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Cancer Research 67, 674-683, January 15, 2007. doi: 10.1158/0008-5472.CAN-06-2907
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Nur77 Agonists Induce Proapoptotic Genes and Responses in Colon Cancer Cells through Nuclear Receptor–Dependent and Nuclear Receptor–Independent Pathways

Sung Dae Cho1, Kyungsil Yoon1, Sudhakar Chintharlapalli1, Maen Abdelrahim1, Ping Lei1, Stanley Hamilton2, Shaheen Khan3, Shashi K. Ramaiah4 and Stephen Safe1,3

1 Institute of Biosciences and Technology, Texas A&M University Health Science Center; 2 Division of Pathology and Laboratory Medicine, University of Texas M.D. Anderson Cancer Center, Houston, Texas; and Departments of 3 Veterinary Physiology and Pharmacology and 4 Pathobiology, Texas A&M University, College Station, Texas

Requests for reprints: Stephen Safe, Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, Veterinary Research Building 410, College Station, TX 77843-4466. Phone: 979-845-5988; Fax: 979-862-4929; E-mail: ssafe{at}cvm.tamu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nerve growth factor–induced B{alpha} (NGFI-B{alpha}, Nur77) is an orphan nuclear receptor with no known endogenous ligands; however, recent studies on a series of methylene-substituted diindolylmethanes (C-DIM) have identified 1,1-bis(3'-indolyl)-1-(phenyl)methane (DIM-C-Ph) and 1,1-bis(3'-indolyl)-1-(p-anisyl)methane (DIM-C-pPhOCH3) as Nur77 agonists. Nur77 is expressed in several colon cancer cell lines (RKO, SW480, HCT-116, HT-29, and HCT-15), and we also observed by immunostaining that Nur77 was overexpressed in colon tumors compared with normal colon tissue. DIM-C-Ph and DIM-C-pPhOCH3 decreased survival and induced apoptosis in RKO colon cancer cells, and this was accompanied by induction of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) protein. The induction of apoptosis and TRAIL by DIM-C-pPhOCH3 was significantly inhibited by a small inhibitory RNA for Nur77 (iNur77); however, it was evident from RNA interference studies that DIM-C-pPhOCH3 also induced Nur77-independent apoptosis. Analysis of DIM-C-pPhOCH3–induced gene expression using microarrays identified several proapoptotic genes, and analysis by reverse transcription-PCR in the presence or absence of iNur77 showed that induction of programmed cell death gene 1 was Nur77 dependent, whereas induction of cystathionase and activating transcription factor 3 was Nur77 independent. DIM-C-pPhOCH3 (25 mg/kg/d) also inhibited tumor growth in athymic nude mice bearing RKO cell xenografts. These results show that Nur77-active C-DIM compounds represent a new class of anti–colon cancer drugs that act through receptor-dependent and receptor-independent pathways. [Cancer Res 2007;67(2):674–83]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Indole-3-carbinol (I3C) conjugates are highly expressed in cruciferous vegetables, such as broccoli, Brussels sprouts, and cauliflower (1, 2), and this compound and its metabolites have been linked to the cancer chemopreventive and chemoprotective effects of vegetables (3–7). I3C is highly unstable in the acid environment of the gut and undergoes heterodimerization and rearrangement to give several products (8), including 3,3'-diindolylmethane (DIM), which also exhibits anticancer activities through modulation of multiple pathways, including activation of the aryl hydrocarbon receptor (AhR; ref. 9) and inhibition of the androgen receptor (AR; ref. 10). Other studies show that DIM inhibits growth and induces apoptosis in many different cancer cell lines, and these effects are associated with altered expression of cell cycle and growth regulatory genes, induction of growth inhibitory factors and apoptosis, modulation of kinase activities, decreased mitochondrial membrane potential, and induction of endoplasmic reticulum (ER) stress (11–19). The effectiveness of DIM as an anticancer agent is due, in part, to activation of one or more of these proapoptotic and/or growth inhibitory pathways.

Recent studies in this laboratory have used DIM as a synthetic scaffold for the synthesis of a series of 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methanes or methylene-substituted DIMs (C-DIM; ref. 20). The addition of the bulky substituted phenyl group abrogated interactions with the AhR and AR; however, initial results of receptor screening assays showed that compounds containing trifluoromethyl (DIM-C-pPhCF3), t-butyl (DIM-C-pPhtBu), and phenyl (DIM-C-pPhC6H5) substituents activated peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}; ref. 20). These PPAR{gamma} agonists inhibited growth and induced apoptosis in breast, pancreatic, colon, bladder, leukemia, and other cancer cell lines through both receptor-dependent and receptor-independent pathways (20–26).

DIM-C-pPhCF3 is a PPAR{gamma} agonist but also activates Nur77 (27), a member of the nerve growth factor–induced B (NGFI-B) subfamily of orphan nuclear receptors that contains three proteins [i.e., NGFI-B{alpha} (Nur77), NGFI-Bß (Nurr1), and NGFI-B{gamma} (Nor1; ref. 27)]. Subsequent screening of a series of C-DIM analogues also identified the p-methoxy (DIM-C-pPhOCH3) and unsubstituted (DIM-C-Ph) analogues as Nur77 agonists, which did not activate PPAR{gamma}. The Nur77-active C-DIMs inhibited growth and induced apoptosis in pancreatic cancer cells, and this result correlated with previous studies showing that overexpression of Nur77 (unliganded)–induced apoptosis in thymocytes (28). In this study, we show that Nur77 is expressed in several colon cancer cell lines, and that Nur77 is also overexpressed in human colorectal tumors compared with non-tumor tissue. Nur77 agonists induce tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) protein in RKO colon cancer cells, and we have used this cell line as a model to investigate the effects of the Nur77 agonists DIM-C-pPhOCH3 and DIM-C-Ph. The results indicate that Nur77 agonists inhibit growth and induce apoptosis and TRAIL in RKO cells, and these responses are inhibited by a small inhibitory RNA for Nur77 (iNur77). DIM-C-pPhOCH3 also inhibits tumor growth in athymic nude mice bearing RKO cell xenografts. Microarray and cell culture studies also show that DIM-C-pPhOCH3 induces receptor-dependent and receptor-independent proapoptotic responses and genes, including programmed cell death gene 1 (PDCD1; Nur77 dependent) and cystathionase {gamma}-lyase (CSE) and activating transcription factor 3 (ATF3; Nur77 independent). This is the first study to show that Nur77 is overexpressed in human tumors, and that Nur77-active C-DIMs induce apoptosis in colon cancer cells through nuclear receptor-dependent and receptor-independent pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Human colon carcinoma cell lines RKO and SW480 were provided by the University of Texas M.D. Anderson Cancer Center, Houston, Texas. HT-29, HCT116, and HCT-15 were obtained from the American Type Culture Collection (Manassas, VA). RKO, SW480, and HT-29 cells were maintained in DMEM/Ham's F-12 (Sigma, St. Louis, MO) without phenol red supplemented with 0.22% sodium bicarbonate, 0.011% sodium pyruvate, and 5% fetal bovine serum (FBS) and 10 ml/L of 100x antibiotics antimycotic solution (Sigma). HCT116 and HCT-15 cells were maintained in RPMI 1640 (Sigma) supplemented with 0.22% sodium bicarbonate, 0.011% sodium pyruvate, 0.45% glucose, 0.24% HEPES, 10% FBS, and 10 ml/L of 100x antibiotics antimycotic solution. Cells were maintained at 37°C in the presence of 5% CO2.

Plasmids and reagents. The GAL4-Nur77 (full-length) chimera and the Nur77 response element-luciferase (NRE-Luc) reporter construct were provided by Dr. Jae W. Lee (Baylor College of Medicine, Houston, TX). The GAL4 reporter containing five GAL4 response elements (pGAL4) was provided by Dr. Marty Mayo (University of North Carolina, Chapel Hill, NC). Antibodies for cleaved poly(ADP-ribose) polymerase (PARP), caspase-3, caspase-8, caspase-9, bax, and bcl-2 were purchased from Cell Signaling (Danvers, MA). Antibodies for Nur77, TRAIL, IgG, and ß-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and the antibody for NAG-1 was from Upstate (Charlottesville, VA). Antibody for cytochrome c was purchased from BD PharMingen (San Diego, CA). Caspase inhibitor was obtained from Alexis Biochemicals (Lausen, Switzerland). ß-Galactosidase (ß-gal) reagent was obtained from Tropix (Bedford, MA). Western Lightning chemiluminescence reagent was from Perkin-Elmer Life Sciences (Boston, MA). For RNA interference assays, we used a nonspecific scrambled (iScr) oligonucleotide as described (29). The small inhibitory RNA for Nur77 (iNur77) was identical to the reported oligonucleotide (30), and this was purchased from Dharmacon Research (Lafayette, CO). Leptomycin B was purchased from Sigma. The C-substituted DIMs (C-DIMs) were synthesized in this laboratory as previously described (20).

Transfection and luciferase assay. RKO cells were plated in 12-well plates at 1 x 105 per well in DMEM/Ham's F12 supplemented with 2.5% charcoal-stripped FBS. After growth for 16 h, various amounts of DNA [i.e., Gal4Luc (0.4 µg), ß-gal (0.04 µg), Gal4-Nur77 (0.04 µg), and Gal4-Nur77(E/F) chimera (0.04 µg)] were transfected by LipofectAMINE plus reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. After 5 h of transfection, cells were treated with complete media containing either vehicle (DMSO) or the indicated ligands for 20 to 22 h. Cells were then lysed with 100 µL of 1x reported lysis buffer (Promega, Madison, WI), and 30 µL of cell extract was used for luciferase and ß-gal assays. Lumicount was used to quantitate luciferase and ß-gal activities, and the luciferase activities were normalized to ß-gal activity. Results are expressed as means ± SD for at least three independent determinations for each treatment group.

WST-1 cell proliferation assay. WST-1 assay purchased from Roche Applied Science (Indianapolis, IN) was used to evaluate the effect of C-DIMs on viable cell number. RKO cells were seeded in 48-well plates and treated with DMSO and different concentrations of test compounds as indicated in DMEM/Ham's F-12 containing 2.5% charcoal-stripped FBS for 24, 48, and 72 h when well plates reached 50% to 60% confluence usually at 24 h after seeding. The cell proliferation reagent WST-1 (50 µL) was added to each well, and cells were incubated for 0.5 h at 37°C, 5% CO2. The absorbance of the samples against a background control as blank was measured at 490 nm using a microtiter plate reader.

Western blot analysis. Cells were treated with the C-DIM compounds, and caspase inhibitor and leptomycin B were added simultaneously. Cell lysates were prepared using lysis buffer [20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 0.1% SDS, 1 mmol/L sodium orthovanadate; 1 mmol/L phenylmethylsulfonyl fluoride, 1 µmol/L leupeptin, and 1 µg/mL aprotinin were added fresh]. After centrifugation of lysate at 15,000 x g for 20 min, the supernatants were recovered, and protein was quantified by the Bradford protein assay using reagent kit from Bio-Rad (Hercules, CA). Protein samples (20–60 µg) were size-separated by electrophoresis on SDS-PAGE under nonreducing conditions. Separated proteins were electroblotted onto nitrocellulose membranes. The blot was blocked by incubating in blocking buffer [5% skim milk, 10 mmol/L Tris (pH 7.5), 10 mmol/L NaCl, and 0.1% Tween 20] for 1 h at room temperature and was incubated with the primary antibody overnight at 4°C. Incubation with a horseradish peroxidase–conjugated anti-mouse or rabbit secondary antibody was then carried out at 37°C for 1 h. Antibody-bound proteins were detected by the enhanced chemiluminescence Western blotting analysis system.

Immunostaining. Cells were fixed immediately in 4% paraformaldehyde, added with 0.3% Triton X-100 (Roche Molecular Biochemicals, Indianapolis, IN) for 10 min, and preincubated for 1 h with 10% normal goat serum (Vector Laboratories, Burlingame, CA). Cells were incubated with anti-Nur77 antibody (1:100) or anti-IgG (1:100) and incubated with FITC-conjugated secondary antibody (1:500; Vector Laboratories). The four-well chambers were mounted with mounting medium (Vector Laboratories) to be viewed on a fluorescence microscope (Olympus). Colon tumor specimens were provided from a selection of tumor and non-tumor tissues obtained at the University of Texas M.D. Anderson Cancer Center. After deparaffinization, tissue sections were subjected to antigen retrieval with 0.1% pepsin in 0.01 N HCl at room temperature for 10 min followed by treatment with 0.1% H2O2 to block endogenous peroxidase activity. Sections were incubated with the normal rabbit IgG or rabbit polyclonal anti-Nur77 antibody (1:100) at 4°C overnight after blocking with normal goat serum at room temperature for 1 h. After washing in PBS, sections were incubated with biotinylated goat anti-rabbit IgG at room temperature for 30 min. Detection was made with Vectastain Elite ABC kit (Vector Laboratories) and 3,3'-diaminobenzidine (Biogenex Laboratories, San Ramon, CA) as the chromagen following manufacturer's protocol. The sections were counterstained with hematoxylin and dehydrated, and coverslips were mounted.

Reverse transcription-PCR. Total RNA was extracted using RNeasy Mini kit (Qiagen, Inc., Valencia, CA), and 1 µg RNA was used to synthesize cDNA using Reverse Transcription System (Promega). The PCR conditions were as follows: initial denaturation at 94°C (2 min) followed by 35 cycles (PDCD1), 28 cycles (CSE), 30 cycles (ATF3), or 26 cycles [glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] of denaturation for 1 min at 94°C; annealing for 1 min at 53°C (PDCD1) and 61°C (CSE, ATF3 and GAPDH); extension at 72°C for 1 min; and a final extension step at 72°C for 5 min. The mRNA levels were normalized using GAPDH as an internal housekeeping gene. Primers obtained from IDT (Coralville, IA) and used for amplification were PDCD1 (sense, 5'-CTGGGCGGTGCTACAACTGGG-3'; antisense, 5'-ATGTGGAAGTCACGCCCGTTGG-3'), 291 bp; CSE (sense, 5'-GGCGATCCATGTGGGCCAGGA-3'; antisense, 5'-ATGTCTCCATGCTTATGGACAAT-3'), 483 bp; ATF3 (sense, 5'-CTGTTGGATAAAGAGGTTTCTCT-3'; antisense, 5'-ATGTCCTCTGCGCTGGAATCAG-3'), 348 bp; GAPDH (sense, 5'-ACGGATTTGGTCGTATTGGGCG-3'; antisense, 5'-CTCCTGGAAGATGGTGATGG-3'), 212 bp. PCR products were electrophoresed on 3% agarose gels containing ethidium bromide and visualized under UV transillumination.

Microarray experiments. Microarray studies focused on early-induced genes, and RKO cells were treated with DMSO or 12.5 µmol/L DIM-C-pPhOCH3 for 2 and 6 h. RNA was isolated as described for the reverse transcription-PCR (RT-PCR) experiment and analyzed for gene expression using the Codelink Whole Genome Bioarrays (300026), and three replicates were determined for each time point and the DMSO control. The microarray data were analyzed using GeneSpring software version 7.2 (Agilent, Palo Alto, CA). The data were normalized in two steps. First, for each array, the expression value of each gene was divided by the median of all the values in that array. Second, for each gene, the expression value in each array was divided by the median value of that gene across all arrays. Genes with low-quality signals were excluded for statistical analysis. One-way ANOVA (assume equal variances) was carried out to identify differentially expressed genes. A gene was said to be differentially expressed if the Benjamini and Hochberg adjusted Ps were <0.05.

Xenograft studies in athymic mice. Male athymic nude mice (Foxn1nu, ages 7–8 weeks) were purchased from Harlan (Indianapolis, IN). The mice were housed and maintained in laminar flow cabinets under specific pathogen-free conditions. A xenograft was established by s.c. injection of in vitro cultured RKO cells (5 x 106 per 150 µL) into the flanks of individual mice. Tumors were allowed to grow for 4 days until tumors were palpable. Mice were then randomized into two groups of six mice per group and dosed by oral gavage with either corn oil or 25 mg/kg/d DIM-C-pPHOCH3 for 21 days. The mice were weighed, and tumor size was measured twice a week with calipers to permit calculation of tumor volumes: V = LW2 / 2, where L and W were length and width, respectively. Final body, organ, and tumor weights were determined at the end of the dosing regiment, and both organ and tumor blocks were obtained for H&E staining and histopathologic analysis.

Quantitative real-time PCR. cDNA was prepared from the RKO, SW480, HCT116, HT-29, and HCT-15 cell lines using Reverse Transcription System (Promega). Each PCR was carried out in triplicate in a 30-µL volume using SYBR Green Mastermix (Applied Biosystems, Foster city, CA) for 15 min at 95°C for initial denaturing, followed by 40 cycles of 95°C for 30 s and 60°C for 1 min in the Applied Biosystems 7900HT Fast Real-time PCR System. The ABI Dissociation Curves software was used following a brief thermal protocol (95°C for 15 s and 60°C for 15 s, followed by a slow ramp to 95°C) to control for multiple species in each PCR amplification. Values for each gene were normalized to expression levels of TATA-binding protein. The primers used for real-time PCR were obtained from Qiagen.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Recent studies in this laboratory have identified C-DIMs containing a p-methoxy group (DIM-C-pPhOCH3) or no substituents (DIM-C-Ph) as Nur77 agonists in pancreatic cancer cells (27). Figure 1A shows that Nur77 protein is expressed RKO, SW480, HCT-116, HT29, and HCT-15 colon cancer cells lines, and these results are consistent with a previous report also showing expression of this receptor in cell lines derived from several different tumors (27). Nur77 expression in human colon tumors and surrounding non-tumor tissue was investigated by immunohistochemical analysis. Figure 1B illustrates the higher levels of brown staining (Nur77) in two colon cancer patients. Nur77 was primarily located in the nucleus in tumor and non-tumor tissue with only weak cytosolic staining. Immunohistochemical staining for Nur77 in 20 colon tumor and non-tumor tissues showed that the percentage of colon tumors that exhibited nondetectable, low, and high Nur77 staining was 5% (1 of 20), 35% (3 of 20), and 60% (12 of 20), respectively, whereas in normal colon tissue, the staining pattern was 30% (6 of 20), 60% (12 of 20), and 10% (2 of 20), respectively (e.g., Fig. 1A). These results clearly show that Nur77 protein is overexpressed in colon tumors. Due to their high transfection efficiency, we also showed that the Nur77-active C-DIMs induce transactivation in HCT-15 cells transfected with GAL4-Nur77/pGAL4 (Fig. 1C) or a construct (pNRE) containing a Nur response element (Fig. 1D). DIM-C-pPhOCH3, DIM-C-Ph, and DIM-C-pPhCF3 all induced transactivation as previously reported (27); however, in the following studies, we used the former two compounds as models because DIM-C-pPhCF3 also activates PPAR{gamma} (20–24).


Figure 1
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  		Figure 1. Nur77 expression and induced transactivation in colon cancer cells and tumors. A, Nur77 expression in colon cancer cells. Whole-cell lysates from RKO, SW480, HCT-116, HT-29, and HCT-15 cells were analyzed by Western blot analysis as described in Materials and Methods. B, immunostaining of Nur77 in human colon tumor and non-tumor tissue. Colon tumor samples were deparaffinized and immunostained using Nur77 antibodies as described in Materials and Methods. Enhanced brown staining for Nur77 was primarily observed in the nuclei from tumor samples and was decreased in non-tumor tissue from the same patients. C, transactivation studies. HCT-15 cells were transfected with GAL4-Nur77/pGAL4 (C) or pNuRE (D) and treated with C-DIM compounds, and induction of luciferase activity was determined as described in Materials and Methods. *, P < 0.05, significant induction.

 
In previous studies, we showed that Nur77 agonists induced TRAIL mRNA and protein, and this response was Nur77 dependent and blocked after cotransfection with a small inhibitory RNA for Nur77 (iNur77; ref. 27). Results of preliminary screening assays showed that induction of TRAIL by Nur77-active C-DIMs in colon cancer cells was maximal in RKO cells, and this cell line was used as a model in our studies on Nur77-active C-DIMs. Results in Fig. 2A and B show that DIM-C-pPhOCH3 and DIM-C-Ph (10 and 15 µmol/L, respectively) inhibit cell growth after treatment for 24, 48, or 72 h, and the maximum inhibitory response was observed after 72 h, where there was considerable cell detachment and dead cells. The growth-inhibitory effects observed for these compounds after 72 h were also accompanied by several markers of apoptosis, including PARP cleavage and cleavage of caspase-3, caspase-9, and caspase-8 (Fig. 2C). PARP cleavage was also observed after treatment of RKO cells for 48 h with DIM-C-Ph and DIM-C-pPhOCH3, and this response was blocked by the pancaspase inhibitor Z-VAD-fmk (Fig. 2D). These results show that Nur77-active C-DIMs inhibit cell proliferation and induce apoptosis in RKO cells. Previous studies reported that apoptosis inducers cause the translocation of Nur77 from the nucleus to the cytosol (30–33), whereas Nur77 agonists work through the nuclear protein in Panc28 cancer cells (27). Results in Fig. 2E illustrate that immunostaining of RKO cells shows that in solvent (DMSO)–treated cells, Nur77 is primarily nuclear with some weak cytosolic staining. After treatment with 12.5 µmol/L DIM-C-pPhOCH3 for 24 h, the pattern of dominant nuclear and weak cytosolic staining is unchanged. After preliminary studies using several concentrations of leptomycin B, a concentration of 0.5 ng/mL did not affect PARP cleavage or nuclear Nur77 expression (Fig. 2F), whereas PARP cleavage was induced at higher doses. It was also shown that apoptosis (PARP cleavage) induced by DIM-C-pPhOCH3 was not affected by leptomycin B, showing that the Nur77-dependent apoptosis was dependent on the nuclear protein. The results are in contrast to the effects of apoptosis inducers that act through cytosolic Nur77, where leptomycin B inhibits their translocation from the nucleus to the cytosol, and this results in activation of cell death pathways (30, 31).


Figure 2
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  		Figure 2. Nur77-active C-DIMs decrease cell proliferation and induce apoptosis in RKO cells and also act through nuclear Nur77. DIM-C-pPhOCH3 (A) and DIM-C-Ph (B) decrease RKO cell proliferation. Cells were treated with DMSO or different concentrations of C-DIMs for 24, 48, or 72 h, and the percentage of cells in the treatment groups compared with the solvent control (DMSO; set at 100%) was determined as described in Materials and Methods. Columns, mean for three replicate experiments for each treatment group; bars, SD. Significant (P < 0.05) growth inhibition was observed for all treatment groups using 10 or 15 µmol/L concentrations of C-DIMs. Dose-dependent induction of apoptosis by C-DIMs (C) and inhibition by Z-VAD-fmk (D). RKO cells were treated with different concentrations of DIM-C-pPhOCH3 or DIM-C-Ph alone or in combination with 10 µmol/L Z-VAD-fmk, and whole-cell lysates were analyzed by Western blot analysis as described in Materials and Methods. c-PARP, cleaved PARP; c-caspase, cleaved caspase. E, immunostaining for Nur77. RKO cells were treated with DMSO or 12.5 µmol/L DIM-C-pPhOCH3 for 24 h and immunostained with IgG or Nur77 antibodies as described in Materials and Methods. F, effects of leptomycin B on PARP cleavage. RKO cells were treated for 24 h with DIM-C-pPhOCH3 alone or in the presence of leptomycin B (0.5 ng/mL), and Western blot analysis of whole-cell lysates was used to detect Nur77, PARP cleavage, and ß-tubulin (loading control) as described in Materials and Methods.

 
Figure 3A and B illustrates the time-dependent induction of TRAIL protein by 12.5 µmol/L DIM-C-pPhOCH3 and 15 µmol/L DIM-C-pH, respectively, and significant induction was first observed after 48 and 24 h, respectively. The concentrations used in this experiment were selected to give a maximal induction response with minimal cytotoxicity. The role of Nur77 in mediating the induced proapoptotic responses was confirmed by RNA interference using transfected iNur77, which showed that Nur77 knockdown decreased Nur77 protein (Fig. 3C and D). Moreover, DIM-C-pPhOCH3–induced TRAIL protein expression and PARP cleavage (Fig. 3C and E) were also significantly inhibited in cells transfected with iNur77. These results show that proapoptotic responses induced by Nur77 agonists were dependent on Nur77 protein.


Figure 3
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  		Figure 3. Nur77-dependent induction of TRAIL and apoptosis in RKO cells. Induction of TRAIL by DIM-C-pPhOCH3 (A) and DIM-C-Ph (B). Cells were treated with Nur77-active C-DIMs for 24, 48, or 72 h, and whole-cell lysates were analyzed for TRAIL or ß-tubulin (loading control) proteins by Western blot analysis as described in Materials and Methods. RNA interference with iNur77 (C–E). RKO cells were transfected with iScr (nonspecific) or iNur77 and treated with DMSO or 12.5 µmol/L DIM-C-pPhOCH3, and whole-cell lysates were analyzed by Western blot analysis for Nur77, PARP cleavage, TRAIL, and ß-tubulin (loading control) proteins as described in Materials and Methods. Columns, mean for three replicate experiments for each treatment group; bars, SD. Protein levels were normalized to ß-tubulin. *, P < 0.05, significant inhibition of Nur77 expression (D). *, significant induction of TRAIL and PARP cleavage; **, decreased expression of these proteins by iNur77 (E).

 
We further investigated the effects of DIM-C-pPhOCH3 on activation of genes in RKO cells using the Amersham Biosciences CodeLink human whole genome array in which each treatment group was determined in triplicate. After treatment with DMSO or 12.5 µmol/L DIM-C-pPhOCH3 for 2 and 6 h, we analyzed gene expression from the microarray data and identified several induced genes associated with apoptosis and ER stress. This latter response was not surprising because DIMs and C-DIMs induce ER stress in other cancer cell lines. In addition, we also observed activation of genes (by ≥2-fold) associated with metabolism and homeostasis, transport, kinase activity and signal transduction, transcription, protein folding, and modification and genes with several other functions (Table 1 ). We further examined three of the most highly induced proapoptotic genes after treatment of RKO cells with DIM-C-pPhOCH3 for 6 h [i.e., PDCD1 (5.2-fold), CSE (5.6-fold), and ATF3 (4.6-fold)]. RT-PCR analysis shows that 12.5 µmol/L DIM-C-pPhOCH3 induced PDCD1, CSE, and ATF3 (Fig. 4A ), and based on replicate (3x) experiments, all three genes are significantly induced in RKO cells (Fig. 4B). In cells cotransfected with iScr or iNur77, the induction of PDCD1 is blocked by iNUR77 (Fig. 4C), whereas induction of CSE and ATF3 are unaffected by iNur77 and represent receptor-independent responses. A comparison of PDCD1 inducibility in five colon cancer cell lines (RKO, SW480, HCT-116, HT-29, and HCT-15) showed that mRNA was induced by DIM-C-pPhOCH3 in RKO, HCT-116, and HT-29 cells but not in SW480 or HCT-15 cells where endogenous expression was relatively high or nondetectable, respectively. The induction of ATF3 and CSE by DIM-C-pPhOCH3 also depended on cell context. ATF3 was constitutively expressed or induced in all cell lines and most prominently induced in HT-29 cells. The pattern of CSE expression was similar to that of PDCD1 except for HCT-116 cells where CSE was constitutively expressed but not induced. Results illustrated in Fig. 4E show that 12.5 µmol/L DIM-C-pPhOCH3 induced apoptosis (PARP cleavage) in RKO, SW480, HCT-116, and HT-29 cells but not in HCT-15 cells. The failure to induce apoptosis in HCT-15 cells correlates with the lack of induction of PDCD1 and CSE in this cell line.


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  		Table 1. Induction of different classes of genes by DIM-C-pPhOCH3 in RKO cells using the Amersham Biosciences CodeLink human whole genome array

 

Figure 4
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  		Figure 4. Induction of gene expression by DIM-C-pPhOCH3. A and B, Induction of PDCD1, CSE, and ATF3 in RKO cells. Cells were treated with DMSO or 12.5 µmol/L DIM-C-pPhOCH3 for 6 h, and PDCD1, CSE, ATF3, and GAPDH (reference mRNA) were determined by RT-PCR as described in Materials and Methods. Induction responses were determined in three separate experiments. *, P < 0.05, significantly induced mRNA expression (normalized to GAPDH). C, effects of iNur77 on induced gene expression. RKO cells were transfected with iScr or iNur77, treated with DMSO or 12.5 µmol/L DIM-C-pPhOCH3 for 6 h, and analyzed by real-time PCR as described in Materials and Methods. Columns, mean for three replicate determinations for each treatment group; bars, SE. *, P < 0.05, significant induction by DIM-C-pPhOCH3; **, significant decrease (for PDCD1) or increase (for ATF3) after transfection with iNur77. D, analysis of DIM-C-pPhOCH3–induced gene expression in multiple colon cancer cell lines. Several different colon cancer cell lines were treated essentially as described above and analyzed by real-time PCR as outlined in Materials and Methods. *, P < 0.05, significantly induced genes. E, induction of PARP cleavage. Cells were treated with 12.5 µmol/L DIM-C-pPhOCH3 for 24 h, and whole-cell lysates were analyzed for the cleaved PARP protein as described in Materials and Methods.

 
The effects of DIM-C-pPhOCH3 (25 mg/kg/d) on colon tumor growth were also investigated in athymic nude mice bearing RKO cell xenografts. Treatment with the Nur77 agonist significantly decreased tumor volumes (Fig. 5A ) and final tumor weights (Fig. 5B) compared with corn oil controls. Extensive examination of body weight, organ weights, and H&E staining showed that there were no significant differences between corn oil– and DIM-C-pPhOCH3–treated animals. Representative H&E-stained histopathology sections of colon tumors from the control (Fig. 5C and D) and treated (Fig. 5E and F) mice were also examined. Tumors from untreated mice consisted of minimally encapsulated (dashed arrow), abundant dense expansile nests of epithelial-to-mesenchymal–appearing cells with marked atypical cytologic features, such as anisocytosis, anisokaryosis, single to multiple variably sized nucleoli, nuclear molding, and binucleation. Mitotic figures were frequently noted within the neoplastic cells (arrows). Some of the neoplastic cells were vacuolated (empty areas) with few areas of necrosis. A fine amount of stroma occasionally dissecting the tumor was also observed. Tumors from treated mice (Fig. 5E and F) consisted of similar population of neoplastic cells. However, there was abundant necrosis noted within the tumor cells, and mitotic figures seemed to significantly decreased. Areas within the circle represent necrotic components of the tumor. TRAIL protein was overexpressed in tumors from treated animals but not in tumors from animals receiving corn oil alone; however, this was not consistently observed in all tumors (data not shown). These studies clearly show that the Nur77 agonist DIM-C-pPhOCH3 exhibits antitumorigenic activity in both in vivo and in vitro models, and these results, coupled with the overexpression of Nur77 in human colon tumors (Fig. 1C), suggest that this orphan receptor is a target for colon cancer chemotherapy.


Figure 5
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  		Figure 5. Inhibition of tumor growth by DIM-C-pPhOCH3. Male athymic nude mice bearing RKO cell xenografts were treated with DIM-C-pPhOCH3, and tumor volumes (A) and weight (B) were determined as described in Materials and Methods. The compound was given daily (25 mg/kg/d) in corn oil by oral gavage, and corn oil served as a solvent control. Body and organ weights were obtained, and tissues (including tumors) were examined histopathologically after H&E staining. H&E staining of tumors from corn oil control (C and D) and DIM-C-pPhOCH3–treated (E and F) mice. Tumor tissues were initially formalin fixed and subsequently prepared for H&E staining.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Nur77 is an NGFI-B orphan receptor and a member of the nuclear receptor superfamily of transcription factors. Nur77 is expressed in multiple tissues; Nurr1 has been detected in thymus osteoblasts, liver, and pituitary gland, and Nor1 is highly expressed in the pituitary gland with low expression in other tissues (34–38). The physiologic roles for NGFI-B proteins are not fully understood; however, gene-targeting knockout experiments show several important functions of these proteins that correlate, in part, with other in vitro and in vivo studies. For example, Nurr1 knockout mice have severe impairments in midbrain neuronal development and dopamine expression, and these animals die soon after birth (39, 40). A recent study reports that Nor1 knockout animals die at gestation day 5, showing an important role for this protein in early embryogenesis (41). In contrast, Nur77 knockout mice do not exhibit a specific phenotype (42), and this may be related to coexpression of both Nur77/Nor1, which exhibit some overlapping functions. Nur77 plays an important role in thymocyte-negative selection and in T-cell receptor–mediated apoptosis in thymocytes, and overexpression of Nur77 in transgenic mice resulted in high levels of apoptosis in thymocytes (43, 44).

Li et al. (31) reported that treatment of LNCaP prostate cancer cells with several apoptosis-inducing agents, such as retinoids, 12-O-tetradecanoylphorbol-13-acetate and tumor necrosis factor-{alpha}, resulted in induction of Nur77 gene expression. Surprisingly, induction of apoptosis and cytochrome c release from the mitochondria was independent of the DNA-binding domain of Nur77. Treatment with leptomycin B (a blocker of nuclear export) inhibited induction of Nur77-dependent apoptosis. Using a series of wild-type and deletion GFP-Nur77 constructs, it was clear that induction of apoptosis by these agents was accompanied by translocation of Nur77 from the nucleus to the mitochondria. Moreover, Nur77 specifically interacts with Bcl-2 and converts Bcl-2 into a proapoptotic factor in HEK293T and HCT-116 cells (30). Nur77 translocation from the nucleus has been observed in several cell lines with inducers of apoptosis (30–33). However, a recent study in colon cancer cells reported that butyrate-induced apoptosis was associated with nuclear to cytoplasmic translocation of Nur77, which is not accompanied by subsequent mitochondrial interactions (45).

A report from this laboratory showed that Nur77 was widely expressed in cancer cell lines, and DIM-C-pPhCF3, DIM-C-Ph, and DIM-C-pPhOCH3 were characterized as Nur77 agonists, which activated Nur77-dependent transactivation in pancreatic cancer cell lines (27). In this study, we used DIM-C-pPhOCH3 and DIM-C-Ph as prototypical Nur77 agonists and show that these compounds activated Nur77 in colon cancer cell lines (Fig. 1C and D). Nur77 was also expressed in RKO, SW480, HCT-116, HT-29, and HCT-15 colon cancer cells (Fig. 1A) and overexpressed in human colorectal tumors compared with non-tumor tissue (Fig. 1B). Results of initial screening studies in colon cancer cells showed that DIM-C-pPhOCH3 and DIM-C-Ph strongly induced TRAIL in RKO cells (8- to 15-fold), and this cell line was used as a model to investigate the growth-inhibitory and apoptotic effects of Nur77-active DIM-C-pPhOCH3 and DIM-C-Ph.

Both Nur77-active C-DIMs decreased cell proliferation and induced caspase-dependent apoptosis in RKO cells (Fig. 2), and this was accompanied by the induction of TRAIL (Fig. 3A and B). Moreover, using RNA interference, we also showed that decreased Nur77 expression by RNA interference was accompanied by significant inhibition of PARP cleavage and induction of TRAIL (Fig. 3C–E). These results parallel previous studies in pancreatic cancer cell lines (27) and confirm that ligand-dependent activation of Nur77 was required for the proapoptotic responses induced by DIM-C-pPhOCH3 and DIM-C-Ph. Moreover, these responses were not blocked by the nuclear export inhibitor leptomycin B (Fig. 2F) as previously reported for several apoptosis-inducing agents (30–33), and Nur77 was primarily a nuclear protein in the presence or absence of agonists (Fig. 2E). In contrast to the important extranuclear function of Nur77 in mediating the induction of cell death by inducers of apoptosis (30–33), Nur77 agonists induce cell death through nuclear pathways that include the induction of TRAIL.

The results of RNA interference studies (Fig. 3C) suggest that although Nur77 plays a role in DIM-C-pPhOCH3–induced apoptosis, it was evident that induction of receptor-independent proapoptotic responses may also be important. Previous studies with PPAR{gamma}-active C-DIMs show that these compounds activate other proapoptotic responses, including induction of ER stress, modulation of kinase pathways, and induction of the transforming growth factor-ß–like peptide NAG-1 (20–24). Initial studies showed that although both DIM-C-pPhOCH3 and DIM-C-Ph induced TRAIL within 24 or 48 h after treatment, respectively (Fig. 3A and B); PARP cleavage was also observed 12 to 24 h after treatment, and we also observed that DIM-C-pPhOCH3 and DIM-C-Ph decreased mitochondrial membrane potential (data not shown). These results suggest that some of the proapoptotic effects of the Nur77-active C-DIMs were receptor independent, and the pathways associated with these responses are currently being investigated.

We also used the Amersham Biosciences CodeLink human whole genome array to further investigate genes induced by DIM-C-pPhOCH3 within 2 and 6 h after treatment. This approach would not detect TRAIL, which is a late-inducible gene, but should identify other genes associated with both receptor-dependent and receptor-independent pathways. Analysis by GeneSpring showed that several different functional categories of genes were induced or decreased after treatment with DIM-C-pPhOCH3 for 2 or 6 h (Table 1, induced genes), and we initially focused on three proapoptotic genes that were the most highly induced genes after 6 h. DIM-C-pPhOCH3 induced expression of PDCD1, CSE, and ATF3 by 5.3-, 5.6-, and 4.6-fold, respectively, as determined in the microarray experiments (in triplicate), and the induction responses were confirmed by quantitative RT-PCR (Fig. 4A and B). PDCD1 has previously been characterized as a proapoptotic gene (46), and, in pancreatic cancer cells, PDCD1 is overexpressed in a well-differentiated cell line compared with a poorly differentiated cell line (47). In this study, we show that induction of PDCD1 by DIM-C-pPhOCH3 is Nur77 dependent (Fig. 4C) and is induced in RKO, HCT-116, and HT-29 but not SW480 or HCT-15 colon cancer cells (Fig. 4D).

ATF3 is a stress-responsive transcription factor induced by different classes of compounds, including nonsteroidal anti-inflammatory drugs in colon and other cancer cell lines, and exhibits growth inhibitory and proapoptotic activity (48–50). In contrast, CSE acts primarily through catalyzing formation of hydrogen sulfide (H2S) from cysteine, and the growth-inhibitory effects of CSE in some cells may be due to the effects of H2S (51, 52). Both ATF3 and CSE are induced by DIM-C-pPhOCH3 in RKO cells (Fig. 4A and B) and are differentially expressed and induced in the five colon cancer cell lines (Fig. 4D). RNA interference studies show that CSE and ATF3 are induced through receptor-independent pathways (Fig. 4C), and these results parallel studies on other responses that are induced by Nur77-active C-DIMs through receptor-dependent and receptor-independent pathways (Fig. 3; data not shown). Interestingly, SW480 and HCT-15 cells were most resistant to DIM-C-pPhOCH3–induced apoptosis (PARP cleavage), and this result correlated with minimal to nondetectable induction of CSE and PDCD1 in both cell lines. Current studies are investigating DIM-C-pPhOCH3–induced genes, which are required for induction of apoptosis by this compound. These results show that Nur77 agonists inhibit RKO cell growth through multiple pathways, and in vivo studies using athymic nude mice bearing RKO cell xenografts show that DIM-C-pPhOCH3 (25 mg/kg/d) also inhibits colon tumor growth and weight (Fig. 5), and this result complements results of in vitro studies with this compound. Moreover, we did not observe any toxicity of this compound based on body or organ weights and extensive histopathology of various organs/tissues (Fig. 5C–F).

In summary, this study shows that Nur77-active C-DIMs inhibit growth and induce apoptosis in colon cancer cell lines, and Nur77 is overexpressed in human colorectal adenocarcinomas. In contrast to apoptosis-inducing agents (30–33) that induce nuclear to cytosolic translocation of Nur77, the receptor-dependent and receptor-independent responses induced by Nur77-active C-DIMs are not related to altered subcellular distribution of Nur77, which is primarily located in the nucleus. Current studies are focused on identification of other critical genes induced by Nur77-active C-DIMs in colon cancer cell lines and on their cell context– and time-dependent contributions to activation of cell death. Moreover, because Nur77 is overexpressed in colon tumors (Fig. 1B), the Nur77-active C-DIMs are also being developed as a novel class of anticancer drugs for treatment of colon cancer.


   Acknowledgments
 
Grant support: NIH grants ES09106 and CA112337 and Texas Agricultural Experiment Station.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/ 8/06. Revised 10/24/06. Accepted 11/15/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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