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Activation and Potentiation of Interferon- Signaling by 3,3'-Diindolylmethane in MCF-7 Breast Cancer Cells

Departments of Nutritional Sciences and Toxicology (J.E.R., L.X., L.F.B.) and Molecular and Cell Biology (U.C., G.L.F.), University of California, Berkeley, California; and Incyte Genomics, Fremont, California (E.L.B.)

Received July 19, 2005; accepted November 1, 2005

	Abstract

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3,3'-Diindolylmethane (DIM), a natural autolytic product in plants of the Brassica genus, including broccoli, cauliflower, and Brussels sprouts, exhibits promising cancer protective activities, especially against mammary neoplasia in animal models. We observed previously that DIM induced a G₁ cell-cycle arrest and strong induction of cell-cycle inhibitor p21 expression and promoter activity in both estrogen-responsive and -independent breast cancer cell lines. We showed recently that DIM up-regulates the expression of interferon (IFN) in human MCF-7 breast cancer cells. This novel effect may contribute to the anticancer effects of DIM because IFN plays an important role in preventing the development of primary and transplanted tumors. In this study, we observed that DIM activated the IFN signaling pathway in human breast cancer cells. DIM activated the expression of the IFN receptor (IFNGR1) and IFN-responsive genes p56- and p69-oligoadenylate synthase (OAS). In cotreatments with IFN, DIM produced an additive activation of endogenous p69-OAS and of an OAS-Luc reporter and a synergistic activation of a GAS-Luc reporter. DIM synergistically augmented the IFN induced phosphorylation of signal transducer and activator of transcription factor 1, further evidence of DIM activation of the IFN pathway. DIM and IFN produced an additive inhibition of cell proliferation and a synergistic increase in levels of major histocompatibility complex class-1 (MHC-1) expression, accompanied by increased levels of mRNAs of MHC-1-associated proteins and transporters. These results reveal novel immune activating and potentiating activities of DIM in human tumor cells that may contribute to the established effectiveness of this dietary indole against various tumors types.

Interferons (IFNs) are a group of immune cytokines with antiviral and cytostatic functions. Type I IFNs, including IFN and IFN, are produced by virus-infected cells. Type II IFN, usually called interferon- (IFN) or the "immune interferon", promotes B cell differentiation into immunoglobulin-producing cells (Boehm et al., 1997). The recently recognized antitumor activity of IFN includes the priming of macrophages for nonspecific tumoricidal activity; the activation of monocytes, natural killer cells, and T cells to increase cytotoxicity against tumor cells; and the inhibition of tumor-induced angiogenesis (Ikeda et al., 2002). The possible therapeutic use of IFN in cancer patients, however, has been limited because of serious side effects (Wimer, 1998).

In a recent report (Xue et al., 2005), we showed that DIM can up-regulate the expression of IFN in the human MCF-7 breast cancer cell line. Using promoter deletions, we showed that the region between –108 and –36 bp in the IFN promoter, which contains two conserved and essential regulatory elements, is required for DIM-induced IFN expression. DIM activates both c-Jun NH₂-terminal kinase and p38 pathways, induces the phosphorylation of c-Jun and ATF-2, and increases the binding of the homodimer or heterodimer of c-Jun/ATF-2 to the proximal AP-1–cAMP response element-binding protein–ATF-binding element. Moreover, studies with specific enzyme inhibitors showed that upstream Ca²⁺-dependent kinase(s) is required for the inducing effects of DIM in MCF-7 cells. These results established that DIM-induced IFN expression in human breast tumor cells is mediated by activation of both c-Jun NH₂-terminal kinase and p38 pathways, which is ultimately dependent on intracellular calcium signaling.

In the present study, we report that DIM activated the IFN signal transduction pathway in human breast cancer cells. DIM treatment increased the expression of IFN receptor-1 (IFNGR1) and the IFN-responsive genes p56 and p69-oligoadenylate synthase (OAS). In addition, DIM produced a synergistic increase in IFN-induced activation of phosphorylation of signal transducer and activator of transcription 1 (STAT-1) and reporter gene expression. Finally, DIM and IFN exhibited additive antiproliferative activities in cultured cells but produced synergistic increases in expression of major histocompatability complex class I (MHC-I). This latter result is consistent with a strong immune potentiation activity of DIM.

	Materials and Methods

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Materials. Dulbecco's modified Eagle's medium, Opti-MEM, and Lipofectamine were supplied by Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) and human recombinant IFN, were supplied by Sigma Chemical Co. (St. Louis, MO). [-³²P]ATP was supplied by New England Nuclear (Boston, MA). DIM was prepared from I3C as described previously (Grose and Bjeldanes, 1992) and recrystallized in toluene. All other reagents were of the highest grade available.

Cell Culture. The human breast adenocarcinoma MCF-7 cell line, obtained from the American Type Culture Collection (Manassas, VA), was grown as adherent monolayers in Dulbecco's modified Eagle's medium, supplemented to 4.0 g/l glucose, 3.7 g/l sodium bicarbonate, and 10% heat-inactivated FBS, in a humidified incubator at 37°C and 5% CO_2, and passaged at approximately 80% confluence. Cultures used in subsequent experiments were at less than 25 passages.

Cell Counting. Cells were harvested by trypsinization and resuspended in complete medium. Aliquots were diluted 50-fold in Isoton II (Beckman Coulter, Fullerton, CA), and 500-µl duplicates were counted in a model Z1 Coulter particle counter and averaged.

Microarray Analysis of Differential Expression. MCF-7 cells were treated with 50 µM DIM for 24 h or with the DMSO vehicle for controls. Poly-A-RNA was isolated by two cycles of purification on oligo-dT cellulose. Labeled cDNA probes were prepared using the fluorescent dyes cyanine 3 and cyanine 5 for the treated and the control samples, respectively, and were hybridized simultaneously to microarrays (Incyte Genomics, Fremont, CA) of 960 selected cDNAs representing human genes involved in cell cycling, apoptosis, signal transduction, motility, adhesion, and angiogenesis. Differential expression was measured by the ratio of the fluorescence intensity at the wavelengths corresponding to the two probes. The samples were spiked with known concentrations of various nonhuman cDNA to serve as positive controls and to correct for variations in hybridization efficiency.

Determination of mRNA by RT-PCR. Cells were lysed by addition of Tri-reagent (Molecular Research Center, Inc., Cincinnati, OH) and chloroform was used for phase separation. After centrifugation, the aqueous upper phase was collected and total RNA was precipitated by isopropanol, washed with 75% ethanol, and dissolved in diethyl pyrocarbonate-treated water. Levels of specific mRNAs were determined by reverse transcription-polymerase chain reaction (RT-PCR) using Invitrogen enzymes and reagents. Reverse transcription was done using a 15-mer oligo-dT primer to generate cDNAs from mRNAs only. The sequences of the PCR primer pairs for specific mRNA are given in Table 1. After the appropriate number of cycles, the PCR products were dyed with ethidium bromide, separated on a 1.5% agarose gel, and photographed under UV light to verify the size of the amplicons and relative abundance of the specific mRNA templates.

Reporter Assays. The interferon-inducible luciferase reporter pGL2-25AS (–972) (OAS-Luc) was a gift from Dr. Georgia Floyd-Smith (Arizona State University, Tempe, AZ) (Floyd-Smith et al., 1999). OAS-Luc contains the promoter and 5'-flanking region (–972) of p69-OAS. The consensus GAS reporter, GAS-Luc, containing four repeats of the GAS element (5'-GATCAGTGATTTCTCGGAAAGAGAG-3') from the IFN consensus sequence binding protein (consensus motif underlined) was a gift from Dr. Keiko Ozato (National Institute of Child Health and Human Development, Bethesda, MD) (Kanno et al., 1993). Reporter plasmids were transiently transfected in MCF-7 cells by lipofection (Lipofectamine; Invitrogen), and after treatments as indicated under Results, luciferase activity in cell lysates was measured as described previously (Riby et al., 2000).

Gel Mobility Shift Assay. Nuclear extracts of cells were prepared as described previously (Riley et al., 2000) at the end of the treatment periods indicated under Results. The complementary 25-mer oligonucleotides 5'-GATCAGTGATTTCTCGGAAAGAGAG-3' and 5'-GATCCTCTCTTTCCGAGAAATCACT-3', containing the palindromic -activated sequence (GAS) consensus motif (underlined), were annealed and 5'end-labeled with [-³²P]ATP using T4 nucleotide kinase. The resulting labeled, double-stranded DNA probe was purified on a Sephadex G50 spin-column, precipitated in ethanol, dissolved in Tris-EDTA buffer, and diluted in 25 mM HEPES, 1 mM dithiothreitol (DTT), 10% glycerol, and 1 mM EDTA to contain approximately 25,000 cpm/µl ³²P. Nuclear extracts (7 µg of proteins), were mixed with 90 ng of poly(dI-dC), 25 mM HEPES, 1 mM DTT, 10% glycerol, 1 mM EDTA, and 160 mM KCl in a total volume of 21 µl. For antibody supershift experiments, 0.5 µg of monoclonal mouse-IgG anti-human-IFN (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the incubation mixture. After incubation for 15 to 20 min at room temperature, 4 µl (100,000 cpm) of end-labeled ³²P-ERE probe was added and incubated for another 15 min at room temperature. After addition of 2.8 µl of 10x Ficoll loading buffer (0.25% bromphenol blue, and 25% Ficoll type 400), 22-µl aliquots were loaded unto a prerun, nondenaturing 4.0% polyacrylamide gel in 67 mM Tris, 33 mM sodium acetate, and 10 mM EDTA, pH 8.0, at 120 V for 2 h. The gel was then dried and autoradiographed using BioMax MR film (Eastman Kodak, Rochester, NY).

Flow Cytometry Analysis of Cell Cycle Measurements. MCF-7 cells were plated at 10,000 cells/well of a six-well tissue culture dish and treated for 6, 24, 48, 72, 96, 120, and 144 h in complete medium. DIM was added to a final concentration of 10 and 30 µM and IFN to 10 ng/ml. Medium was changed every 24 h. After treatment, cells were washed with phosphate-buffered saline and hypotonically lysed in 1 ml of DNA staining solution (0.5 mg/ml propidium iodide, 0.1% sodium citrate, and 0.05% Triton X-100). Cell debris was removed by filtration through 60-µm nylon mesh (Sefar America Inc., Kansas City, MO). Nuclear-emitted fluorescence with wavelengths of >585 nm was measured with a Coulter Elite instrument. Ten thousand nuclei were analyzed from each sample at a rate of 300 to 500 nuclei/second. The percentages of cells within the G₁,S, and G₂/M phases of the cell cycle were determined by analysis with the Multicycle software MPLUS (Phoenix Flow Systems) in the Cancer Research Laboratory Microchemical Facility of the University of California, Berkeley.

Flow Cytometry Analysis of MHC-I Expression. Flow cytometric analysis of membrane immunofluorescence was performed as follows: MCF-7 cells were pretreated with 30 µM DIM separately for 6, 24, or 48 h and then treated with different concentrations of IFN for another 16 h. Cells were harvested, washed with phosphate-buffered saline (PBS), and then 10⁶ cells in 90 µl of PBS were incubated with 10 µl of FITC-conjugated HLA-ABC mouse monoclonal antibody or negative control FITC-conjugated IgG2b antibody (Chemicon International, Temecula, CA) on ice for 30 min. After washing twice with PBS, the fluorescence density was measured using a Coulter Elite instrument and analyzed with WinMDI 2.8 software provided by Duke University (Durham, NC). The expression of HLA-1complex was calculated as the percentage of antibody-positive cells (%PC) and mean fluorescence value (MFV).

Western Blot Analysis. After the indicated treatments, cells were harvested in radioimmunoprecipitation assay buffer (150 mM NaCl, 0.5% deoxycholate, 0.1% Nonidet P-40, 0.1% SDS, and 50 mM Tris) containing protease and phosphatase inhibitors (50 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 0.1 µg/ml NaF, 1 mM dithiothreitol, 0.1 mM sodium orthovanadate, and 0.1 mM glycerophosphate). Equal amounts of total cellular protein were mixed with loading buffer (25% glycerol, 0.075% SDS, 1.25 ml of 14.4 M 2-mercaptoethanol, 10% bromphenol blue, and 3.13% stacking gel buffer) and fractionated by electrophoresis on 10% polyacrylamide/0.1% SDS resolving gels. Rainbow marker (GE Healthcare, Little Chalfont, Buckinghamshire, UK) was used as the molecular weight standard. Proteins were electrically transferred to nitrocellulose membranes (Micron Separations, Inc., Westborough, MA) and blocked overnight at 4°C with 5% non-fat dry milk in 1x Western wash buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20). Blots were subsequently incubated with antibodies against IFNGR1, phosphorylated STAT1 (P-STAT1), or STAT1 protein purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody concentration was 2 µg/ml in Western wash buffer. Immunoreactive proteins were detected after 1 h of incubation at room temperature with horseradish peroxidase-conjugated secondary antibodies. Goat anti-rabbit antibodies were used as secondary antibodies (Bio-Rad) after being diluted 1:3000 in wash buffer. Blots were treated with enhanced chemiluminescence reagents (PerkinElmer Life and Analytical Sciences, Boston, MA), and fluorescence was detected using BioMax MR film (Eastman Kodak). Equal protein loading was ascertained by Ponceau S staining of blotted membranes and by reprobing the membranes with antitubulin antibody.

Statistical Analyses. Statistically significant differences were determined by two-way analysis of variance using the SigmaStat software (Systat Software, Inc., Point Richmond, CA).

	Results

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DIM Activates Transcription of IFN and Related Genes in MCF-7 Human Breast Tumor Cells
An initial cDNA gene expression microarray screen of human breast cancer MCF-7 cells treated with DIM, followed by confirmation of results using semiquantitative RT-PCR, established the transcriptional activation of IFN-IFNGR1-OAS family member protein 69 (p69-OAS) and interferon-inducible protein 56 (p56) (Fig. 1). The expressions of IFN, p69-OAS, and p56 were fully induced after 6 h of treatment, whereas maximum IFNGR1 induction was reached after 48 h of treatment. As shown previously (Xue et al., 2005), the level of mRNA for IFN returned to near control level after 48 h of DIM treatment. The mRNA levels for p56, IFNGR1 and p69-OAS remained elevated after 48 h of treatment. Maximum induction for IFN, IFNGR1, p69-OAS, and p56 were 4.6-, 3.8-, 3.2-, and 4.0-fold, respectively. The mRNAs for IFN and IFN were not detectable by RT-PCR analysis in DIM-treated or -untreated MCF-7 cells (data not shown).

View larger version (67K): [in this window] [in a new window]   Fig. 1. Transcriptional activation of IFN and related genes by DIM. Cells were treated with 50 µM DIM for the indicated times. The mRNA levels were measured by RT-PCR. Quantitative detection of amplicons required 40 PCR cycles for IFN and 17 cycles for the other genes. Images of the ethidium bromide-stained gels were inverted (negative black/white) for presentation. Molecular weights are compared with a 100-bp ladder (darker band is 600 bp) to confirm identity of the amplicons. GAPDH was used as the control. Densitometry results are presented as -fold induction over the 0-h treatment after correction for GAPDH. Results are presented as the mean ± S.D. of three separate experiments. *, statistically significant difference with time 0 control (p < 0.05).

View larger version (37K): [in this window] [in a new window]   Fig. 2. Western blot analysis of INFGR1. Cells were treated with DMSO (control) or 50 µM DIM for 6, 24, or 48 h. The relative abundance of IFNGR1 was corrected for variations in tubulin, as a loading control between samples. A representative Western blot from three separate experiments is shown.

DIM Augments IFN-Induced Expression of the Endogenous p69-OAS Gene and Associated Reporter Gene Constructs

View larger version (38K): [in this window] [in a new window]   Fig. 3. DIM activates transcription of IFN-responsive genes. A and B, transcriptional activation of endogenous p69-OAS was measured by RT-PCR. Cells were treated with 10, 25, or 50 µM DIM alone or in combination with 10 ng/ml IFN for 24 h. In addition (B), some plates received 50 µM cycloheximide (CHX) 1 h before the beginning of the treatments to inhibit protein synthesis. Results are presented as the mean ± S.D. of three separate experiments. *, statistically significant difference with the corresponding DMSO control for each concentration of DIM (p < 0.05); **, significant difference with IFN alone (p < 0.05). C, cells transfected with the p69OAS-Luc reporter were treated with IFN (1.0–30 ng/ml) alone or in combination with DIM 50 µM, for 24 h. Transcriptional activity is expressed as -fold induction over the DMSO treated control. Results are presented as the mean ± S.D. of three separate experiments. *, statistically significant difference with the DMSO control (p < 0.05). D, cells transfected with the 4xGAS-Luc reporter were treated with IFN (10 pg/ml–1 ng/ml) alone or in combination with 50 µM DIM, for 24 h. Transcriptional activity is expressed as -fold induction over the DMSO-treated control. Results are presented as the mean ± S.D. of three separate experiments. *, statistically significant difference with the DMSO control (p < 0.05).

We determined next whether the inducing effects of DIM on endogenous p69-OAS gene expression required concurrent protein synthesis. Results presented in Fig. 3B indicate that cotreatments with the translation inhibitor cycloheximide blocked the inducing effects of DIM treatments as well as the enhancing effects of DIM on IFN-induced expression of endogenous p69-OAS.

Transfected Reporter Gene Constructs. To examine whether the inducing effects of DIM operate at the transcriptional level, the activities of two luciferase reporter constructs, OAS-Luc and 4GAS-Luc were measured after treatment with DIM. OAS-Luc contains the promoter and 5'-flanking region (–972) of p69-OAS and GAS-Luc contains four repeats of the consensus GAS element. MCF-7 cells transiently transfected with these reporter constructs were treated with IFN, DIM, or a combination of both for 24 h. As shown in Fig. 3C, DIM treatment induced expression of the OAS-Luc reporter to a maximum of approximately 2.3-fold, a level of induction that was somewhat less than the level we observed for induction of the corresponding endogenous gene of over 3-fold. IFN induced transcriptional activity of this reporter in a concentration-dependent manner to a maximum of only approximately 1.8-fold, which is considerably less than the response of the endogenous gene of approximately 5.0-fold. In marked contrast to the maximum effects of combined treatments of the inducers on endogenous gene expression (an increase of nearly 12-fold compared with vehicle-treated cells), the reporter gene construct responded with less than a 3-fold increase over control cells. These results show that whereas the reporter and endogenous gene responded similarly to treatment with DIM by itself, the reporter was considerably less responsive than the endogenous gene to IFN.

Finally, studies with cells transfected with the 4GAS-Luc reporter (Fig. 3D) showed an inducing effect of DIM that was similar to the responses of the p69-OAS gene but a more robust response to IFN that was accompanied by a consistent synergistic response to cotreatments with the two inducers. Thus, treatment with DIM (50 µM) by itself again produced a 2- to 3-fold increase in activity of this reporter. IFN treatments, however, produced a robust concentration-dependent increase in reporter activity to approximately 40-fold induction compared with vehicle-treated controls. Cotreatments with DIM and IFN produced a roughly 2-fold synergistic activation of the 4GAS-Luc reporter at all IFN concentrations examined, reaching a maximum induction of approximately 75-fold.

Taken together, these results show that DIM by itself produced similar levels of activation of endogenous and transfected reporter genes that are differentially responsive to IFN. The results show further that the inducing effects of DIM are mediated by a short-lived transcription factor or require de novo synthesis of an intermediate regulatory protein.

Synergistic Effects of DIM on IFN-Mediated Signal Transduction
To probe further the role of GAS element activation in transcriptional activation by DIM, we examined the effect of this indole on downstream events in the GAS signaling pathway. For this purpose, the effects of DIM on phosphorylation of STAT-1 were determined, and the binding of the activated STAT-1 dimer to the cognate GAS responsive element were examined. Phosphorylation of STAT-1 by Janus-activated kinase (JAK) is the first step in IFN signaling after binding to IFNGR1. For these studies, cells were pretreated with DIM for 6 or 24 h followed by a 15-min exposure to IFN. STAT-1 phosphorylation levels in lysates of DIM-treated cells were compared with levels in control cells that received IFN but had not been treated with DIM. Phosphorylation was measured by Western blot analysis using an antibody specific to STAT-1 phosphorylated on Tyr-701 residue. Results presented in Fig. 4 show that whereas DIM treatment by itself for 6 or 24 h did not induce STAT-1 phosphorylation, pretreatment with DIM augmented IFN-induced STAT-1 phosphorylation by more than 4-fold after 24-h DIM pretreatment. These treatments did not significantly affect the levels of inactive (total) STAT-1.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Effects of DIM on STAT-1 activation. Cells were pretreated with 50 µM DIM for 0, 6, or 24 h. After treatments, some plates were also treated with 10 ng/ml IFN for 15 min. Phosphorylated STAT-1 and total STAT-1 were measured by Western analysis. Three replicate blots were analyzed by densitometry. Results are presented as the mean ± S.D. of three separate experiments. A representative Western blot from three separate experiments is shown. *, statistically significant difference with the IFN alone without pretreatment with DIM (p < 0.05); **, significant difference between 6 and 24 h of DIM pretreatments (p < 0.05).

View larger version (38K): [in this window] [in a new window]   Fig. 5. Gel mobility shift analysis of STAT-1 binding to GAS. Cells were pretreated with 50 µM DIM for the indicated periods and then with 10 ng/ml IFN for 30 min. Nuclear extracts were incubated with a 32P-labeled DNA probe containing a GAS consensus sequence. The identity of the DNA binding protein was confirmed by supershifting with a STAT-1 antibody. Results are representative of three separate experiments.

Taken together, these results show that DIM can augment IFN-induced STAT-1 activation, dimerization, and binding to the GAS element in DNA. We obtained no evidence for STAT-1 activation by DIM by itself, however, under these assay conditions.

Effects of DIM and IFN on Proliferation and Cell Cycling
Possible functional consequences of the synergistic effects of DIM and IFN on gene expression were examined at the level of proliferation rates in MCF-7 cells. Cell proliferation was measured over a 4-day treatment period with treatments with DIM, IFN, or both substances and compared with a DMSO control. As shown in Fig. 6, with heat-treated serum, IFN alone reduced cell proliferation by as much as 70% compared with controls. DIM treatments reduced proliferation by as much as 60%. With the exception of the highest concentration of IFN, combined treatments with DIM and IFN resulted in roughly additive increases in the cytostatic activity compared with treatment with DIM by itself. These effects were accompanied by morphological changes characteristic of apoptosis (data not shown). We observed no significant effect of IFN on proliferation of cells grown in unheated serum (data not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 6. Cell proliferation. Plates were seeded with 4 x 104 cells and incubated overnight to allow attachment before starting the treatments. Treatments were DMSO as a control, 10 or 25 µM DIM, and 1, 10, or 100 ng/ml IFN alone or a combination of DIM in triplicate wells. A, cells were counted 4 days after the beginning of treatments. B, thymidine incorporation was measured after 24 h from the beginning of treatments. Results are presented as the mean ± S.D. of three separate experiments. *, statistically significant difference with the corresponding DMSO control for each concentration of IFN (p < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 7. Flow cytometry. The percentage of cells in the G1 phase of the cell cycle was measured in cells treated with DIM, IFN, or combinations of the two for 6 h or 1, 2, or 3 days. Results are presented as the mean ± S.D. of three separate experiments. *, statistically significant difference with the corresponding DIM treatment without IFN at the same time point is noted (p < 0.05).

DIM Potentiates IFN-Induced Expression of MHC-I Complex in MCF-7 Cells
Induced expression of the MHC-I complex is well established as an important downstream target of IFN-mediated signal transduction. To examine the possible synergistic effects of DIM on this highly significant metabolic product of IFN signaling, we tested the effects of cotreatment with DIM on IFN-induced expression of MHC-I complex in MCF-7 cells.

Flow cytometry analysis of cell surface MHC-I expression was conducted using FITC-conjugated HLA-ABC antibody. In an initial control experiment, cultured MCF-7 cells were pretreated with 30 µM DIM for 48 h and then with or without 10 ng/ml IFN for another 16 h. Analyses using a negative control antibody, FITC-conjugated mouse IgG2b, indicated no significantly induced signal (data not shown). In a subsequent experiment in which cells were treated with a range of IFN concentrations and analyzed with the anti-MHC-ABC antibody, a strong signal indicative of IFN-induced MHC-I expression (Fig. 8) was detectable at 0.1 ng/ml treatment, and the level seemed to plateau at 10 ng/ml. In cells treated with vehicle control only, MHC-I expression was not detected in greater than 99% of cells. In another control experiment, treatment of cells with 30 µM DIM induced MFV of approximately 60 after 48 h in only 2% of the cells. Pretreatment with 30 µM DIM for 48 h followed by treatment with 0.1 ng/ml IFN produced a further strong increase in %PC from 12.97% to 40.98%, but had no significant effect on MFV (Fig. 8D). Exposure to DIM of cells treated with 10 ng/ml of IFN, approximately 95% of which expressed MHC-I produced a strong increase in MFV from 72 to 131. As expected, addition of an IFN blocking antibody into the medium before the treatments abrogated the inducing effects of cotreatments of IFN and DIM on MHC-I expression (data not shown), confirming the requirement of the cytokine for the observed effects. In an analogous experiment, we observed no effect of DIM or IFN on MHC-II expression in the MCF-7 cells (data not shown). These results show that whereas DIM by itself produces little or no effect on expression of MHC-I, this indole strongly augments the proportion of cells that express this protein complex, as well as the maximum level of MHC-I per cell, in response to IFN.

View larger version (28K): [in this window] [in a new window]   Fig. 8. The expression level of MHC-I complex on the MCF-7 cell surface. A, cells were treated with different concentrations of IFN for 16 h. B and C, cells were pretreated with DMSO (control) or 30 µM DIM for 48 h, followed by 0.1 ng/ml of IFN (B) or 10 ng/ml of IFN (C) for another 16 h. One million cells were incubated in 90 µl of PBS with 10 µl of FITC-conjugated anti-HLA-ABC mouse monoclonal antibody. The fluorescence intensity was measured by flow cytometry using a Coulter Elite instrument and analyzed by WinMDI 2.8 software provided by Duke University. D, the expression of HLA class I complex was calculated as %PC (open bars) and MFV (cross-hatched bars).

DIM Enhances IFN-Induced Transcription of MHC-I Components and Transporters in MCF-7 Cells
Because the level of the IFN-induced MHC-I protein complex on the surface of MCF-7 cells could be enhanced by pretreatment with DIM, we examined whether expressions of the corresponding genes for the complex and its transporters were also increased. Results of RT-PCR analyses of human MHC-I, including HLA-A, HLA-B, HLA-C, and -microtubulin, and of two important associated transporters, TAP1 and TAP2, are presented in Fig. 9. The expression levels of all six genes were not significantly enhanced by DIM compared with vehicle-treated MCF-7 cells. As expected, the expressions of these genes were increased 2- to 8-fold by treatment with IFN. The results show that pretreatment of cells with DIM further increased the mRNA levels of these genes by at least 2-fold above the levels induced by IFN by itself, which equates to increases in mRNA levels of 4- to 16-fold above background levels.

View larger version (33K): [in this window] [in a new window]   Fig. 9. DIM further increases IFN-induced transcription of MHC-I components and two associated transporter proteins. Cells were pretreated with 30 µM DIM for 48 h, and 10 ng/ml of IFN was added into the medium for another 16 h. The mRNA levels were measured by RT-PCR. GAPDH was used as the control. Densitometry results are presented as -fold induction over the DMSO treatment after correction for GAPDH. Results are presented as the mean ± S.E. of three separate experiments. *, statistically significant difference from DMSO control (p < 0.05).

	Discussion

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The results of these studies indicate that DIM is an immunomodulator that not only can induce expression of IFN in breast tumor cells as shown in our previous report (Xue et al., 2005) but also can modify the response of the cells to exogenous exposure to this cytokine. We measured the levels of secreted IFN protein in conditioned medium, using a very sensitive ELISA assay, and found that after 3 days of DIM treatment, IFN had accumulated to a concentration of less than 100 pg/ml, whereas it was undetectable in DMSO controls (data not shown). Therefore, the inducing effects on the IFN-responsive genes and reporters do not seem to result from DIM-induced secretion of IFN, because this concentration is below the effective concentration of IFN treatments. Furthermore, it is unknown whether the immunoreactive material found in the conditioned medium had retained biological activity. The results of the present study show that in MCF-7 cells DIM can 1) induce the expression of IFN, the IFN receptor, and two IFN-inducible genes, 2) induce the expression of IFN-inducible reporter gene constructs, 3) synergistically augment IFN-mediated activation of the STAT-1 signal transduction pathway, 4) additively augment the cytostatic effects of IFN in cultured cells, and 5) synergistically augment the expression of IFN-induced MHC-I protein complex and associated mRNAs.

The signaling cascade regulated by IFN has been examined in considerable detail in many laboratories. Binding of IFN to the highly specific IFNGR1 on the membrane of target cells activates a phosphorylation cascade involving JAK1, JAK2, and STAT-1. Phosphorylated STAT-1 homodimerizes, translocates to the nucleus, and binds to the IFN-activated sequence in the promoter of IFN-inducible genes, and activates transcription. Activation of one such gene, p69-OAS, plays an important role in the prevention of replication of viral RNA in infected cells. In addition, p69-OAS has been identified recently as an inhibitor of cell growth and a pro-apoptotic protein related to Bcl-2 (Ghosh et al., 2001). Our results confirm that IFN can activate the JAK/STAT pathway in breast tumor cells and show that each of the steps in IFN-induced gene activation is synergistically augmented by treatments with DIM.

The effects of DIM are clearly distinguishable from the effects of other cytostatic agents that induce IFN expression. In one series of studies, the synergistic effects of retinoic acid (RA) and IFN in breast cancer cells were described. At first, a synergistic antiproliferative effect was observed after sequential treatment of cells with IFN and RA. The effect seemed to result from an IFN-mediated augmentation of RA cytostatic activity. IFN was shown to increase RA cytostatic potency by increasing levels of expression of retinoic acid receptor- and decreasing expression of cellular RA-binding proteins (Widschwendter et al., 1995). Thereafter, the effects of RA on IFN signaling were also examined in breast tumor cells. The results showed that RA could synergistically augment the effects of IFN on gene transcription in MCF-7 cells by a mechanism that involved RA-mediated augmentation of STAT1 expression (Kolla et al., 1996). In another series of studies, the effects of combined treatments of breast cancer cells with IFNs and the estrogen antagonist tamoxifen were examined. Cotreatments with these substances caused an augmentation of the expression of certain IFN-stimulated genes, including the transcription factors ISGF-3 and GAF (Lindner et al., 1997). Tamoxifen by itself, however, produced no effect on the expression of these transcription factors. Thus, our results with DIM show significant differences from the reported effects of RA and tamoxifen on IFN action. We observed only an additive inhibitory effect of DIM and IFN on MCF-7 cell proliferation, whereas the effect with RA was synergistic. Also, in contrast to RA, although DIM synergistically augmented IFN signal transduction, this indole did not increase STAT1 expression in the absence of IFN treatment. Finally, DIM treatment by itself clearly induced expression of IFNGR1, IFN, and OAS-related genes, effects that were not reported for RA or tamoxifen.

Our observation of a synergistic effect of DIM on IFN-induced expression of MHC-I is potentially of considerable importance because this complex plays a central role in tumor immunosurveillance. MHC-I molecules are required for the presentation of tumor-associated antigen to cytotoxic T lymphocytes. Decreased expression of MHC antigen is thought to protect tumor cells from immunosurveillance (Goodenow et al., 1985; Marincola et al., 2000). Therefore, loss or down-regulation of MHC-I has been shown to be a frequent event in breast tumorigenesis. Indeed, several studies with murine models of induced carcinogenesis have confirmed the role of down-regulation of MHC-I antigens in increasing the tumorigenic and metastatic potential of tumors (Tanaka et al., 1988). Conversely, induced MHC-I expression is thought to be important in antitumorigenic properties of IFN (David-Watine et al., 1990) and certain small molecule cancer therapeutic agents, including the nucleotide analog 5-azacytidine, cytosine, arabinoside, 5-fluorouracil, retinoids, vitamin D3, as well as the plant alkaloid vincristine (Geissmann et al., 2003; Ohtsukasa et al., 2003). In contrast to the activities of these other MHC-I inducers, we observed little or no effect of DIM by itself, whereas we observed a clear synergistic augmentation by DIM of IFN-induced MHC-I expression. Significant also was our observation that after treatments with the higher concentrations of DIM, the level of MHC-I expressed/cell was increased to levels that were unattainable by treatments with IFN by itself. The roles in these synergies of DIM-induced increases in expression of IFNGR1 or possibly of modified MHC-I protein processing and trafficking are under investigation in our laboratories.

A comparison between our current results with DIM and our recently published studies with I3C (Chatterji et al., 2004) shows some distinct differences in the activity of DIM and its in vivo precursor in their effects on IFN signaling. Similar to our current results with DIM, we showed in the previous work that I3C also can affect IFN signaling in MCF-7 cells by a mechanism that involves a strong and rapid increase in expression of IFNGR1 and synergistic activation of STAT1 signaling. Similar effects of DIM and I3C were also observed on cell proliferation and cell cycling. In contrast to the present results with DIM, however, we did not observe in the previous studies increased expression by I3C of the IFN-stimulated genes (p56 and p69-OAS). These results are consistent with the hypothesis that the effects of I3C on IFNGR1 expression are mediated by its slow conversion to DIM during incubation with the cells. Indeed, we have shown previously that DIM accumulates in the nucleus of cultured MCF-7 cells treated with I3C (Staub et al., 2002). We speculate that our failure to detect I3C-induced expression of p56 and p69-OAS in the previous studies may be due to insufficient intracellular concentrations of DIM or, more interestingly, to a requirement for extracellular exposure to DIM for induced expression of these genes. Further studies are in progress to test these possibilities.

Our results may provide a rationale for explaining the clinical effectiveness of indole treatments in the control of recurrent respiratory papillomatosis (RRP). I3C and DIM are said to be the most popular adjunct therapies for this disorder because of their effectiveness and low level of toxicity (Auborn, 2002; Wiatrak, 2003). RRP is caused by certain types of human papilloma viruses (Coll et al., 1997; Rosen et al., 1998), and a hallmark of this disease is the tendency of the papillomas to recur after surgical removal (Gissmann et al., 1982; Kashima et al., 1996). One report indicated that most patients (55.4%) responded to the treatment of I3C/DIM by slowing down the recurrence rate, and recurrence of the disease was completely inhibited in 19% of patients (Yuan et al., 1999). Previously suggested modes of action of I3C/DIM in the control of RRP include induction of a better estrogen metabolite balance (Yuan et al., 1999), inhibition of cell proliferation (Cover et al., 1998), and induction of apoptosis (Hong et al., 2002,Hong et al., 2002). Our results suggest that I3C/DIM may function by yet another mechanism, that of immune potentiation. If confirmed by on going studies in our laboratories, this activity might be useful not only in the prevention of recurrent papillomas but also in treatment of papillomatosis and prevention of malignant conversion of a broad range of tumor types.

In summary, our studies have shown that DIM can induce the expression of IFN, IFNGR1, and two IFN-responsive genes in cultured human breast tumor cells. The inducing effects on the IFN responsive genes do not seem to result from DIM-induced secretion of IFN, but they are dependent on de novo protein synthesis. DIM synergistically augments IFN-induced STAT-1 signaling, which, in combination with the increased expression of IFNGR1, may be responsible for an increase in MHC-I expression per cell that is not attainable by treatment with IFN by itself. However, the antiproliferative effects of DIM and IFN in MCF-7 cells are only additive, suggesting that these substances may affect cell cycling by unrelated mechanisms. Thus, these studies provide evidence that DIM is a novel immune potentiator that can induce IFN expression and potentiate IFN activities in breast tumor cells. Further studies of the immune function of DIM are in progress.

	Acknowledgements

 
We express our appreciation to members of both the Bjeldanes and Firestone laboratories for helpful comments throughout the duration of this work. We are also thankful to Hector Nolla for assistance in flow cytometry assay.

	Footnotes

 
This research was supported by the Department of Defense, Army Breast Cancer Research Program grant DAMDI7-96-1-6149 and by National Institutes of Health grant CA69056.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.017053.

ABBREVIATIONS: DIM, 3,3'-diindolylmethane; I3C, indole-3-carbinol; IFN, interferon; bp, base pair(s); ATF, activating transcription factor; AP-1, activator protein 1; IFNGR1, interferon- receptor-1; STAT, signal transducer and activator of transcription; RT, reverse transcription; PCR, polymerase chain reaction; GAS, interferon-–activated sequence; DTT, dithiothreitol; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; HLA, human leukocyte antigen; %PC, percentage of positive cells; MFV, mean fluorescence value; OAS, oligoadenylate synthase; JAK, Janus-activated kinase; DMSO, dimethyl sulfoxide; RA, retinoic acid; RRP, recurrent respiratory papillomatosis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Address correspondence to: Leonard F. Bjeldanes, Department of Nutritional Sciences and Toxicology, 119 Morgan Hall, University of California, Berkeley, CA 94720-3104. E-mail:lfb{at}nature.berkeley.edu

	References

Top Abstract Materials and Methods Results Discussion References

 
Auborn KJ (2002) Therapy for recurrent respiratory papillomatosis. Antivir Ther 7: 1–9.[Medline]

Boehm U, Klamp T, Groot M, and Howard JC (1997) Cellular responses to interferon-gamma. Annu Rev Immunol 15: 749–795.[CrossRef][Medline]

Chatterji U, Riby JE, Taniguchi T, Bjeldanes EL, Bjeldanes LF, and Firestone GL (2004) Indole-3-carbinol stimulates transcription of the interferon gamma receptor 1 gene and augments interferon responsiveness in human breast cancer cells. Carcinogenesis 25: 1119–1128.[Abstract/Free Full Text]

Chen I, McDougal A, Wang F, and Safe S (1998) Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane. Carcinogenesis 19: 1631–1639.[Abstract/Free Full Text]

Coll DA, Rosen CA, Auborn K, Potsic WP, and Bradlow HL (1997) Treatment of recurrent respiratory papillomatosis with indole-3-carbinol. Am J Otolaryngol 18: 283–285.[CrossRef][Medline]

Cover CM, Hsieh SJ, Tran SH, Hallden G, Kim GS, Bjeldanes LF, and Firestone GL (1998) 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. J Biol Chem 273: 3838–3847.[Abstract/Free Full Text]

David-Watine B, Israel A, and Kourilsky P (1990) Regulatory elements involved in the liver-specific expression of the mouse MHC class I Q10 gene: characterization of a new TATA-binding factor. Immunol Today 11: 286–292.[CrossRef][Medline]

Floyd-Smith G, Wang Q, and Sen GC (1999) Transcriptional induction of the p69 isoform of 2',5'-oligoadenylate synthetase by interferon-beta and interferon-gamma involves three regulatory elements and interferon-stimulated gene factor 3. Exp Cell Res 246: 138–147.[CrossRef][Medline]

Geissmann F, Revy P, Brousse N, Lepelletier Y, Folli C, Durandy A, Chambon P, and Dy M (2003) Retinoids regulate survival and antigen presentation by immature dendritic cells. J Exp Med 198: 623–634.[Abstract/Free Full Text]

Ghosh A, Sarkar SN, Rowe TM, and Sen GC (2001) A specific isozyme of 2'-5' oligoadenylate synthetase is a dual function proapoptotic protein of the Bcl-2 family. J Biol Chem 276: 25447–25455.[Abstract/Free Full Text]

Gissmann L, Diehl V, Schultz-Coulon HJ, and zur Hausen HJ (1982) Molecular cloning and characterization of human papilloma virus DNA derived from a laryngeal papilloma. J Virol 44: 393–400.[Abstract/Free Full Text]

Goodenow RS, Vogel JM, and Linsk RL (1985) Histocompatibility antigens on murine tumors. Science (Wash DC) 230: 777–783.[Abstract/Free Full Text]

Grose KR and Bjeldanes LF (1992) Oligomerization of indole-3-carbinol in aqueous acid. Chem Res Toxicol 5: 188–193.[CrossRef][Medline]

Grubbs CJ, Steele VE, Casebolt T, Juliana MM, Eto I, Whitaker LM, Dragnew KH, Kellof GJ, and Lubet RL (1995) Chemoprevention of chemically-induced mammary carcinogenesis by indole-3-carbinol. Anticancer Res 15: 709–716.[Medline]

Hong C, Firestone GL, and Bjeldanes LF (2002) Bcl-2 family-mediated apoptotic effects of 3,3'-diindolylmethane (DIM) in human breast cancer cells. Biochem Pharmacol 63: 1085–1097.[CrossRef][Medline]

Hong C, Kim HA, Firestone GL, and Bjeldanes LF (2002) 3,3'-Diindolylmethane (DIM) induces a G₁ cell cycle arrest in human breast cancer cells that is accompanied by Sp1-mediated activation of p21(WAF1/CIP1) expression. Carcinogenesis 23: 1297–1305.[Abstract/Free Full Text]

Ikeda H, Old LJ, and Schreiber RD (2002) The roles of IFN gamma in protection against tumor development and cancer immunoediting. Cytokine Growth Factor Rev 13: 95–109.[CrossRef][Medline]

Kanno Y, Kozak CA, Schindler C, Driggers PH, Ennist DL, Gleason SL, Darnell JE Jr, and Ozato K (1993) The genomic structure of the murine ICSBP gene reveals the presence of the gamma interferon-responsive element, to which an ISGF3 alpha subunit (or similar) molecule binds. Mol Cell Biol 13: 3951–3963.[Abstract/Free Full Text]

Kashima HK, Mounts P, and Shah K (1996) Recurrent respiratory papillomatosis. Obstet Gynecol Clin North Am 23: 699–706.[Medline]

Kolla V, Lindner DJ, Xiao W, Borden EC, and Kalvakolanu DV (1996) Modulation of interferon (IFN)-inducible gene expression by retinoic acid. Up-regulation of STAT1 protein in IFN-unresponsive cells. J Biol Chem 71: 10508–10514.

Lindner DJ, Kolla V, Kalvakolanu DV, and Borden EC (1997) Tamoxifen enhances interferon-regulated gene expression in breast cancer cells. Cell Biochem 167: 169–177.

Marincola FM, Jaffee EM, Hicklin DJ, and Ferrone S (2000) Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance. Adv Immunol 74: 181–273.[Medline]

Ohtsukasa S, Okabe S, Yamashita H, Iwai T, and Sugihara KJ (2003) Increased expression of CEA and MHC class I in colorectal cancer cell lines exposed to chemotherapy drugs. J Cancer Res Clin Oncol 129: 719–726.[CrossRef][Medline]

Riby JE, Chang GH, Firestone GL, and Bjeldanes LF (2000) Ligand-independent activation of estrogen receptor function by 3,3'-diindolylmethane in human breast cancer cells. Biochem Pharmacol 60: 167–177.[CrossRef][Medline]

Rosen CA, Woodson GE, Thompson JW, Hengesteg AP, and Bradlow HL (1998) Preliminary results of the use of indole-3-carbinol for recurrent respiratory papillomatosis. Otolaryngol Head Neck Surg 118: 810–815.[CrossRef][Medline]

Shertzer HG (1983) Protection by indole-3-carbinol against covalent binding of benzo[a]pyrene metabolites to mouse liver DNA and protein. Food Chem Toxicol 21: 31–35.[CrossRef][Medline]

Shertzer HG (1984) Indole-3-carbinol protects against covalent binding of benzo-[a]pyrene and N-nitrosodimethylamine metabolites to mouse liver macromolecules. Chem Biol Interact 48: 81–90.[CrossRef][Medline]

Staub RE, Feng C, Onisko B, Bailey GS, Firestone GL, and Bjeldanes LF (2002) Fate of indole-3-carbinol in cultured human breast tumor cells. Chem Res Toxicol 15: 101–109.[CrossRef][Medline]

Tanaka K, Yoshioka T, Bieberich C, and Jay G (1988) Role of the major histocompatibility complex class I antigens in tumor growth and metastasis. Annu Rev Immunol 6: 359–380.[CrossRef][Medline]

Wattenberg LW and Loub WD (1978) Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by naturally occurring indoles. Cancer Res 38: 1410–1413.[Abstract/Free Full Text]

Wiatrak BJ (2003) Overview of recurrent respiratory papillomatosis. Curr Opin Otolaryngol Head Neck Surg 11: 433–441.[Medline]

Widschwendter M, Daxenbichler G, Dapunt O, and Marth C (1995) Effects of retinoic acid and gamma-interferon on expression of retinoic acid receptor and cellular retinoic acid-binding protein in breast cancer cells. Cancer Res 55: 2135–2139.[Abstract/Free Full Text]

Wimer BM (1998) Implications of the analogy between recombinant cytokine toxicities and manifestations of hantavirus infections. Cancer Biother Radiopharm 13: 193–207.[Medline]

Xue L, Firestone GL, and Bjeldanes LF (2005) DIM stimulates IFNgamma gene expression in human breast cancer cells via the specific activation of JNK and p38 pathways. Oncogene 24: 2343–2353.[CrossRef][Medline]

Yuan F, Chen DZ, Liu K, Sepkovic DW, Bradlow HL, and Auborn K (1999) Antiestrogenic activities of indole-3-carbinol in cervical cells: implication for prevention of cervical cancer. Anticancer Res 19: 1673–1680.[Medline]

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