1,1-Bis(3 -Indolyl)-1-(p-substitutedphenyl)methanes Inhibit Growth, Induce Apoptosis, and Decrease the Androgen Receptor in LNCaP Prostate Cancer Cells through Peroxisome Proliferator-Activated Receptor -Independent Pathways

1,1-Bis(3 -indolyl)-1-(p-substitutedphenyl)methanes (C-DIMs) containing para-trifluoromethyl, t-butyl, and phenyl groups are a novel class of peroxisome proliferator-activated receptor (PPAR) agonists. In LNCaP prostate cancer cells, these compounds induce PPAR -dependent transactivation, inhibit cell proliferation, and induce apoptosis. In addition, these PPAR agonists modulate a number of antiproliferative and proapoptotic responses, including induction of p27, activating transcription factor 3, and nonsteroidal anti-inflammatory drugactivated gene-1 and down-regulation of cyclin D1 and caveolin-1. Moreover, the PPAR antagonist 2-chloro-5-nitrobenzanilide (GW9662) does not inhibit these effects. The C-DIM compounds also abrogate androgen receptor (AR)-mediated signaling and decrease prostate-specific antigen (PSA) and AR protein expression, and these responses were PPAR -independent. The effects of C-DIMs on AR and PSA were due to decreased AR and PSA mRNA expression in LNCaP cells. Thus, this series of methylene-substituted diindolylmethane derivatives simultaneously activate multiple pathways in LNCaP cells, including ablation of androgen-responsiveness and down-regulation of caveolin-1. Both of these responses are associated with activation of proapoptotic pathways in this cell line. Peroxisome proliferator-activated receptor (PPAR) is a member of the PPAR subfamily of nuclear receptors that bind and are activated by lipids, prostaglandins, and structurally diverse synthetic compounds (Rosen and Spiegelman, 2001; Willson et al., 2001; Lee et al., 2003). 15-Deoxyprostaglandin J2, fatty acids, and stress-induced nitrolinoleic acid are biochemicals that activate PPAR ; however, their role as endogenous ligands for this receptor is unknown. The synthetic thiazolidinediones troglitazone, rosiglitazone, and proglitazone are PPAR agonists, and the latter two compounds are currently being used as insulin-sensitizing drugs for the treatment of type II diabetes (Staels and Fruchart, 2005; Boden and Zhang, 2006). PPAR agonists are structurally diverse and include flavonoids, phosphonophosphates, chromane carboxylic acids, indole derivatives, and triterpenoids such as 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and related compounds (Suh et al., 1999; Rieusset et This study was supported by National Institutes of Health Grants ES09106 and CA112337 and the Texas Agricultural Experiment Station. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.028696. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor; CDDO, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; DIM-C-pPhC6H5, 1,1-bis(3 -indolyl)-1-(p-phenyl)methane; DIM-C-pPhCF3, 1,1-bis(3 -indolyl)-1-(p-trifluoromethyl)methane; DIM-C-pPhtBu, 1,1-bis(3 -indolyl)-1-(pt-butyl)methane; DIM-C-pPhOCH3, 1,1-bis(3 -indolyl)-1-(p-methyl)methane; DIM-C-pPhOH, 1,1-bis(3 -indolyl)-1-(p-hydroxyl)methane; C-DIMs, 1,1-bis(3 -indolyl)-1-(p-substitutedphenyl)methanes; NAG-1, nonsteroidal anti-inflammatory drug activated gene-1; AR, androgen receptor; PSA, prostate-specific antigen; FBS, fetal bovine serum; luc, luciferase; ERK, extracellular signal-regulated kinase; ATF3, activating transcription factor 3; EGR-1, early growth response factor 1; PPRE, peroxisome proliferator response element; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; TBST, Tris-buffered saline/Tween 20; BLOTTO, bovine lacto transfer optimizer; PCR, polymerase chain reaction; DHT, dihydrotestosterone; PI3K, phosphatidylinositol-3-kinase; PCK, protein kinase C; GF109203X, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; PD98059, 2 -amino-3 -methoxyflavone; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; GW9662, 2-chloro-5-nitrobenzanilide; SP600125, 1,9-pyrazoloanthrone. 0026-895X/07/7102-558–569$20.00 MOLECULAR PHARMACOLOGY Vol. 71, No. 2 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 28696/3171559 Mol Pharmacol 71:558–569, 2007 Printed in U.S.A. 558 http://molpharm.aspetjournals.org/content/suppl/2006/11/09/mol.106.028696.DC1 Supplemental material to this article can be found at: at A PE T Jornals on O cber 0, 2017 m oharm .aspeurnals.org D ow nladed from al., 2002; Berger et al., 2003; Place et al., 2003; Qin et al., 2003; Koyama et al., 2004; Acton et al., 2005; Liu et al., 2005; Schopfer et al., 2005). PPAR is overexpressed in tumors compared with nontumor tissues, and this receptor is also expressed in several different cancer cell lines derived from hematopoietic and nonhematopoietic tumors (Ikezoe et al., 2001). Laboratory animal studies demonstrate that PPAR agonists are highly effective antitumor agents with potential for their development as clinical drugs for cancer chemotherapy (Grommes et al., 2004). Research in this laboratory has identified 1,1-bis(3 -indolyl)-1-(p-substitutedphenyl)methanes containing para-trifluoromethyl (DIM-C-pPhCF3), t-butyl (DIM-C-pPhtBu), and phenyl (DIM-C-pPhC6H5) substituents as a new class of PPAR agonists that inhibit cancer cell proliferation and tumor growth in vivo (Chintharlapalli et al., 2004, 2005a, 2006; Hong et al., 2004; Contractor et al., 2005; Abdelrahim et al., 2006; Kassouf et al., 2006). These PPAR -active methylene-substituted diindolylmethanes [1,1-bis(3 -indolyl)-1-(psubstitutedphenyl)methanes; C-DIMs] induce PPAR -dependent transactivation in breast, colon, pancreatic, and bladder cancer lines; however, their growth inhibitory and proapoptotic effects are cell context-dependent. For example, some growth inhibitory responses including induction of p21 in pancreatic cancer cells and induction of the tumor suppressor gene caveolin-1 in colon and bladder cancer were PPAR dependent and inhibited by PPAR antagonists or small inhibitory RNA for PPAR (Chintharlapalli et al., 2004, 2006; Hong et al., 2004; Kassouf et al., 2006). In contrast, several proapoptotic responses induced by PPAR -active CDIMs were PPAR -independent, and these include endoplasmic reticulum stress-induced activation of death receptor 5 and induction of nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), a member of the transforming growth factor family (Chintharlapalli et al., 2005a, 2006; Abdelrahim et al., 2006). Other structural classes of PPAR agonists induce receptor-dependent and -independent effects, and these multiple modes of action can be advantageous for development of anticancer drugs. PPAR agonists inhibit growth and induce apoptosis in prostate cancer cells, and they also affect androgenic responsiveness in androgen receptor (AR)-positive prostate cancer cells (Kubota et al., 1998; Mueller et al., 2000; Moretti et al., 2001; Segawa et al., 2002; Jiang et al., 2004; Jarvis et al., 2005; Yang et al., 2006). For example, troglitazone decreases basal and androgen-induced prostate-specific antigen (PSA) expression in LNCaP cells at concentrations 10 M, and AR protein levels are decreased at higher concentrations ( 20 M) (Yang et al., 2006). Although the mechanisms of these troglitazone-induced responses are unknown, other PPAR -inactive analogs of troglitazone were active, indicating that these responses were PPAR -independent. In this study, we show that PPAR -active C-DIMs induce growth inhibitory and proapoptotic responses in LNCaP cells and also decrease PSA and AR expression through PPAR -independent pathways. The antiandrogenic effects are due, in part, to decreased PSA and AR mRNA levels and reporter gene activity in cells transfected with constructs containing PSA (pPSA-luc) and AR (pAR-luc) promoter inserts. The CDIM compounds offer important clinical advantages for treatment of prostate cancer through their activation of multiple responses linked to antiandrogenicity, growth inhibition, and cell death. Materials and Methods Cell Lines, Antibodies, and Reagents. Human prostate cancer cell line LNCaP was obtained from American Type Culture Collection (Manassas, VA). LNCaP cells were maintained in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 0.22% sodium bicarbonate, 0.011% sodium pyruvate, 0.45% glucose, 0.24% HEPES, 10% FBS, and 10 l/l 100 antibiotic antimycotic solution (Sigma-Aldrich). Cells were maintained at 37°C in the presence of 5% CO2. Rosiglitazone was purchased from LKT Laboratories, Inc. (St. Paul, MN). Antibodies for Sp1, poly(ADP-ribose) polymerase, cyclin D1, p27, AR, pERK, ERK, ATF3, and caveolin-1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). PSA was obtained from Dako Denmark A/S (Glostrup, Denmark). NAG-1 was from Upstate Biotechnology (Charlottesville, VA), and EGR-1 was from Cell Signaling Technology Inc. (Danvers, MA). Monoclonal -actin antibody was purchased from Sigma-Aldrich. Reporter lysis buffer and luciferase reagent for luciferase studies were purchased from Promega (Madison, WI). -Galactosidase reagent was obtained from Tropix (Bedford, MA). Lipofectamine reagent was supplied by Invitrogen (Carlsbad, CA). Western Lightning chemiluminescence reagents were from PerkinElmer Life and Analytical Sciences (Boston, MA). The C-substituted diindolylmethanes were prepared in this laboratory by condensation of indole with p-substituted benzaldehydes, and compounds were 95% pure by gas chromatography-