Activation of Nur77 by selected 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes induces apoptosis through nuclear pathways.

Nur77 is an orphan receptor and a member of the nerve growth factor-I-B subfamily of the nuclear receptor family of transcription factors. Based on the results of transactivation assays in pancreatic and other cancer cell lines, we have now identified for the first time Nur77 agonists typified by 1,1-bis(3-indolyl)-1-(p-anisyl)methane that activate GAL4-Nur77 chimeras expressing wild-type and the ligand binding domain (E/F) of Nur77. In Panc-28 pancreatic cancer cells, Nur77 agonists activate the nuclear receptor, and downstream responses include decreased cell survival and induction of cell death pathways, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, the transactivation and apoptotic responses are also induced in other pancreatic, prostate, and breast cancer cells that express Nur77. In Panc-28 cells, small inhibitory RNA for Nur77 reverses ligand-dependent transactivation and induction of TRAIL and PARP cleavage. Nur77 agonists also inhibit tumor growth in vivo in athymic mice bearing Panc-28 cell xenografts. These results identify compounds that activate Nur77 through the ligand binding domain and show that ligand-dependent activation of Nur77 through nuclear pathways in cancer cells induces cell death and these compounds are a novel class of anticancer agents.

The nuclear receptor superfamily of eukaryotic transcription factors encompasses steroid hormone and other nuclear receptors for which ligands have been identified and orphan receptors with no known ligands (1)(2)(3)(4)(5)(6)(7). Nuclear receptors share common structural features that include an N-terminal A/B domain, containing activation function-1 (AF-1), 1 and a C-ter-minal E domain, which contains AF-2 and the ligand binding domain (LBD). Nuclear receptors also have a DNA binding domain (C domain), a variable hinge (D domain), and C-terminal F regions. Ligand activation of class 1 steroid hormone receptors induces homo-or heterodimer formations, which interact with consensus or nonconsensus palindromic response elements. In contrast, class 2 receptors form heterodimers with the retinoic X receptor as a common partner, whereas class 3 and 4 orphan receptors act as homodimers or monomers and bind to direct response element repeats or single sites, respectively. The DNA binding domains of nuclear receptors all contain two zinc finger motifs that interact with similar half-site motifs; however, these interactions vary with the number of half-sites (1 or 2), their orientation, and spacing. Differences in nuclear receptor action are also determined by their other domains, which dictate differences in ligand binding, receptor dimerization, and interaction with other nuclear cofactors.
Most orphan receptors were initially cloned and identified as members of the nuclear receptor family based on their domain structure and endogenous or exogenous ligands have subsequently been identified for many of these proteins (5)(6)(7). The nerve growth factor I-B (NGFI-B) family of orphan receptors were initially characterized as immediate early genes induced by nerve growth factor in PC12 cells, and the three members of this family include NGFI-B␣ (Nur77), NGFI-B␤ (Nurr1), and NGFI-B␥ (Nor1) (8 -10).
Nur77 plays an important role in thymocyte-negative selection and in T-cell receptor-mediated apoptosis in thymocytes (11,12), and overexpression of Nur77 in transgenic mice resulted in high levels of apoptosis in thymocytes (13,14). In cancer cells, several mechanisms for Nur77-mediated apoptosis have been described, and differences between studies may be due to the apoptosis-inducing agent or cell line (15)(16)(17)(18)(19)(20)(21). For example, the retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2naphthalene carboxylic acid (CD437) and 12-O-tetradecanoylphorbol-13-acetate (TPA) induce translocation of Nur77 from the nucleus to the mitochondria where Nur77 binds Bcl-2 to form a pro-apoptotic complex (15,16). In contrast, it has been suggested that TPA-induced Nur77 in LNCaP prostate cancer cells activates transcription of E2F1, which is also pro-apoptotic (21). These studies are examples of ligand-independent pathways where Nur77 expression is induced and/or Nur77 protein undergoes intracellular translocation, because ligands for this receptor have hitherto not been reported. This report shows that 1,1-bis(3Ј-indolyl)-1-(p-substitutedphenyl)methanes containing trifluoromethyl, hydrogen, and methoxy substituents induce Nur77-dependent transactivation in Panc-28 pancreatic and other cancer cell lines. Nur77 agonists also induce typical cellular signatures of apoptosis, including PARP cleavage and induction of TRAIL, and both ligand-dependent transactivation and induction of apoptosis were associated with the action of nuclear Nur77. This study shows for the first time that ligand-dependent activation of the orphan receptor Nur77 induces apoptosis in cancer cells, suggesting that Nur77 agonists represent a new class of anticancer drugs.
Transfection Assays-Transfection assays were essentially carried out as previously described using Lipofectamine Plus reagent (Invitrogen), and luciferase activities were normalized to ␤-galactosidase activity. For RNA interference studies, cells were transfected with small inhibitor RNAs for 36 h to ensure protein knockdown prior to the standard transfection and treatment protocols (22,23). Results are expressed as means Ϯ S.E. for at least three replicate determinations for each treatment group.
Cell Growth and Apoptosis Assays-The different cancer cell lines were cultured under standardized conditions. Panc-28 cells were grown in DMEM/Ham's F-12 media containing 2.5% charcoal-stripped fetal bovine serum, and cells were treated with Me 2 SO and different concentrations of test compounds as indicated. For longer term cell survival studies, the media was changed every second day, and values were presented for a 4-day experiment. For all other assays, cytosolic, nu-clear fractions, or whole cell lysates were obtained at various time points, analyzed by Western blot analysis, and bands were quantitated as previously described (22,23). Immunocytochemical analysis was determined using Nur77 antibodies as previously reported (23).
Gel Shift Assay-Cells were seeded in DMEM/F-12 medium supplemented with 2.5% charcoal-stripped serum and treated with 10 M DIM-C-pPhOCH 3 for 30 min. Nuclear extracts were obtained using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Chemical Co.). Oligonucleotides were synthesized, purified, and annealed, and 5 pmol of specific oligonucleotides was 32 P-labeled at the 5Ј-end using T 4 polynucleotide kinase and [␥-32 P]ATP. Nuclear extracts were incubated in HEPES with ZnCl 2 and 1 g of polydeoxyinosine-deoxycytidine for 5 min; 100-fold excess of unlabeled wild-type or mutant oligonucleotides were added for competition experiments and incubated for 5 min. The mixture was incubated with labeled DNA probe for 15 min on ice. The reaction mixture was loaded onto a 5% polyacrylamide gel and ran at 150 V for 2 h. The gel was dried, and protein-DNA complexes were visualized by autoradiography using a Storm 860 PhosphorImager (Amersham Biosciences).
Annexin-V Staining-Detection of phosphatidylserine on the outside of the cell membrane, a unique and early marker for apoptosis, was performed using a commercial kit (Vybrant Apoptosis Assay Kit #2, Molecular Probes, Eugene, OR). Panc-28 cells were cultured as described above, and treated with 10 M DIM-C-pPhOCH 3 or camptothecin for 6, 12, and 24 h. Binding of annexin V-Alexa-488 conjugate and propidium iodide (PI) was performed according to the manufacturer's instructions. After binding and washing, cells were observed under phase contrast and epifluorescent illumination using a 495 nm excitation filter and a 520 nm absorption filter for annexin V-Alexa 488 and a 546 nm excitation filter and a 590 nm absorption filter for PI. Healthy cells were unstained by either dye; cells in early stages of apoptosis were stained only by annexin V, whereas dead cells were stained by annexin V and PI. The assay was repeated on three separate Panc-28 cell preparations.
Quantitative Real-time PCR-cDNA was prepared from the Panc-28 cell line using a combination of oligodeoxythymidylic acid (Oligo-d(T) 16 ), and dNTP mix (Applied Biosystems) and Superscript II (Invitrogen). Each PCR was carried out in triplicate in a 20-l volume using Sybr Green Mastermix (Applied Biosystems) 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 ABI Prism 7700 Sequence Detection System. The ABI Dissociation Curves software was used following a brief thermal protocol (95°C 15 s and 60°C 20 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 sequences of the primers used for reverse transcription-PCR were as follows: TRAIL forward, 5Ј-CGT GTA CTT TAC CAA CGA GCT GA-3Ј, reverse, 5Ј-ACG GAG TTG CCA CTT GAC TTG-3Ј; and TATA-binding protein forward, 5Ј-TGC ACA GGA GCC AAG AGT GAA-3Ј, reverse, 5Ј-CAC ATC ACA GCT CCC CAC CA-3Ј.
Xenograft Experiment-Male athymic nude mice (BALB/c, ages 8 -12 weeks) were purchased from Harlan (Indianapolis, IN). The mice were housed and maintained in laminar flow cabinets under specific pathogen-free conditions. Panc-28 cells were harvested from subconfluent cultures by trypsinization and washed. Panc-28 cells (2 ϫ 10 6 ) were injected subcutaneously into each mouse on both flanks using a 30gauge needle. The tumors were allowed to grow for 11 days until tumors were palpable. Mice were then randomized into two groups of seven mice per group and dosed by oral gavage with either corn oil or DIM-C-pPhOCH 3 every second day. The volume of corn oil was 75 l, and the dose of DIM-C-pPhOCH 3 was 25 mg/kg/day. The mice were weighed, and tumor areas were also measured every other day. Final body and tumor weights were determined at the end of the dosing regiment, and selected tissues were further examined by routine H & E staining and immunohistochemical analysis for apoptosis using the TUNEL assay.

Nur77
Expression and Structure-dependent Activation by Csubstituted DIMs-Studies in this laboratory have been investigating the anticarcinogenic activities of a series of ringsubstituted 3,3Ј-diindolylmethanes (DIMs) and methylene (C)-substituted DIMs, and many of these compounds were active in vivo and in cell culture assays (22, 24 -26). Some members of a series of C-substituted DIMs activated peroxisome proliferator-activated receptor ␥ (PPAR␥) but not PPAR␣, retinoic acid receptor, retinoic X receptor, estrogen receptor ␣, or the aryl hydrocarbon receptor. Previous studies have linked Nur77 to decreased cell survival and activation of cell death pathways by apoptosis-inducing agents in some cancer cell lines (15)(16)(17)(18)(19)(20)(21), and we therefore investigated expression of Nur77 in cancer cell lines and the effects of a series of eleven C-substituted DIMs on Nur77 activation/translocation. Fig. 1A FIG. 1. Nur77 expression and activation in cancer cell lines. A, Nur77 protein expression. Whole cell lysates from 12 different cancer cell lines were analyzed for Nur77, Nurr1, and Nor1 by Western blot analysis as described under "Materials and Methods." Nor1 protein was not detected in these cell lines. Activation of Gal4-Nur77 (B) and NuRE (C) Panc-28 cells were treated with 10 or 20 M of the various compounds, transfected with GAL4-Nur77/pGAL4 or NuRE, and luciferase activity was determined as described under "Materials and Methods." D, Nur77 activation by isomeric DIM-C-PhOCH 3 compounds. Panc-28 cells were treated with 10 or 20 M of the DIM-C-PhOCH 3 isomers, transfected with GAL4-Nur77/pGAL4 and luciferase activity determined as described under "Materials and Methods." Results are expressed as means Ϯ S.E. for at least three separate determinations for each treatment group, and significant (p Ͻ 0.05) induction is indicated by an asterisk. The compounds in B and C were 1,1-bis(3Ј-indolyl)-1-(p-substitutedphenyl)methanes and the p-substituent X is shown directly in the figure. D compares the activity of the p-substituted methoxy derivative (CIM-pPhOCH 3 ) with the meta (DIM-C-mPhOCH 3 ) and ortho (DIM-C-oPhOCH 3 ) isomers.
summarizes Western blot analysis of Nur77 in whole cell lysates from 12 different cancer cell lines derived from pancreatic, prostate, breast, colon, and bladder tumors. Only the 253 JB-V-33 bladder cancer cell line exhibited relatively low expression of Nur77, and the antibodies and electrophoretic conditions gave two immunostained bands as previously reported in other studies. Western blot analysis of the other NGFI-B proteins showed variable expression of Nurr1, and Nor1 was not detectable in these cancer cell lines (data not shown). Similar results were also obtained in Jurkat T-cell leukemia cells (data not shown). Structure-dependent activation of Nur77 by a series of eleven C-substituted DIMs was investigated in Panc-28 cells transfected with a GAL4-Nur77 (fulllength) chimera and a reporter construct containing five GAL4 response elements linked to a luciferase reporter gene (pGAL4). The results (Fig. 1B) showed that three compounds containing p-trifluoromethyl (DIM-C-pPhCF 3 ) and methoxy (DIM-C-pPhOCH 3 ) substituents or the unsubstituted phenyl group (DIM-C-Ph) activated luciferase activity. Similar results were also obtained in Panc-28 cells transfected with a construct containing a Nur response element (NurRE) (Fig. 1C), and these same compounds also activated GAL4-Nur77/pGAL4 and NurRE in MiaPaCa-1 pancreatic, HCT-15 colon, and MCF-7 breast cancer cells (data not shown). The structure-dependent activation of Nur77 was also investigated using DIM-C-pPhOCH 3 as a model, and the position of the methoxyl group was changed to the meta (DIM-C-mPhOCH 3 ) and ortho (DIM-C-oPhOCH 3 ) positions (Fig. 1D). Only the para-substituted compound was active. We also investigated N-methyl and 2-methyl indole ring-substituted analogs of DIM-C-pPhOCH 3 , DIM-C-Ph, and DIM-C-pPhCF 3 , and these compounds did not activate Nur77 (data not shown). These results demonstrate that activation of Nur77 by C-DIMs was structure-dependent and sensitive to substitution on the phenyl and indole rings. Thus, at least three C-substituted DIMs activate Nur77; one of these compounds (DIM-C-pPhCF 3 ) also activates PPAR␥ (22,26), whereas DIM-C-pPhOCH 3 and DIM-C-Ph are PPAR␥-inactive (22). DIM-C-pPhOH was inactive in both transactivation assays and, at higher concentrations, decreased activity lower than observed in solvent (Me 2 SO) control.
Characterization and Interactions of C-DIMs That Activate and Inhibit Nur77-mediated Transactivation-The role of the LBD or E/F region in ligand-induced transactivation of Nur77 was investigated in Panc-28 cells transfected with pGAL4 and a chimeric GAL4-Nur77(E/F) construct containing only the E/F domain of Nur77. Treatment of Panc-28 cells with different concentrations (5-15 M) of DIM-C-pPhCF 3 , DIM-C-pPhOCH 3 , and DIM-C-Ph induced luciferase activity, whereas no response was observed in cells treated with Nur77-inactive DIM-C-pPhOH ( Fig. 2A). These results are the first to identify a series of compounds that directly activate Nur77(LBD)dependent transactivation in Panc-28 or any other cancer cell line. The role of Nur77 in mediating transactivation was further investigated in Panc-28 cells treated with 10 or 20 M DIM-C-pPhOCH 3 or DIM-C-Ph and transfected with pNurRE, a nonspecific "scrambled" small inhibitory RNA (iScr), or small inhibitory RNA for Nur77 (iNur77). The results (Fig. 2B) showed decreased Nur77 protein in whole cell lysates and a 90 -100% decrease in ligand-induced transactivation over the different concentrations of compounds, thus confirming the role of Nur77 in mediating this response. As noted above, one compound that contained a p-hydroxy substituent (DIM-C-pPhOH) did not induce activity (Fig. 1B) and DIM-C-pPhOH was further investigated as a potential Nur77 antagonist. Panc-28 cells were transfected with GAL4-Nur77/pGAL4 and cotreated with DIM-C-pPhOH and Nur77 agonists DIM-C-pPhCF 3 , DIM-C-pPhOCH 3 , and DIM-C-pH (Fig. 2C). The results show that DIM-C-pPhOH antagonizes activation of Nur77 by all three C-DIM compounds. The structural specificity of Nur77 antagonists was further investigated using meta-hydroxy (DIM-C-mPhOH) and ortho-hydroxy (DIM-C-oPhOH) analogs. DIM-C-mPhOH (10 or 20 M) did not inhibit DIM-C-pPhOCH 3 -or DIM-C-Ph-induced transactivation (Fig. 2D). DIM-C-oPhOH also did not exhibit Nur77 antagonist activity (Fig. 2E); however, high doses (20 M) of both Nur77 agonists and DIM-C-oPhOH were toxic. Thus, activation of Nur77 by C-DIMs was E/F domain-dependent and Nur77 activation was inhibited by DIM-C-pPhOH; moreover, both activation and inhibition of Nur77-mediated transactivation was dependent on the structure of the C-DIM compounds.
Nur77 DNA Binding and C-DIM-induced Nur77-coactivator Interactions-Incubation of nuclear extracts from Panc-28 cells treated with Me 2 SO or DIM-C-pPhOCH 3 with 32 P-labeled NBRE and NurRE (lanes 1 and 2, and 5 and 6, respectively) gave retarded bands in EMSA assays (Fig. 3A). Retarded band intensities were decreased after incubation with 100-fold ex- These results show that nuclear extracts containing Nur77 bind NurRE and NBRE as dimers and monomers, respectively, and this corresponds to their migration in an electrophoretic mobility shift assay. Extracts from cells treated with Nur77active C-substituted DIMs gave retarded band intensities similar to those observed for solvent-treated extracts suggesting minimal ligand-dependent loss of nuclear Nur77 in these cells. The retarded band pattern corresponds to that observed in previous studies using nuclear extracts from cells or in vitro translated Nur77 (27,28).
Effects of Nur77-active C-DIMs on Cell Survival and Apoptosis and Role of Nuclear Nur77-In several cancer cell lines transfected with Nur77-GFP constructs, treatment with apoptosis and differentiation-inducing agents results in rapid translocation of Nur77 into the cytosol/mitochondria (15)(16)(17)(18)(19)(20). Similar results have been observed in BGC-823 human gastric cancer cells where endogenous Nur77 is nuclear and TPA induced Nur77 translocation into the cytosol, and this was accompanied by apoptosis but not by Nur77-dependent transactivation (17). Results summarized in Fig. 4A show immunostaining of Nur77 in the nucleus of Panc-28 cells treated with Me 2 SO and Nur77-active DIM-C-pPhCF 3 , DIM-C-pPhOCH 3 , and DIM-C-Ph for 6 h, and comparable results were obtained in Panc-28, MiaPaCa, and LNCaP cells after treatment for 6 or 12 h (data not shown). In all cases, Nur77 remained in the nucleus, and cells exhibited a compacted nuclear staining pattern typically observed in cells activated for cell death pathways. In a separate experiment, Panc-28 cells were treated with 10 or 20 M DIM-C-pPhCF 3 , DIM-C-pPhOCH 3 , and DIM-C-Ph or 10 M DIM-C-pPhOH for 12 h, and Nur77 protein levels were determined by Western blot analysis of cytosolic and nuclear extracts (Fig. 4B). These results also confirm that Nur77, in the presence or absence of C-substituted DIM agonists, is a nuclear protein and ligand-induced Nur77 translocation from the nucleus is not observed. Sp1 is a nuclear protein and was used as a control to ensure efficient separation of the two extracts, and Sp1 was identified only in the nuclear fraction (Fig. 4B).
Nur77 agonists significantly decreased survival of Panc-28 cells (Fig. 5A), and IC 50 values for DIM-C-pPhCF 3 , DIM-C-pPhOCH 3 , and DIM-C-Ph were between 1 and 5 M, whereas DIM-C-pPhOH did not affect cell survival. At longer time points (4 and 6 days), DIM-C-pPhOH slightly inhibited cell proliferation; however, induction of cell death was not observed for this compound at concentrations as high as 20 M. De- FIG. 2. A, activation of GAL4-Nur77(E/F)/pGAL4. The effects of the various compounds was essentially determined as described in Fig. 1; however, a truncated GAL4 chimera expressing only the E/F domain of Nur77 was used in this experiment. B, effects of iNur77 on transactivation. Cells were treated with DIM-C-pPhOCH 3 or DIM-C-Ph, transfected with NuRE and iNur77 or iScr (nonspecific), and luciferase activity determined as described. Similar results were obtained for DIM-C-pPhCF 3 . Nur77 antagonist activity of DIM-C-pPhOH (C), DIM-C-mPhOH (D), and DIM-C-oPhOH (E). Cells were transfected with GAL4-Nur77/pGAL4 and different concentrations of the DIM-C-PhOH isomers and the Nur77 agonists, and luciferase activity determined as described under "Materials and Methods." Significant (p Ͻ 0.05) inhibition of transactivation is indicated (**). Results are expressed as means Ϯ S.E. for at least three separate determinations for each treatment group, and significant (p Ͻ 0.05) induction (*) or inhibition by iNur77 or DIM-C-pPhOH (**) is indicated.

FIG. 3. DNA binding of Nur77 and ligand-induced coactivator-Nur77 interactions.
A, gel mobility shift assay. Cells were treated with Me 2 SO (DMSO) or Nur77 agonists for 0.5 h, nuclear extracts were incubated with 32 P-labeled NurRE and NBRE, and formation of retarded bands was determined in a gel mobility shift assay as described under "Materials and Methods." Arrows denote the specifically bound bands. GAL4-coactivator interactions with VP-Nur77(E/F) in Panc-28 cells were treated with DIM-C-pPhCF 3 (B), DIM-C-pPhOCH 3 (C), and DIM-C-Ph (D). Cells were transfected with the pGAL4, VP-Nur77(E/F), and GAL4-coactivator/repressor (chimera) constructs and treated with the Nur77 agonists, and luciferase activity was determined as described under "Materials and Methods." Significant (p Ͻ 0.05) induction of luciferase activity is indicated (*), and results are expressed as means Ϯ S.E. for three separate determinations for each treatment group. creased cell survival is also observed for agents that induce apoptosis and/or Nur77 nuclear to cytosolic translocation in cancer cells (15)(16)(17)(18)(19)(20). Results illustrated in Fig. 5B show that treatment of Panc-28 cells with Nur77 agonists induced cleavage of PARP, whereas the Nur77-inactive DIM-C-pPhOH did not induce this response. PARP cleavage is associated with activation of cell death pathways; however, this was not accompanied by changes in levels of bax (Fig. 5B) or bcl-2 proteins (data not shown). Moreover, treatment of Panc-28 cells with 10 and 20 M DIM-C-pPhOCH 3 for 8 and 12 h showed a time-and dose-dependent increase of annexin V-stained cells using a green fluorescent Alexa Fluor 488 probe (Fig. 5C). The effects of camptothecin (positive control for apoptosis) and DIM-C-pPhOCH 3 were comparable. After treatment with DIM-C-pPhOCH 3 for 6 h, annexin V-stained cells were significantly increased, plasma membrane blebbing was observed, and there was minimal PI staining. However, after 12 h, PI staining was increased. Induction of PARP cleavage by Nur77 agonists was also observed in other pancreatic (MiaPaCa-2), prostate (LN-CaP), and breast (MCF-7) cancer cell lines (Fig. 5D). Induction of PARP cleavage by the Nur77-active compounds in Panc-28 cells was not accompanied by changes in Nur77 expression (Fig. 4B), and this was in contrast to TPA, which activates nuclear pathways by inducing Nur77 expression (21). Using a protocol comparable to that outlined in Fig. 5B, the induction of PARP cleavage by the Nur77 agonists in Panc-28 cells was not affected by the nuclear export inhibitor leptomycin B (LMB) (1 ng/ml) (Fig. 5E). LMB alone slightly induced PARP cleavage and, for some cells cotreated with LMB plus Nur77 agonists, there was enhanced PARP cleavage. In contrast, previous studies showed that LMB inhibits apoptosis in cells treated with apoptosis-inducing agents that activate nuclear-cytosol/mitochondrial translocation of Nur77 (15,16). These results demonstrate that activation of nuclear Nur77 by C-substituted DIMs induces apoptosis in Panc-28 and other cancer cell lines; however, evidence for activation of the intrinsic apoptotic pathways was not observed.
Nur77-active C-DIMs Induce TRAIL-In thymocytes, there is evidence that Nur77-induced apoptosis is linked to transcriptional activation (32), and microarray studies in thymocytes undergoing Nur77-dependent apoptosis identified several apoptosis-related genes, including fasL and TRAIL (33). Results in Fig. 6A show that Nur77 agonists that induce PARP cleavage also induce TRAIL (but not fasL) protein expression in Panc-28 cells, suggesting that this response may be a direct or indirect downstream target of Nur77 agonists in cancer cells. The Nur77-inactive DIM-C-pPhOH did not induce TRAIL. In addition, DIM-C-pPhOCH 3 or DIM-C-Ph induced TRAIL mRNA levels in Panc-28 cells (Fig. 6B). Because TRAIL activates the extrinsic apoptosis pathway and activation of caspase 8, we also investigated the effect of a caspase 8 inhibitor (z-IETDfmk) and the pan-caspase inhibitor (z-VAD-fmk) on induction of PARP cleavage by Nur77 agonists (Fig. 6C). The results show that both inhibitors blocked (60 -90%) induction of PARP cleavage by Nurr7 agonists.
The role of Nur77 in mediating induction of TRAIL and PARP cleavage by DIM-C-pPhOCH 3 was further investigated in Panc-28 cells transfected with nonspecific RNA (iScr) and iNur77 (Fig. 6D). Levels of Nur77, PARP cleavage, and TRAIL proteins were determined by Western blot analysis of whole cell extracts, and the results showed that iNurr significantly decreased levels of all three proteins. In addition, cotreatment of Panc-28 cells with DIM-C-pPhOH 3 or DIM-C-Ph and the Nur77 antagonist DIM-C-pPhOH (Fig. 6E) showed that the latter compound also inhibited induction of PARP cleavage and TRAIL protein expression induced by Nur77 agonists. These results demonstrate that Nur77 agonists induce apoptosis pathways in cancer cells through transcriptional (nuclear) FIG. 4. Nuclear localization of Nur77. A, immunostaining. Panc-28 cells were treated with Me 2 SO or 10 M Nur77 agonists for 6 h, and cells were immunostained for Nur77 as described under "Materials and Methods." Nur77 staining was not observed in cells treated with nonspecific IgG. B, nuclear localization in subcellular fractions. Panc-28 cells were treated with the various compounds for 12 h and Nur77 protein expression in cytosolic, and nuclear extracts were determined by Western blot analysis. Sp1 protein and a nonspecific (NS) band serve as loading controls, and Sp1, a nuclear protein, also serves as a control for separation of nuclear and cytosolic extracts. mechanisms, and at least one of the induced proteins (TRAIL) activates an extrinsic apoptotic pathway. In summary, selected C-substituted DIMs have now been identified as ligands for the orphan receptor Nur77, and activation of this receptor is associated with decreased cancer cell survival, induction of TRAIL, and apoptosis.

FIG. 6. Induction of TRAIL and PARP cleavage in Panc-28 cells is dependent on Nur77.
A, induction of TRAIL. Cells were treated with Nur77 agonists and levels of TRAIL protein in whole cell lysates were determined by Western blot analysis as described under "Materials and Methods." Results are expressed as means Ϯ S.E. for three separate determinations and significant (p Ͻ 0.05) induction is indicated. B, induction of TRAIL mRNA. Panc-28 cells were treated with 10 or 20 M DIM-C-pPhOCH 3 or DIM-C-Ph for 12 h, and TRAIL mRNA levels were determined by reverse transcription-PCR as described under "Materials and Methods." Results are expressed as means Ϯ S.E. for three separate determinations for each treatment group as significant (p Ͻ 0.05) is indicated by an asterisk. C, effects of caspase inhibitors. Cells were treated and analyzed essentially as described in A; however cells were also cotreated with caspase 8 and pan-caspase inhibitors z-IETD or z-VAD-fmk, respectively. Both inhibitors partially decreased PARP cleavage. D, effects of iNur77 on TRAIL expression and PARP cleavage. Panc-28 cells were treated with Me 2 SO or 10 M DIM-C-pPhOCH 3 for 24 h, transfected with iNur77 or iScr (nonspecific), and whole cell lysates were analyzed by Western blot analysis as described under "Materials and methods. " Results are expressed as means Ϯ S.E. for three separate determinations for each treatment group and significant (p Ͻ 0.05) induction (*) or inhibition by iNur77 (**) is indicated. E, inhibition of induced PARP cleavage and TRAIL by DIM-C-pPhOH. Panc-28 cells were treated with 10 M DIM-C-pPhOCH 3 or DIM-C-Ph alone or in the presence of 20 M DIM-C-pPhOH, and PARP cleavage and TRAIL protein expression were determined by Western blot analysis as described under "Materials and Methods."

Inhibition of Tumor Growth in Athymic Nude Mice Bearing
Panc-28 Cell Xenografts-Approximately 11 days after injection of Panc-28 cells, palpable tumors were detected and the mice were administered corn oil (control) or DIM-C-pPhOCH 3 (in corn oil) at a dose of 25 mg/kg/day, which was given by oral gavage. The animals were treated every second day, and tumor areas were determined over the duration of the experiment. The results (Fig. 7A) showed that DIM-C-pPhOCH 3 significantly inhibited tumor growth (area), and this was also complemented by a parallel decrease in tumor weights (Fig. 7B). Analysis of tumors from control and treated animals (TUNEL assay) indicated similar levels of apoptosis. Animal weight gain and organ weights were comparable in both treatment groups, and there were no apparent signs of toxicity in the DIM-C-pPhOCH 3 -treated mice compared with the corn oil controls. The mouse brain and muscle express relatively high levels of Nur77 (34), and examination of brain regions by H&E staining did not indicate any differences between the control (corn oil) and DIM-C-pPhOCH 3 -treated animals.

DISCUSSION
Nur77 is widely expressed in multiple tissues and has been identified as a critical mediator of T-cell receptor-dependent apoptosis in T-lymphocytes and T-hybridoma cells (35,36). Expression of dominant negative or antisense Nur77 blocked T-cell receptor-mediated apoptosis in T-hybridoma cells and extensive apoptosis of thymocytes was observed in transgenic mice overexpressing full-length Nur77 (13,14,35,36). Activation of cell death in macrophages is associated with increased expression of Nur77, and decreased cell death was observed in Nur77-deficient macrophages (37). A recent study (38) show that cadmium acetate induced apoptosis in WI-38 human lung fibroblasts and A549 human lung carcinoma cells, and this was also accompanied by induction of Nur77. Moreover, transfection with dominant-negative Nur77 protected the cells against cadmium-induced apoptosis.
Ongoing studies in the laboratory with a series of C-substituted DIMs indicate that these compounds inhibit growth or induce cell death of multiple cancer cell lines, and some of these analogs, including DIM-C-pPhCF 3 , activate PPAR␥ (22,26). However, several PPAR␥-inactive C-substituted DIMs also decreased cell survival of several cancer cell lines (e.g. Fig. 5A) and inhibited carcinogen-induced mammary tumor growth in female Sprague-Dawley rats (data not shown). Nur77 was considered as a possible target for C-DIM compounds based on results of several studies with retinoids, apoptosis, and differentiation-inducing agents that also inhibit cell growth and activate extranuclear Nur77 (15)(16)(17)(18)(19)(20). Initial studies confirmed Nur77 protein expression in 12 prostate, colon, bladder, pancreatic, and breast cancer cell lines (Fig. 1A). Results of screening a panel of structurally diverse C-substituted DIMs shows that DIM-C-pPhCF 3 and two PPAR␥-inactive analogs, DIM-C-pPhOCH 3 and DIM-C-Ph, activate Nur77-dependent transactivation in Panc-28 and other cancer cell lines transfected with GAL4-Nur77 (full-length) or NuRE (Fig. 1, B and C). Moreover, ligand-induced transcriptional activation is observed with GAL4-Nur77(E/F) chimeras (Fig. 2C) in which only the ligand binding domain of Nur77 is expressed. The role of Nur77 in mediating ligand-dependent transactivation was confirmed in studies showing that these responses were inhibited by either iNur77 (small inhibitory RNA) (Fig. 2B) or DIM-C-pPhOH, which exhibited Nur77 antagonist activity (Fig. 2C). Previous studies on the crystal structure of the mouse Nurr1 LBD (39) and the Drosophila Nurr1 homolog DHR38 (40) show that the ligand binding pocket is occupied by bulky hydrophobic amino acid side chains. Moreover, due to the high sequence homology among NGFI-B family proteins, it has been suggested that Nur77, Nurr1, and Nor-1 may represent a class of orphan receptors that function independently of ligand binding (41). In contrast, this study shows that selected C-DIM compounds uniquely induce nuclear Nur77-mediated transactivation; this induction response is observed through the E/F domain of Nur77 (Fig. 1C) and can be inhibited by DIM-C-pPhOH, a Nur77 antagonist (Figs. 2 and 5). Moreover, both activation of Nur77 and Nur77 antagonist activities by C-DIMs were highly structure-dependent ( Figs. 1 and 2). These results suggest that the active C-DIM compounds interact with the E/F domain of Nur77 and induce conformational changes, resulting in binding to the ligand binding pocket or other sites within the C-terminal region of Nur77. Currently, we are examining critical ligand interaction sites within the E/F domain of Nur77 by deletion/mutation analysis and by crystallization of the Nur77 LBD in the presence or absence of the C-DIM ligands.
Several studies have reported activation of Nur77-dependent transactivation in different cell lines, and these responses primarily involve the AF-1 domain of Nur77 and activation by kinases (28,42,43). For example, induction of Nur77-dependent transactivation was observed for the coactivator ASC-2 in CV-1 and HeLa cells; however, this effect was dependent on calcium/calmodulin-dependent protein kinase IV and did not involve direct ASC-2-Nur77 interactions (42). Transactivation mediated by Nur77 homodimers is enhanced by protein kinase A and SRC1-3 in CV-1 and AtT-20 cells and these responses FIG. 7. Inhibition of tumor growth by DIM-C-pPhOCH 3 . Tumor areas (A) and weights (B). Male athymic nude mice bearing Panc-28 cell xenografts were treated every second day with corn oil and DIM-C-pPhOCH 3 in corn oil as described under "Materials and Methods." The overall dose of DIM-C-pPhOCH 3 was 25 mg/kg/day. Significant (p Ͻ 0.05) inhibition of tumor areas and weights is indicated by an asterisk. H&E stainings of muscle and brain tissue from control and treated animals were similar, and tumors from control and treated animals exhibited apoptosis (data not shown).
were AF-1-dependent (28). Another report also confirmed that Nur77 transactivation in C2C12 and COS-1 cells was enhanced by SRCs and other coactivators, and involved direct interactions of coactivators with the A/B (and not E/F) domain of Nur77 (43). These observations are consistent with the crystal structure of Nurr1, which lacks the "classical binding site for coactivators" (39). However, ligand-dependent activation of Nur77 E/F domain observed in this study (Fig. 1C) should also be accompanied by interactions with some nuclear receptor coactivators/coregulators. Initial studies showed that, in the absence of ligand, VP-Nur77(E/F) did not interact with GAL-4-coactivators (PGF-1, CARM-1, SRC1-3, and TRAP220) or GALR-SMRT chimeras (data not shown); however, DIM-C-pPhCF 3 , DIM-C-pPhOCH 3 , and DIM-C-Ph induced interactions between several common nuclear receptor coactivators (PGC-1, SRC-1, and TRAP220) and the LBD (E/F) of Nur77 in mammalian two-hybrid assays (Fig. 3, B-D). These results are consistent with other studies on activation of nuclear receptors by ligands and their interactions with specific coactivators through binding receptor E/F domains. For example, our recent studies with PPAR␥-active C-substituted DIMs in colon cancer cells show that ligand-induced PPAR␥(E/F domain)-coactivator interactions in mammalian two-hybrid assays primarily involved PGC-1 (26), whereas C-DIM-induced Nur77-coactivator interactions in Panc-28 cells involve multiple coactivators. Although the crystal structure of unliganded Nurr1 shows that this receptor does not contain a classic coactivator interaction site in the E/F domain (helix 12), novel coactivator interaction surfaces have recently been identified between helices 11 and 12 in Nurr1 (44). This region is similar in human Nur77, and current studies are investigating C-DIM-induced interaction surfaces between the E/F domain of Nur77 and coactivators. In summary, the transactivation and coactivator-Nur77 interactions induced by DIM-C-pPhCF 3 , DIM-C-pPhOCH 3 , and DIM-C-Ph are consistent with results obtained for other ligand-activated nuclear receptors suggesting that selected Csubstituted DIMs are a novel class of compounds that induce E/F domain-dependent activation of Nur77.
Treatment of Panc-28 cells with Nur77-active C-substituted DIMs agonists decreased cell survival (Fig. 4A) and induced nuclear condensation within 48 and 24 h, respectively, and this is typically observed in cells undergoing cell death. We therefore further examined Nur77-mediated induction of PARP cleavage, which is a well characterized downstream marker of activated cell death pathways. PARP cleavage was induced in Panc-28 cells treated with Nur77 agonists (Fig. 5B), and similar results were observed in other pancreatic, prostate, and breast cancer cell lines (Fig. 5D). Annexin V staining was also observed in Panc-28 cells treated with Nur77-active C-DIMs (Fig. 5C), and these data further confirm induction of apoptosis in these cancer cell lines. Previous studies report that induction of cell death pathways by apoptosis-inducing agents in some cancer cell lines is accompanied by translocation of Nur77 from the nucleus to the cytosol/mitochondria, and this has been linked to cytochrome c release and direct interaction of Nur77 with bcl-2 (15)(16)(17)(18)(19)(20). In contrast, we observed that treatment of Panc-28 cells with Nur77-active C-DIMs resulted only in formation of a nuclear complex (Fig. 4, A and B). Moreover, inhibition of nuclear export of Nur77 by LMB did not affect PARP cleavage induced by Nur77-active C-DIMs (Fig. 5E), suggesting that this response is mediated through nuclear Nur77. This nuclear pathway for induction of apoptosis is in contrast to the effects observed for TPA and CD437, which induce nuclear export of Nur77 in cancer cell lines, and inhibition of Nur77 nuclear export by LMB, which inhibits induction of apoptosis (15,16). These results clearly distinguish between the induc-tion of cell death pathways in cancer cells through ligand-dependent activation of nuclear Nur77 (this study) and through induction of Nur77 nuclear translocation (15)(16)(17)(18)(19)(20).
Overexpression of Nur77 in thymocytes induces expression of several genes associated with apoptosis (33), and at least one of the genes, TRAIL (protein and mRNA), is also induced by Nur77 agonists in Panc-28 cells (Fig. 6, A and B). RNA interference assays with iNur77 (Fig. 6D) and inhibition studies with the Nur77 antagonist DIM-C-pPhOH (Fig. 6E) demonstrate that induction of TRAIL and PARP cleavage by DIM-C-pPhOCH 3 and DIM-C-Ph are Nur77-dependent. Thus, the nuclear action of Nur77 agonists in cancer cell lines is comparable to the transcriptionally dependent pathway observed in T-cells overexpressing Nur77 (33). TRAIL typically activates caspase 8, and the extrinsic pathways of apoptosis and the caspase 8 inhibitor z-IETD-fmk significantly blocks (Ͼ60%) induction of PARP cleavage by Nur77 agonists (Fig. 5C). The pan-caspase inhibitor z-VAD-fmk blocked Ͼ90% of induced PARP cleavage suggesting that, although TRAIL may be a major Nur77-induced gene in Panc-28 cells, other pro-apoptotic genes may also be induced; these are currently being investigated. We also observed in xenograft experiments that DIM-C-pPhOCH 3 inhibited tumor growth in athymic nude mice bearing Panc-28 cell xenografts (Fig. 7).
In summary, results of this study have identified a novel group of C-substituted DIMs that activate the orphan receptor Nur77 through the E/F domain. These results are in contrast to previous reports showing that kinase/coactivator-dependent activation of Nur77 was primarily AF-1-dependent (23,42,43). It has also been reported that nuclear receptor coactivators interact with the N-terminal A/B but not E/F domains of Nur77, and in the absence of C-DIM compounds, coactivator-Nur(E/F) interactions were not observed in this study. However, DIM-C-pPhCF 3 , DIM-C-Ph, and DIM-C-pPhOCH 3 induced coactivator interactions with the E/F domain of Nur77 (Fig. 3), and this was consistent with Nur77 (nuclear)-dependent transactivation. Activation of Nur77 by selected C-DIMs is associated with decreased cancer cell survival, induction of apoptosis, induced expression of the apoptosis gene/protein TRAIL, and inhibited tumor growth in vivo. These results suggest that C-DIM ligands that activate Nur77 are a potential new class of anticancer agents. Their activities and mechanisms of action in other cancer cell lines are currently being investigated.