Activation of Nur77 by Selected 1,1-Bis(3′-indolyl)-1-(p-substituted phenyl)methanes Induces Apoptosis through Nuclear Pathways
2005; Elsevier BV; Volume: 280; Issue: 26 Linguagem: Inglês
10.1074/jbc.m500107200
ISSN1083-351X
AutoresSudhakar Chintharlapalli, Robert C. Burghardt, Sabitha Papineni, Shashi K. Ramaiah, Kyungsil Yoon, Stephen Safe,
Tópico(s)Macrophage Migration Inhibitory Factor
ResumoNur77 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. 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 (1Tsai M.J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2702) Google Scholar, 2Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6110) Google Scholar, 3Beato M. Herrlich P. Schutz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1639) Google Scholar, 4Olefsky J.M. J. Biol. Chem. 2001; 276: 36863-36864Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 5Enmark E. Gustafsson J.A. Mol. Endocrinol. 1996; 10: 1293-1307Crossref PubMed Scopus (192) Google Scholar, 6Giguere V. Endocr. Rev. 1999; 20: 689-725Crossref PubMed Scopus (720) Google Scholar, 7Mohan R. Heyman R.A. Curr. Top. Med. 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Nuclear receptors share common structural features that include an N-terminal A/B domain, containing activation function-1 (AF-1), 1The abbreviations used are: AF, activation function; LBD, ligand binding domain; NGFI-B, nerve growth factor I-B; CD437, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid; TPA, 12-O-tetradecanoylphorbol-13-acetate; PARP, poly(ADP-ribose) polymerase; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; LMB, leptomycin B; DMEM, Dulbecco's modified Eagle's medium; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; DIM, 3,3′-diindolylmethane; C, methylene; PPAR, peroxisome proliferator-activated receptor; NurRE, Nur77 response element; NBRE, Nur77 binding response element; z, benzyl-oxycarbonyl; fmk, fluoromethyl ketone; H&E, hematoxylin & eosin. and a C-terminal 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 (5Enmark E. Gustafsson J.A. Mol. Endocrinol. 1996; 10: 1293-1307Crossref PubMed Scopus (192) Google Scholar, 6Giguere V. Endocr. Rev. 1999; 20: 689-725Crossref PubMed Scopus (720) Google Scholar, 7Mohan R. Heyman R.A. Curr. Top. Med. Chem. 2003; 3: 1637-1647Crossref PubMed Scopus (76) Google Scholar). 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-Bα (Nur77), NGFI-Bβ (Nurr1), and NGFI-Bγ (Nor1) (8Milbrandt J. Neuron. 1988; 1: 183-188Abstract Full Text PDF PubMed Scopus (531) Google Scholar, 9Ryseck R.P. Macdonald-Bravo H. Mattei M.G. Ruppert S. Bravo R. 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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 (15Li H. Kolluri S.K. Gu J. Dawson M.I. Cao X. Hobbs P.D. Lin B. Chen G. Lu J. Lin F. Xie Z. Fontana J.A. Reed J.C. Zhang X. Science. 2000; 289: 1159-1164Crossref PubMed Scopus (589) Google Scholar, 16Lin B. Kolluri S.K. Lin F. Liu W. Han Y.H. Cao X. Dawson M.I. Reed J.C. Zhang X.K. Cell. 2004; 116: 527-540Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 17Wu Q. Liu S. Ye X.F. Huang Z.W. Su W.J. Carcinogenesis. 2002; 23: 1583-1592Crossref PubMed Scopus (105) Google Scholar, 18Holmes W.F. Soprano D.R. Soprano K.J. Oncogene. 2003; 22: 6377-6386Crossref PubMed Scopus (74) Google Scholar, 19Holmes W.F. Soprano D.R. Soprano K.J. J. Cell. Biochem. 2003; 89: 262-278Crossref PubMed Scopus (62) Google Scholar, 20Wilson A.J. Arango D. Mariadason J.M. Heerdt B.G. Augenlicht L.H. Cancer Res. 2003; 63: 5401-5407PubMed Google Scholar, 21Mu X. Chang C. J. Biol. Chem. 2003; 278: 42840-42845Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). For example, the retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene 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 (15Li H. Kolluri S.K. Gu J. Dawson M.I. Cao X. Hobbs P.D. Lin B. Chen G. Lu J. Lin F. Xie Z. Fontana J.A. Reed J.C. Zhang X. Science. 2000; 289: 1159-1164Crossref PubMed Scopus (589) Google Scholar, 16Lin B. Kolluri S.K. Lin F. Liu W. Han Y.H. Cao X. Dawson M.I. Reed J.C. Zhang X.K. Cell. 2004; 116: 527-540Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar). In contrast, it has been suggested that TPA-induced Nur77 in LNCaP prostate cancer cells activates transcription of E2F1, which is also pro-apoptotic (21Mu X. Chang C. J. Biol. Chem. 2003; 278: 42840-42845Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). 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(3′-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. Cell Lines and Reagents—Panc-28, Panc-1, MiaPaCa-2, LNCaP, MCF-7, HT-29, and HCT-15 cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA). RKO, DLD-1, and SW-480 colon cancer cells were provided by Dr. S. Hamilton, and KU7 and 253-JB-V-33 bladder cells were provided by Dr. A. Kamat (M. D. Anderson Cancer Center, Houston, TX). The C-substituted DIMs were synthesized in this laboratory as previously described (22Qin C. Morrow D. Stewart J. Spencer K. Porter W. Smith III, R. Phillips T. Abdelrahim M. Samudio I. Safe S. Mol. Cancer Ther. 2004; 3: 247-259Crossref PubMed Scopus (42) Google Scholar). Antibodies for PARP (sc8007), Sp1 (sc-59), and TRAIL (sc7877) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Nur77 (IMG-528) from Imgenex (San Diego, CA). The GAL4 reporter containing five GAL4 response elements (pGAL4) was provided by Dr. Marty Mayo (University of North Carolina, Chapel Hill, NC). The GAL4-Nur77 (full-length) and GAL4-Nur77 (E/F) chimeras were provided by Dr. Jae W. Lee (Baylor College of Medicine, Houston, TX) and Dr. T. Perlmann (Ludwig Institute for Cancer Research, Stockholm, Sweden), respectively, and Dr. Lee also provided the Nur77 response element-luciferase (NurRE-Luc) reporter construct. The GAL-4-coactivator fusion plasmids pMSRC1, pMSRC2, pMSRC3, pMDRIP205, and pMCARM-1 were kindly provided by Dr. Shigeaki Kato (University of Tokyo, Tokyo, Japan). For RNA interference assays, we used a nonspecific scrambled (iScr) oligonucleotide as described (23Abdelrahim M. Samudio I. Smith R. Burghardt R. Safe S. J. Biol. Chem. 2002; 277: 28815-28822Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The small inhibitory RNA for Nur77 (iNur77) was identical to the reported oligonucleotide (16Lin B. Kolluri S.K. Lin F. Liu W. Han Y.H. Cao X. Dawson M.I. Reed J.C. Zhang X.K. Cell. 2004; 116: 527-540Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar), and these were purchased from Dharmacon Research (Lafayette, CO). Leptomycin B (LMB) was obtained from Sigma, and caspase inhibitors were purchased from BD Pharmingen. The following oligonucleotides were prepared by IDT (Coralville, IA) and were used in gel mobility shift assays; NBRE, 5′-GAT CCT CGT GCG AAA AGG TCA AGC GCT A-3′; NurRE, 5′-GAT CCT AGT GAT ATT TAC CTC CAA ATG CCA GGA-3′. Transfection Assays—Transfection assays were essentially carried out as previously described using Lipofectamine Plus reagent (Invitrogen), and luciferase activities were normalized to β-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 (22Qin C. Morrow D. Stewart J. Spencer K. Porter W. Smith III, R. Phillips T. Abdelrahim M. Samudio I. Safe S. Mol. Cancer Ther. 2004; 3: 247-259Crossref PubMed Scopus (42) Google Scholar, 23Abdelrahim M. Samudio I. Smith R. Burghardt R. Safe S. J. Biol. Chem. 2002; 277: 28815-28822Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Results are expressed as means ± S.E. for at least three replicate determinations for each treatment group. Mammalian Two-hybrid Assay—Panc-28 cells were plated in 12-well plates at 1 × 105 cells/well in DMEM/F-12 media supplemented with 2.5% charcoal-stripped fetal bovine serum. After growth for 16 h, various amounts of DNA, i.e. Gal4Luc (0.4 μg), β-gal (0.04 μg), VP-Nur77(E/F) (0.04 μg), pMSRC1 (0.04 μg), pMSRC2 (0.04 μg), pMSRC3 (0.04 μg), pMDRIP205 (0.04 μg), and pMCARM-1 (0.04 μg) were transfected by Lipofectamine (Invitrogen) according to the manufacturer's protocol. After 5 h of transfection, the transfection mix was replaced with complete media containing either vehicle (Me2SO) or the indicated ligand for 20-22 h. Cells were then lysed with 100 ml of 1× reporter lysis buffer, and 30 μl of cell extract was used for luciferase and β-galactosidase assays. Lumicount was used to quantitate luciferase and β-galactosidase activities, and the luciferase activities were normalized to β-galactosidase activity. 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 Me2SO 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, nuclear fractions, or whole cell lysates were obtained at various time points, analyzed by Western blot analysis, and bands were quantitated as previously described (22Qin C. Morrow D. Stewart J. Spencer K. Porter W. Smith III, R. Phillips T. Abdelrahim M. Samudio I. Safe S. Mol. Cancer Ther. 2004; 3: 247-259Crossref PubMed Scopus (42) Google Scholar, 23Abdelrahim M. Samudio I. Smith R. Burghardt R. Safe S. J. Biol. Chem. 2002; 277: 28815-28822Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Immunocytochemical analysis was determined using Nur77 antibodies as previously reported (23Abdelrahim M. Samudio I. Smith R. Burghardt R. Safe S. J. Biol. Chem. 2002; 277: 28815-28822Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Gel Shift Assay—Cells were seeded in DMEM/F-12 medium supplemented with 2.5% charcoal-stripped serum and treated with 10 μm DIM-C-pPhOCH3 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 32P-labeled at the 5′-end using T4 polynucleotide kinase and [γ-32P]ATP. Nuclear extracts were incubated in HEPES with ZnCl2 and 1 μg 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 μm DIM-C-pPhOCH3 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-μl 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, 5′-CGT GTA CTT TAC CAA CGA GCT GA-3′, reverse, 5′-ACG GAG TTG CCA CTT GAC TTG-3′; and TATA-binding protein forward, 5′-TGC ACA GGA GCC AAG AGT GAA-3′, reverse, 5′-CAC ATC ACA GCT CCC CAC CA-3′. 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 × 106) were injected subcutaneously into each mouse on both flanks using a 30-gauge 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-pPhOCH3 every second day. The volume of corn oil was 75 μl, and the dose of DIM-C-pPhOCH3 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 C-substituted DIMs—Studies in this laboratory have been investigating the anticarcinogenic activities of a series of ring-substituted 3,3′-diindolylmethanes (DIMs) and methylene (C)-substituted DIMs, and many of these compounds were active in vivo and in cell culture assays (22Qin C. Morrow D. Stewart J. Spencer K. Porter W. Smith III, R. Phillips T. Abdelrahim M. Samudio I. Safe S. Mol. Cancer Ther. 2004; 3: 247-259Crossref PubMed Scopus (42) Google Scholar, 24McDougal A. Sethi-Gupta M. Ramamoorthy K. Sun G. Safe S. Cancer Letts. 2000; 151: 169-179Crossref PubMed Scopus (62) Google Scholar, 25McDougal A. Wilson C. Safe S. Cancer Lett. 1997; 120: 53-63Crossref PubMed Scopus (62) Google Scholar, 26Chintharlapalli S. Smith III, R. Samudio I. Zhang W. Safe S. Cancer Res. 2004; 64: 5994-6001Crossref PubMed Scopus (70) Google Scholar). Some members of a series of C-substituted DIMs activated peroxisome proliferator-activated receptor γ (PPARγ) but not PPARα, retinoic acid receptor, retinoic X receptor, estrogen receptor α, 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 (15Li H. Kolluri S.K. Gu J. Dawson M.I. Cao X. Hobbs P.D. Lin B. Chen G. Lu J. Lin F. Xie Z. Fontana J.A. Reed J.C. Zhang X. Science. 2000; 289: 1159-1164Crossref PubMed Scopus (589) Google Scholar, 16Lin B. Kolluri S.K. Lin F. Liu W. Han Y.H. Cao X. Dawson M.I. Reed J.C. Zhang X.K. Cell. 2004; 116: 527-540Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 17Wu Q. Liu S. Ye X.F. Huang Z.W. Su W.J. Carcinogenesis. 2002; 23: 1583-1592Crossref PubMed Scopus (105) Google Scholar, 18Holmes W.F. Soprano D.R. Soprano K.J. Oncogene. 2003; 22: 6377-6386Crossref PubMed Scopus (74) Google Scholar, 19Holmes W.F. Soprano D.R. Soprano K.J. J. Cell. Biochem. 2003; 89: 262-278Crossref PubMed Scopus (62) Google Scholar, 20Wilson A.J. Arango D. Mariadason J.M. Heerdt B.G. Augenlicht L.H. Cancer Res. 2003; 63: 5401-5407PubMed Google Scholar, 21Mu X. Chang C. J. Biol. Chem. 2003; 278: 42840-42845Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), 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 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 (full-length) 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-pPhCF3) and methoxy (DIM-C-pPhOCH3) 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-pPhOCH3 as a model, and the position of the methoxyl group was changed to the meta (DIM-C-mPhOCH3) and ortho (DIM-C-oPhOCH3) 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-pPhOCH3, DIM-C-Ph, and DIM-C-pPhCF3, 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-pPhCF3) also activates PPARγ (22Qin C. Morrow D. Stewart J. Spencer K. Porter W. Smith III, R. Phillips T. Abdelrahim M. Samudio I. Safe S. Mol. Cancer Ther. 2004; 3: 247-259Crossref PubMed Scopus (42) Google Scholar, 26Chintharlapalli S. Smith III, R. Samudio I. Zhang W. Safe S. Cancer Res. 2004; 64: 5994-6001Crossref PubMed Scopus (70) Google Scholar), whereas DIM-C-pPhOCH3 and DIM-C-Ph are PPARγ-inactive (22Qin C. Morrow D. Stewart J. Spencer K. Porter W. Smith III, R. Phillips T. Abdelrahim M. Samudio I. Safe S. Mol. Cancer Ther. 2004; 3: 247-259Crossref PubMed Scopus (42) Google Scholar). DIM-C-pPhOH was inactive in both transactivation assays and, at higher concentrations, decreased activity lower than observed in solvent (Me2SO) 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 μm) of DIM-C-pPhCF3, DIM-C-pPhOCH3, 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 μm DIM-C-pPhOCH3 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-pPhCF3, DIM-C-pPhOCH3, 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 μm) did not inhibit DIM-C-pPhOCH3- or DIM-C-Ph-induced transactivation (Fig. 2D). DIM-C-oPhOH also did not exhibit Nur77 antagonist activity (Fig. 2E); however, high doses (20 μm) 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 Me2SO or DIM-C-pPhOCH3 with 32P-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 excess NurRE (lane 3) or NBRE (lane 7) but not by mutant NurRE (lane 4) or mutant NBRE (lane 8) oligonucleotides. 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 Nur77-active 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 (27Philips A. Lesage S. Gingras R. Maira M.H. Gauthier Y. Hugo P. Drouin J. Mol. Cell. Biol. 1997; 17: 5946-5951Crossref PubMed Scopus (317) Google Scholar, 28Maira M. Martens C. Batsche E. Gauthier Y. Drouin J. Mol. Cell. Biol. 2003; 23: 763-776Crossref PubMed Scopus (126) Google Scholar). Ligand-dependent activation of nuclear receptors is dependent on interaction of the bound receptor with coactivators (29Rosenfeld M.G. Glass C.K. J. Biol. Chem. 2001; 276: 36865-36868Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 30Xu J. Li Q. Mol. Endocrinol. 2003; 17: 1681-1692Crossref PubMed Scopus (401) Google Scholar, 31Smith C.L. O'Malley B.W. Endocrine Rev. 2004; 25: 45-71Crossref PubMed Scopus (810) Google Scholar), and Fig. 3 (B-D) summarizes results of a mammalian two-hybrid assay in Panc-28 cells transfected with VP-Nur77 (ligand binding domain) and GAL4-coactivator chimeras. Ligand-induced Nur77-coactivator interactions were determined using a construct (pGAL4) containing 5 GAL4 response elements. Coactivators used in this study include SRC-1, SRC-2 (TIFII), SRC-3 (AIB1), PGC-1, TRAP220, and CARM-1. A GAL4-repressor (SMRT) chimera was also included in the assay. All three ligands induced transactivation in cells transfected with GAL4-SRC-1, GAL4-PGC-1, and GAL4-TRAP220 chimeras. DIM-C-pPhOCH3-induced transactivation in cells transfected with GAL4-SRC-3 and GAL4-CARM-1 was slightly activated by DIM-C-pPhOCH3 and DIM-C-pPhCF3. The results demonstrate that there were some ligand-dependent differences in transactivation observed for GAL4-
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