seven exons that span 200 kb. The six introns range in size from 2,785 to 58,259 bp, and we identified and cloned 6,000 bp of the Cdk6 5' promoter flanking region (
28).
To functionally define the cis-acting region of the Cdk6 promoter that confers responsiveness to I3C, deletion fragments ranging from -2,464 to -196 bp (3' termini at +24) were cloned into the promoterless pGL2-basic luciferase reporter, and each of the promoter-reporter plasmids was stably transfected into MCF-7 cells ( 28). After 3 wk of selection (to form a population of transfected cells, not individual cell clones), Cdk6 promoter activity and the cell-cycle arrest were examined in each stable pool of transfected cells treated with or without I3C for 48 h. This analysis uncovered a 167-bp I3C-responsive region of the Cdk6 promoter between -805 and -638 bp of the Cdk6 promoter that mediates the I3C downregulation of Cdk6 promoter activity ( 28). I3C causes a near-maximal inhibition of Cdk6 promoter activity, and this transcriptional response is specific for the indole antiproliferative pathway ( Fig. 1A). The I3C downregulation of Cdk6 promoter activity is not an indirect consequence of the growth-arrested state of the cells, because treatment with tamoxifen has no effect on this promoter ( Fig. 1A). Also, treatment with tryptophol ( 28), which is closely related to I3C and contains an ethanol group instead of a methanol group in the 3-carbon position, neither alters Cdk6 promoter activity nor induces a G1 arrest of MCF-7 cells ( Fig. 1A).
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B (NF-B), Ets and Sp1 families of transcription factors. Mutagenic analysis (
28) of each of these three transcription factor consensus sequences in the context of the -920-bp Cdk6-luciferase reporter plasmid reveals that both the Sp1- and Ets-like sites, which are spaced 5 bp apart, are necessary for the I3C downregulation of Cdk6 promoter activity in stably transfected MCF-7 breast cancer cells (
Fig. 1B). I3C does not alter total cellular Sp1 levels and fails to inhibit reporter plasmid activity of MCF-7 cells transfected with an Sp1-chloramphenicol acetyl transferase (CAT) reporter construct that encodes three consensus Sp1 DNA-binding sites, which suggests that I3C responsiveness requires the context of the Cdk6 promoter.
Electrophoretic mobility-shift analysis (
28) of the protein-DNA complexes formed with nuclear proteins isolated from I3C-treated and -untreated cells in combination with supershift assays that used Sp1 antibodies demonstrate that the Sp1-binding site in the Cdk6 promoter forms a specific I3C-responsive DNA-protein complex that contains the Sp1 transcription factor (
Fig. 2). A competitive gel-shift analysis was performed in which mutated promoter fragments were tested for their ability to disrupt the gel-shifted complex that forms on the wild-type Cdk6 promoter. Mutations in Ets or NF-B sites have no effects on the strong competition for formation of the I3C-regulated protein-DNA complex on wild-type sequences, whereas consistent with the supershift results, mutations in the Sp1 site ablate the ability of the promoter fragment to compete. Thus, the DNA-binding capabilities of Sp1 but not Ets are required for the selective interactions of the putative protein complex with the Cdk6 promoter. Taken together, our results establish that the I3C treatment downregulates Cdk6 transcription by selective disruption of the interactions of Sp1 with a composite DNA-binding site within the Cdk6 promoter.
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Breast cancer cells were incubated with [3H]I3C to examine potential I3C-derived products that form in the cell cultures. Gas chromatography and mass spectrometry analysis reveal that I3C is surprisingly inert to metabolism by these cells; it displays a half-life in medium of 40 h. A fraction of [3H]I3C is converted intracellularly into [3H]DIM (a dimerization product of I3C), and the newly formed [3H]DIM accumulates in the nucleus. This suggests that DIM may have a role in the cellular biological activities of I3C (
42) (summarized in
Fig. 3). Both glutathione and protein thiol adducts of I3C (which are inactive degradative products) are the primary conversion products identified after 16 h. These results strongly suggest that the indole effects observed in cultured cells are likely due to the cellular activities of both I3C and DIM.
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The potential antiproliferative effects of DIM were examined in human breast cancer cell lines by characterizing the incorporation of [3H]thymidine and by flow cytometry for DNA content (summarized in Table 1). DIM treatment strongly inhibits breast cancer cell proliferation, and similar to I3C, induces G1 cell-cycle arrest in both estrogen-responsive and -nonresponsive cells ( 37). Also, similar to I3C, higher concentrations of DIM induce breast cancer cells to undergo programmed cell death as monitored by externalization of phosphatidylserine, chromatin condensation and DNA fragmentation ( 38). As part of this response, DIM treatment decreases expression of the apoptosis inhibitory protein Bcl-2 and increases the level of the Bax proapoptotic protein ( 38).
I3C and DIM transcriptional action mechanism: stimulation of p21Waf1/Cip1 promoter activity through a promoter region that contains Sp1 DNA elements
In MCF-7 breast cancer cells, treatment with either I3C or DIM strongly stimulates promoter activity of p21Waf1/Cip1 under conditions in which these indoles induce a G1 cell-cycle arrest ( 37). This stimulation in promoter activity accounts for the indole-mediated increase in p21Waf1/Cip1 transcript and protein levels and implies a transcriptional mechanism of action. Furthermore, indoles directly stimulate p21Waf1/Cip1 transcript levels, because the response can be observed in the absence of de novo protein synthesis ( 37) ( Fig. 4A). Using the same general strategies described above for Cdk6, the cis-acting indole-responsive elements in the p21Waf1/Cip1 promoter are characterized by transfection of serial 5' deletions of the promoter linked to X CAT-reporter plasmids. The promoter-reporter plasmids were initially tested for indole-responsive activities in transfected cells treated for 24 h with or without DIM. The DIM-responsive region is found to be located within the -291-bp p21Waf1/Cip1 promoter fragment ( Fig. 4B). Interestingly, this region contains six consensus elements for the Sp1 transcription factor, which together with our information on the Cdk6 gene promoter suggests Sp1 as a possible candidate for indole regulation of the p21Waf1/Cip1 promoter. I3C has essentially the same effect as DIM in stimulating p21Waf1/Cip1 promoter activity. Thus, p21Waf1/Cip1 is an important indole target gene in human reproductive cells.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Our hypothesis is that I3C and the DIM converted from I3C intracellularly induce a G1 cell-cycle arrest of human breast cancer cells by interacting with specific target protein(s) that subsequently control the transcription of key cell-cycle components that directly participate in the G1 cell-cycle arrest ( Fig. 5). We recently detected a nuclear I3C-binding activity (unpublished result), and conceivably, I3C and DIM can bind to the same intracellular target protein and result in indole-specific cellular effects. Alternatively, I3C and DIM could potentially interact with distinct target proteins to activate antiproliferative pathways in reproductive cancer cells. I3C and DIM appear to have distinct but overlapping effects on the transcriptional control of cell-cycle genes in breast cancer cells. Treatment with I3C but not DIM selectively disrupts the interactions of the Sp1 transcription factor with a composite DNA-binding site within the Cdk6 promoter. In contrast, either I3C or DIM stimulates the interactions of Sp1 with the p21Waf1/Cip1 promoter, which implicates a key role for this transcription factor in regulated cell-cycle control by indoles ( Fig. 5). The level of total functional Sp1 is not altered in indole-treated cells. Thus, the most straightforward interpretation of our results is that the Sp1 transcription factor is one of the downstream targets of the indole-signaling pathway in the context of the Cdk6 and p21Waf1/Cip1 promoters, which is a necessary step to arrest the growth of breast cancer cells.
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The proliferation of eukaryotic cells is regulated by an intricate network of growth-inhibitory and -stimulatory signal transduction pathways that generally converge on individual cellular components that control the cell cycle ( 45, 50). The unique feature of the indole antiproliferative signaling pathway in breast cancer cells is that expression of Cdk6 is downregulated under conditions in which p21Waf1/Cip1 gene expression is stimulated, which results in the loss of both Cdk6 and Cdk2 cellular activity. The transcription of both G1 acting–cell cycle genes is regulated in opposite directions by changes in Sp1 interactions with the corresponding promoters. Our results are consistent with numerous studies that demonstrate that regulated changes in the expression and/or activity of cell-cycle components that act within G1 are closely associated with alterations in the proliferation rate of normal and transformed reproductive epithelial cells ( 51– 54). For example, up to 45% of human breast cancers show an aberrant expression and/or amplification of cyclins D1 or E ( 55– 58). The estrogen-induced activation of Cdk4 and Cdk2 during the progression of human breast cancer cells from the G1 to the S phase is accompanied by increased expression of cyclin D1 and decreased association of Cdk inhibitors with the cyclin E–Cdk2 complex ( 59).
Our evidence uncovers a previously uncharacterized indole-signaling pathway in breast cancer cells that regulates the expression and activity of a distinct combination of cell-cycle components that is different from the actions of the estrogen antagonist tamoxifen. The observed synergism between indoles and tamoxifen suggests that I3C- and DIM-based compounds have the potential for use in combinatory therapies with estrogen antagonists for steroid-responsive breast cancers. Recently, we observed that I3C and DIM induce a G1 cell-cycle arrest of human LNCaP prostate cancer cells that is accompanied by the same selective downregulation of Cdk6 gene expression and strong stimulation of p21 gene expression as observed with human breast cancer cells. Although the mechanism is largely unknown, there is strong evidence that similar dietary factors contribute to both breast and prostate cancers. Furthermore, there are many common epidemiological features between breast and prostate cancer that include parallel incidence rates in various countries, lifetime risks, death rates, ethnic trends and the dual occurrence of the two cancers in some families alone ( 1, 60). Thus, our studies implicate the existence of a transcriptional indole-signaling pathway that targets cell-cycle gene promoters in several different types of human reproductive cancer cells. A key future focus of our work is to identify the nuclear indole target protein, because this indole target protein and its downstream transcriptional regulators can potentially be used to develop novel I3C-based anticancer therapeutics for reproductive cancer cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 This work was supported by grant CA-69056 from the National Institutes of Health, by grant BC990908 awarded by the Department of Defense, Army Breast Cancer Research Program and by grant 5JB-0016 awarded by the California Breast Cancer Research Program.
4 Abbreviations used: Cdk, cyclin-dependent kinase; DIM, 3-3'-diindolylmethane; I3C, indole-3-carbinol; Rb, retinoblastoma protein; RT-PCR, reverse transcriptase–polymerase chain reaction.
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