Activation of Nerve Growth Factor-Induced B by Methylene- Substituted Diindolylmethanes in Bladder Cancer Cells Induces Apoptosis and Inhibits Tumor Growth

Nerve growth factor-induced B (NGFI-B) genes are orphan nuclear receptors, and NGFI-B (Nur77, TR3) is overexpressed in bladder tumors and bladder cancer cells compared with nontumorous bladder tissue. 1,1-Bis(3 -indolyl)-1-(p-methoxyphenyl)-methane (DIM-C-pPhOCH3) and 1,1-bis(3 -indolyl)-1(p-phenyl)methane have previously been identified as activators of Nur77, and both compounds inhibited growth and induced apoptosis of UC-5 and KU7 bladder cancer cells. The proapoptotic effects of methylene-substituted diindolylmethanes (C-DIMs) were unaffected by cotreatment with leptomycin B and were dependent on nuclear Nur77, and RNA interference with a small inhibitory RNA for Nur77 (iNur77) demonstrated that C-DIM-induced activation of apoptosis was Nur77-dependent. Microarray analysis of DIM-C-pPhOCH3-induced genes in UC-5 bladder cancer cells showed that this compound induced multiple Nur77-dependent proapoptotic or growth inhibitory genes including tumor necrosis factor-related apoptosisinducing ligand (TRAIL), cystathionase, p21, p8, and sestrin-2. DIM-C-pPhOCH3 (25 mg/kg/d) also induced apoptosis and inhibited tumor growth in athymic nude mice bearing KU7 cells as xenografts, demonstrating that Nur77-active C-DIMs exhibit potential for bladder cancer chemotherapy by targeting Nur77, which is overexpressed in this tumor type. The nuclear receptor family of transcription factors includes the steroid and thyroid hormones, vitamin D, retinoid and ecdysone receptors, ligand-activated orphan receptors, and orphan receptors with no known ligands (Milbrandt, 1988; Mangelsdorf et al., 1995). Nuclear receptors influence diverse aspects of normal physiology in multiple tissues, and several receptors are drug targets for treating several diseases, including cancer. Nerve growth factor-induced B (NGFI-B) is part of a subfamily of orphan nuclear receptors; members of this subfamily include Nur77 (NGFI-B , TR3), Nurr1 (NGFI-B ), and Nor1 (NGFI-B ). 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 (Milbrandt, 1988; Bandoh et al., 1997; Maruyama et al., 1997). The physiological roles for NGFI-B proteins are not fully understood; however, gene targeting knockout experiments demonstrate several important functions for these proteins. For example, Nurr1 knockout mice have severe This research was supported by the National Institutes of Health National Cancer Institute [01R01-CA124998], the Korea Research Foundation Grant, MOEHRD Basic Research Promotion Fund [Grant KRF-2008-331-E00260]. S.D.C. and S.-O.L. contributed equally to this work. 1 Current affiliation: Eli Lilly and Company, Oncology Division, Indianapolis, IN (Current affiliation). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.109.061143. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: NGFI-B, nerve growth factor-induced B; C-DIM, methylene-substituted diindolylmethane; DIM-C-pPhOCH3, 1,1-bis(3 indolyl)-1-(p-methoxyphenyl)methane; DIM-C-pPhC, 1,1-bis(3 -indolyl)-1-(p-chlorophenyl)methane; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; IgG, immunoglobulin G; iNur77, small inhibitory RNA for Nur77; iScr, nonspecific scrambled oligonucleotide; NAG-1, nonsteroidal anti-inflammatory drug activated gene; DMSO, dimethyl sulfoxide; RT-PCR, reverse transcriptase-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; Z-VADfmk, N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone; PARP, poly(ADP-ribose) polymerase. 0026-895X/10/7703-396–404$20.00 MOLECULAR PHARMACOLOGY Vol. 77, No. 3 Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics 61143/3564861 Mol Pharmacol 77:396–404, 2010 Printed in U.S.A. 396 http://molpharm.aspetjournals.org/content/suppl/2009/12/18/mol.109.061143.DC1 Supplemental material to this article can be found at: at A PE T Jornals on M ay 3, 2017 m oharm .aspeurnals.org D ow nladed from impairments in midbrain neuronal development and dopamine expression, and these animals die soon after birth (Zetterstrom et al., 1997; Saucedo-Cardenas et al., 1998; DeYoung et al., 2003); Nor1 knockout animals die at gestational day 5 (DeYoung et al., 2003), whereas Nur77 knockout mice do not exhibit a specific phenotype (Lee et al., 1995), and this may be related to coexpression of both Nur77/Nor1, which exhibit some overlapping functions. Several studies suggest that in cancer cells, Nur77 plays a role in cell death pathways activated by apoptosisinducing agents (Li et al., 2000; Wu et al., 2002; Holmes et al., 2003a,b; Mu and Chang, 2003; Wilson et al., 2003a; Lin et al., 2004). Li et al. (2000) reported that treatment of LNCaP prostate cancer cells with agents such as retinoids, 12-O-tetradecanoylphorbol-13-acetate, and tumor necrosis factor 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, and treatment with leptomycin B (a blocker of nuclear export) inhibited induction of Nur77-dependent apoptosis. Induction of apoptosis was accompanied by translocation of Nur77 from the nucleus to the mitochondria, and Nur77 specifically interacted with Bcl-2 and converted Bcl-2 into a proapoptotic factor in human embryonic kidney 293T and HCT116 cells (Lin et al., 2004). However, a study in colon cancer cells reported that butyrate-induced apoptosis was associated with nuclear-to-cytoplasmic translocation of Nur77, which was not accompanied by subsequent mitochondrial interactions (Wilson et al., 2003a). Studies in this laboratory have characterized a series of methylene-substituted diindolylmethane (C-DIM) analogs as activators of orphan receptors (Chintharlapalli et al., 2004, 2005a; Qin et al., 2004; Kassouf et al., 2006; Cho et al., 2007; Inamoto et al., 2008). Two of these compounds, 1,1-bis(3 -indolyl)-1-(p-methoxyphenyl)methane (DIM-CpPhOCH3) and 1,1-bis(3 -indolyl)-1-(p-phenyl)methane (DIM-C-pPh), activate Nur77 in colon and pancreatic cancer cells (Chintharlapalli et al., 2005a; Cho et al., 2007), and 1,1-bis(3 -indolyl)-1-(p-chlorophenyl)methane (DIMC-pPhCl) activates NGFI-B (Nurr1) orphan receptor in bladder cancer cells (Inamoto et al., 2008). Nur77 and Nurr1 are widely expressed in bladder cancer cells (Inamoto et al., 2008), and we now show overexpression of Nur77 in bladder tumors. Nur77-active C-DIMs, such as DIM-C-pPhOCH3, inhibit bladder cancer cell growth and induce apoptosis. DIM-C-pPhOCH3 activates nuclear Nur77, and results of RNA interference studies show that induction of apoptosis is due to induction of several proapoptotic genes and proteins, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). DIMC-pPhOCH3 inhibited bladder tumor growth in a xenograft mouse model, suggesting that C-DIM-dependent activation of Nur77 is a promising new mechanism-based pathway for developing new agents for bladder cancer chemotherapy. Materials and Methods Cells, Biochemicals, and Antibodies. Human bladder cancer cell lines KU7 and UC-5 were provided by author A.M.K. Cells were maintained in Dulbecco’s modified Eagle’s medium/Ham’s F-12 (Sigma, St. Louis, MO) without phenol red supplemented with 0.22% sodium bicarbonate, 0.011% sodium pyruvate, 5% fetal bovine serum, and 10 ml/liter of 100 antibiotic/antimycotic solution (SigmaAldrich). Cells were maintained at 37°C in the presence of 5% CO2. Antibodies for cleaved PARP and cleaved caspase 8 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies for Nur77, TRAIL, Sp1, IgG, and -tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Nur77 antibodies from Imgenex (San Diego, CA) were also used for immunostaining and Western blots, and results were similar to those with Nur77 antibodies from Santa Cruz. Caspase inhibitor (Z-VAD-fmk) was obtained from Alexis Biochemicals (Lausen, Switzerland). Western Lightning chemiluminescence reagent was from PerkinElmer Life Sciences (Waltham, MA). For RNA interference assays, we used a nonspecific scrambled (iScr) oligonucleotide as described previously (Abdelrahim et al., 2002). The small inhibitory RNA for Nur77 (iNur77) was identical to the reported oligonucleotide (Lin et al., 2004), and these were purchased from Dharmacon RNA Technologies (Lafayette, CO). Leptomycin B was purchased from Sigma. The C-DIMs were synthesized in this laboratory as described previously (Qin et al., 2004). Cell Proliferation Assay. KU-7 and UC-5 cells were seeded in 12-well plates in Dulbecco’s modified Eagle’s medium/Ham’s F-12 containing 2.5% charcoal-stripped fetal bovine serum for 24 and 48 h until plates reached 50 to 60% confluence, which was usually observed 24 h after seeding. Cells were then treated with different concentrations of the test compounds (in DMSO) or DMSO alone. KU7 and UC-5 cells were counted to evaluate the effect of C-DIMs and DMSO (solvent control) on viable cell number using a particle counter (Z1; Beckman Coulter, Fullerton, CA). Each experiment was carried out in triplicate, and results are expressed as means S.D. for each treatment group. Western Blot Analysis. Cells were treated with the C-DIM compounds, and a caspase inhibitor and leptomycin B were added simultaneously. For RNA interference studies, the oligonucleotides (iScr or iNur77) were transfected into bladder cancer cells; after 36 h, cells were treated with DMSO or C-DIM compounds as described previously (Cho et al., 2007). Cells were collected, and cell lysates were prepared using lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1 mM sodium orthovanadate; 1 mM phenylmethylsulfonyl fluoride, 1 M leupeptin, and 1 g/ml aprotinin). After centrifugation of lysates at 15,000g for 20 min, the supernatants were recovered, and protein was quantified by the Bradford protein assay using reagent kit from Bio-Rad Laboratories (Hercules, CA). Protein samples (20 to 60 g) were sizeseparated by electrophoresis on SDS-polyacrylamide gels under nonreducing conditions. Separated proteins were electroblotted onto nitrocellulose membranes. The blot was blocked by incubating in blocking buffer (5% skim milk, 10 mM Tris, pH 7.5, 10 mM NaCl, and 0.1% Tween 20) for 1 h at 20°C 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 were incubated with fluorescein isothiocyanate-conjugated secondary antibody (1:200; Vector Laboratories, Burlingame, CA). The four-well chambers were mounted with mounting medium (Vector Laboratories) and viewed on a fluorescence microscope (Olympus). Reverse Transcriptase-Polymerase Chain Reaction. Total RNA was extracted using RNeasy Mini Kit (Qiagen Inc., Valencia, CA), and 1 g of RNA was used to synthesize cDNA using reverse transcription system (Promega, Madison, WI). The PCR conditions were as follows: initial denaturation at 94°C (2 min) followed by 28 Activation of Nuclear Nur77 Induces Apoptosis 397 at A PE T Jornals on M ay 3, 2017 m oharm .aspeurnals.org D ow nladed from cycles (NAG-1 and CSE), 30 cycles (p8 and Sestin-2), or 26 cycles (GAPDH) of denaturation for 1 min at 94°C, annealing for 1 min at 58°C (NAG-1) or 61°C (CSE, p8, Sestin-2, 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 Integrated DNA Technologies, Inc. (Coralville, IA) and used for amplification were as follows: NAG-1: sense, 5 -GTG CTC ATT CAA AAG ACC GAC ACC G-3 ; antisense, 5 -ATA CAC AGT TCC ATC AGA CCA GCC CC-3 ; p8: sense, 5 -ATG GCC ACC TTC CCA CCA GCA-3 ; antisense, 5 -TCA GCG CCG TGC CCC TCG CT-3 ; CSE: sense, 5 -GGC GAT CCA TGT GGG CCA GGA-3 ; antisense, 5 -ATG TCT CCA TGC TTA TGG ACA AT-3 ; sestrin-2: sense, 5 -GAC TCC GAG TGC CGC GCA GAG-3 ; antisense, 5 -ATG GCG GGC GGC AGC CAT GAT-3 ; and GAPDH: sense, 5 -ACG GAT TTG GTC GTA TTG GGC G-3 ; antisense, 5 CTC CTG GAA GAT GGT GAT GG-3 . PCR products were electrophoresed on 1% agarose gels containing ethidium bromide and visualized under UV transillumination. Quantitative Real-Time PCR. cDNA was prepared from the total RNA of cells using Reverse Transcription System (Promega). Each PCR was carried out in triplicate in a 20l 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 fast real-time PCR system (7900HT; Applied Biosystems). The ABI Dissociation Curves software was used after 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 sequences of the primers used for real-time PCR were as follows: p21: sense 5 -GGC AGA CCA GCA TGA CAG ATT TC-3 ; antisense, 5 -CGG ATT AGG GCT TCC TCT TGG-3 ; p8: sense, 5 -CTA TAG CCT GGC CCA TTC CT-3 ; antisense, 5 -TCT CTC TTG GTG CGA CCT TT-3 ; sestrin 2: sense, 5 -CAA GCT CGG AAT TAA TGT GCC-3 ; antisense, 5 -CTC ACA CCA TTA AGC ATG GAG-3 ; and TATA-binding protein: sense, 5 -TGC ACA GGA GCC AAG ATG GAA-3 ; antisense, 5 -CAC ATC ACA GCT CCC CAC CA-3 . The PCR primers for CSE, NAG1, and Nur77 were purchased from Qiagen. Microarray Experiments. Microarray studies focused on earlyinduced genes, and UC-5 cells were treated with DMSO or 15 M DIM-C-pPhOCH3 for 2 and 6 h. RNA was isolated as described for the 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 Technologies, Santa Clara, 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 p values were less than 0.05. Xenograft Studies in Athymic Mice. Male athymic nude mice (Foxn1, aged 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 subcutaneous injection of in vitro cultured KU-7 cells (10 cells/150 l) into the flanks of individual mice. Tumors were allowed to grow for 7 days until tumors were palpable. Mice were then randomized into two groups of five mice per group and dosed by oral gavage with either corn oil or 25 mg/kg/day DIM-C-pPHOCH3 for 17 days. The mice were weighed, and tumor size was measured twice a week with calipers to permit calculation of tumor volumes, V L W/2, where L and W were length and width. Final body, organ, and tumor weights were determined at the end of the dosing regimen, and both organ and tumor blocks were obtained for hematoxylin and eosin staining and histopathological analysis. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling Assay. For the TUNEL assay, tumor tissue was fixed in formalin and embedded in paraffin, TUNEL staining was carried out using DeadEnd Colorimetric TUNEL System (Promega). Paraffin-embedded sections (4–6 m thick) were processed per manufacturer’s protocol. In brief, sections were deparaffinized in xylene and then treated with a graded series of alcohol [100, 95, 85, 70, and 50% ethanol (v/v) in double-distilled water] and rehydrated in PBS, pH 7.5. Tissues were then treated with proteinase K solution for permeabilization and then refixed with 4% paraformaldehyde solution. Slides were then treated with recombinant terminal deoxynucleotidyl transferase reaction mix and incubated at 37°C for 1 h. Reaction was terminated by immersing the slides in 2 standard saline citrate solutions for 15 min at room temperature. After blocking the endogenous peroxidases activity (by 0.3% hydrogen peroxide), slides were washed with phosphate-buffered saline and then incubated with streptavidin horseradish peroxidase solution for 30 min at room temperature. After washing, slides were incubated with 3,3 -diaminobenzidine (substrate) solution until a light brown background appeared (10 min) and then rinsed several times in deionized water. After mounting, slides were observed by light microscope. Statistical Analysis. Statistical significance was assessed using Student’s t test. A value of P 0.05 compared with solvent control was considered statistically significant.