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• W2032131631 abstract "3,3′-Diindolylmethane (DIM) is a major digestive product of indole-3-carbinol, a potential anticancer component of cruciferous vegetables. Our results indicate that DIM exhibits potent antiproliferative and antiandrogenic properties in androgen-dependent human prostate cancer cells. DIM suppresses cell proliferation of LNCaP cells and inhibits dihydrotestosterone (DHT) stimulation of DNA synthesis. These activities were not produced in androgen-independent PC-3 cells. Moreover, DIM inhibited endogenous PSA transcription and reduced intracellular and secreted PSA protein levels induced by DHT in LNCaP cells. Also, DIM inhibited, in a concentration-dependent manner, the DHT-induced expression of a prostate-specific antigen promoter-regulated reporter gene construct in transiently transfected LNCaP cells. Similar effects of DIM were observed in PC-3 cells only when these cells were co-transfected with a wild-type androgen receptor expression plasmid. Using fluorescence imaging with green fluorescent protein androgen receptor and Western blot analysis, we demonstrated that DIM inhibited androgen-induced androgen receptor (AR) translocation into the nucleus. Results of receptor binding assays indicated further that DIM is a strong competitive inhibitor of DHT binding to the AR. Results of structural modeling studies showed that DIM is remarkably similar in conformational geometry and surface charge distribution to an established synthetic AR antagonist, although the atomic compositions of the two substances are quite different. Taken together with our published reports of the estrogen agonist activities of DIM, the present results establish DIM as a unique bifunctional hormone disrupter. To our knowledge, DIM is the first example of a pure androgen receptor antagonist from plants. 3,3′-Diindolylmethane (DIM) is a major digestive product of indole-3-carbinol, a potential anticancer component of cruciferous vegetables. Our results indicate that DIM exhibits potent antiproliferative and antiandrogenic properties in androgen-dependent human prostate cancer cells. DIM suppresses cell proliferation of LNCaP cells and inhibits dihydrotestosterone (DHT) stimulation of DNA synthesis. These activities were not produced in androgen-independent PC-3 cells. Moreover, DIM inhibited endogenous PSA transcription and reduced intracellular and secreted PSA protein levels induced by DHT in LNCaP cells. Also, DIM inhibited, in a concentration-dependent manner, the DHT-induced expression of a prostate-specific antigen promoter-regulated reporter gene construct in transiently transfected LNCaP cells. Similar effects of DIM were observed in PC-3 cells only when these cells were co-transfected with a wild-type androgen receptor expression plasmid. Using fluorescence imaging with green fluorescent protein androgen receptor and Western blot analysis, we demonstrated that DIM inhibited androgen-induced androgen receptor (AR) translocation into the nucleus. Results of receptor binding assays indicated further that DIM is a strong competitive inhibitor of DHT binding to the AR. Results of structural modeling studies showed that DIM is remarkably similar in conformational geometry and surface charge distribution to an established synthetic AR antagonist, although the atomic compositions of the two substances are quite different. Taken together with our published reports of the estrogen agonist activities of DIM, the present results establish DIM as a unique bifunctional hormone disrupter. To our knowledge, DIM is the first example of a pure androgen receptor antagonist from plants. Prostate cancer is the second leading cause of cancer-related mortality in American men, with more than 40,000 deaths in 1997 (1Wingo P.A. Landis S. Ries L.A. CA-Cancer J. Clin. 1997; 47: 239-242Crossref PubMed Scopus (94) Google Scholar). One of every four cancers diagnosed is of prostatic origin, making prostate cancer the most commonly diagnosed cancer (2Small E.J. Drugs Aging. 1998; 13: 71-81Crossref PubMed Scopus (23) Google Scholar). Although the incidence of prostate cancer in Japanese and Chinese men is remarkably low compared with the incidence in American males, after migration to the US, the risk of later generations of Asian immigrants rises to levels that are similar to American males (3Shimizu H. Ross R.K. Bernstein L. Yatani R. Henderson B.E. Mack T.M. Br. J. Cancer. 1991; 63: 963-966Crossref PubMed Scopus (748) Google Scholar, 4Haenszel W. Kurihara M. J. Natl. Cancer Inst. 1968; 40: 43-68PubMed Google Scholar). The differences in prostate cancer diagnosed among various population groups suggest that factors in the environment, lifestyles, and diet play a role in prostate cancer initiation and/or progression. One possible contributor to the lower prostate cancer rates in Asian men is the higher consumption of phytochemical-rich vegetables that is typical of this population (5Denis L. Morton M.S. Griffiths K. Eur. Urol. 1999; 35: 377-387Crossref PubMed Scopus (191) Google Scholar, 6Kolonel L.N. Hankin J.H. Whittemore A.S. Wu A.H. Gallagher R.P. Wilkens L.R. John E.M. Howe G.R. Dreon D.M. West D.W. Paffenbarger Jr., R.S. Cancer Epidemiol. Biomark. Prev. 2000; 9: 795-804PubMed Google Scholar). Consumption of cruciferous vegetables, including broccoli, Brussels sprouts, kale, and cauliflower, has been associated with a decreased risk of various human cancers. The strongest associations are with cancers of the breast, endometrium, colon, and prostate (7Terry P. Wolk A. Persson I. Magnusson C. J. Am. Med. Assoc. 2001; 285: 2975-2977Crossref PubMed Google Scholar, 8Terry P. Vainio H. Wolk A. Weiderpass E. Nutr. Cancer. 2002; 42: 25-32Crossref PubMed Scopus (70) Google Scholar, 9Voorrips L.E. Goldbohm R.A. van Poppel G. Sturmans F. Hermus R.J. van den Brandt P.A. Am. J. Epidemiol. 2000; 152: 1081-1092Crossref PubMed Scopus (252) Google Scholar, 10Kristal A.R. Lampe J.W. Nutr. Cancer. 2002; 42: 1-9Crossref PubMed Scopus (236) Google Scholar). Incorporation of Brassica plants in feed reduces spontaneous and carcinogen-induced tumorigenesis in experimental animals, with the greatest protective effects seen in mammary tumors (11Bradlow H.L. Michnovicz J. Telang N.T. Osborne M.P. Carcinogenesis. 1991; 12: 1571-1574Crossref PubMed Scopus (278) Google Scholar, 12Grubbs C.J. Steele V.E. Casebolt T. Juliana M.M. Eto I. Whitaker L.M. Dragnev K.H. Kelloff G.J. Lubet R.L. Anticancer Res. 1995; 15: 709-716PubMed Google Scholar, 13Chen I. McDougal A. Wang F. Safe S. Carcinogenesis. 1998; 19: 1631-1639Crossref PubMed Scopus (258) Google Scholar). A major active compound in cruciferous vegetables, indole-3-carbinol, along with its primary digestive derivative, 3,3′-diindolylmethane (DIM), 1The abbreviations used are: DIM, 3,3′-diindolylmethane; AR, androgen receptor; ARE, androgen response element; DHT, 5α-dihydrotestosterone; GFP, green fluorescent protein; MMTV-Luc, murine mammary tumor virus-luciferase; PSA, prostate-specific antigen; CREB, cAMP-response element-binding protein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; DCC, dextrancoated charcoal. exhibit promising cancer-protective properties in vivo and in vitro. These compounds reduced the incidence of dimethylbenzanthracene-induced mammary tumors in rats, benzo(a)pyrene-induced tumors of the forestomach in mice, and benzo(a)pyrene-induced pulmonary adenomas in mice (14Wattenberg L.W. Loub W.D. Cancer Res. 1978; 38: 1410-1413PubMed Google Scholar, 15Wattenberg L.W. J. Environ. Pathol. Toxicol. 1980; 3: 35-52PubMed Google Scholar). Indole-3-carbinol has been shown to inhibit proliferation of both breast (16Cover C.M. Hsieh S.J. Tran S.H. Hallden G. Kim G.S. Bjeldanes L.F. Firestone G.L. J. Biol. Chem. 1998; 273: 3838-3847Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 17Ge X. Fares F.A. Yannai S. Anticancer Res. 1999; 19: 3199-3203PubMed Google Scholar) and prostate cancer cells (18Chinni S.R. Li Y. Upadhyay S. Koppolu P.K. Sarkar F.H. Oncogene. 2001; 20: 2927-2936Crossref PubMed Scopus (286) Google Scholar, 19Chinni S.R. Sarkar F.H. Clin. Cancer Res. 2002; 8: 1228-1236PubMed Google Scholar) by blocking the cell cycle and inducing apoptosis. In addition, DIM inhibited proliferation and induced programmed cell death in human breast tumor cells in culture (20Hong C. Kim H.A. Firestone G.L. Bjeldanes L.F. Carcinogenesis. 2002; 23: 1297-1305Crossref PubMed Scopus (169) Google Scholar, 21Ge X. Yannai S. Rennert G. Gruener N. Fares F.A. Biochem. Biophys. Res. Commun. 1996; 228: 153-158Crossref PubMed Scopus (171) Google Scholar). The cancer-preventive effects of DIM, especially on hormone-mediated breast cancer, and the effects of indole-3-carbinol on prostate cancer cells led us to investigate the effects and mechanism of action of DIM against proliferation of prostate tumor cells. To examine the androgen antagonist effects of DIM, we conducted a series of cell proliferation and gene activation studies in androgen-dependent (LNCaP) and androgen-independent (PC-3) human prostate cancer cell lines. LNCaP cells were derived from lymph node metastasis, and PC-3 cells were derived from bone metastasis (22Veldscholte J. Berrevoets C.A. Ris-Stalpers C. Kuiper G.G. Jenster G. Trapman J. Brinkmann A.O. Mulder E. J. Steroid Biochem. Mol. Biol. 1992; 41: 665-669Crossref PubMed Scopus (369) Google Scholar, 23Webber M.M. Bello D. Quader S. Prostate. 1997; 30: 58-64Crossref PubMed Scopus (157) Google Scholar, 24Wang M. Stearns M.E. Differentiation. 1991; 48: 115-125Crossref PubMed Scopus (114) Google Scholar, 25Kaighn M.E. Narayan K.S. Ohnuki Y. Lechner J.F. Jones L.W. Investig. Urol. 1979; 17: 16-23PubMed Google Scholar). We found that DIM is a strong antiandrogen that inhibited androgen-dependent tumor cell growth and competitively inhibited androgen receptor translocation and signal transduction. In addition, DIM down-regulated prostate-specific antigen (PSA) expression at the transcriptional level. Results from androgen receptor (AR) competitive binding assays, nuclear translocation studies, and structural modeling computations suggest that DIM disrupts AR function in a manner similar to a chemically dissimilar synthetic antiandrogen, Casodex. Our results identify DIM as a structurally novel, naturally occurring, pure androgen antagonist of potential cancer preventive and therapeutic usefulness for prostate cancer. Materials—Dulbecco's modified Eagle's medium (DMEM), Opti-MEM, and LipofectAMINE reagent were supplied by Invitrogen. Phenol red-free DMEM base, fetal bovine serum (FBS), calf serum, cyproterone acetate (6-chloro-1β,2β-dihydro-17-hydroxy-3′H-cyclopropa-(1Wingo P.A. Landis S. Ries L.A. CA-Cancer J. Clin. 1997; 47: 239-242Crossref PubMed Scopus (94) Google Scholar, 2Small E.J. Drugs Aging. 1998; 13: 71-81Crossref PubMed Scopus (23) Google Scholar)-pregna-1,4,6-triene-3,20-dione acetate) and 5α-dihydrotestosterone were supplied by Sigma. Casodex was provided as a gift from Astra-Zeneca. Dextran-coated charcoal-FBS (DCC-FBS) was from Hyclone (Logan, UT). [γ-32P]ATP, [3H]DHT, and [3H]thymidine were supplied by PerkinElmer Life Sciences. AR rabbit (sc-816, sc-815) polyclonal IgGs and PSA mouse (sc-7316) and goat (sc-7638) mono- and polyclonal IgGs were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). PSA total (M86506M) and free (M86806M) monoclonal antibodies were from Biodesign International (Saco, ME). DIM was prepared from indole-3-carbinol as described (26Bradfield C.A. Bjeldanes L.F. J. Toxicol. Environ. Health. 1987; 21: 311-323Crossref PubMed Scopus (180) Google Scholar, 27Grose K.R. Bjeldanes L.F. Chem. Res. Toxicol. 1992; 5: 188-193Crossref PubMed Scopus (195) Google Scholar, 28Bjeldanes L.F. Kim J.Y. Grose K.R. Bartholomew J.C. Bradfield C.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9543-9547Crossref PubMed Scopus (494) Google Scholar) and recrystallized in toluene. All other reagents were of the highest grade available. Cell Culture—The human prostate adenocarcinoma cell lines LNCaP-FGC and PC-3 were obtained from the American Type Culture Collection (Manassas VA). They were grown as adherent monolayers in 10% FBS-DMEM, supplemented with 4.0 g/liter glucose and 3.7 g/liter sodium bicarbonate in a humidified incubator at 37 °C and 5% CO2, and passaged at ∼80% confluency. Cultures used in subsequent experiments were at less than 40 passages. Cells grown in stripped conditions were in 5% DCC-FBS-DMEM base supplemented with 4.0 g/liter glucose, 3.7 g/liter sodium bicarbonate, and 0.293 g/liter l-glutamine. Cell Growth—Before the beginning of the treatments, cells were depleted of androgen for 4–7 days in medium composed of DMEM base without phenol red and with 4.0 g/liter glucose and 3.7 g/liter sodium bicarbonate. During the depletion period, medium was changed every 48 h. Treatments were administered by the addition of 1 μl of a 1,000-fold concentrated solution of DIM in Me2SO/ml of medium. Once the treatment period started, medium was changed daily to counter possible loss of readily metabolized compounds. Cell Counting—Cells were harvested by trypsinization and resuspended in culture medium. Aliquots were diluted 50-fold in Isoton II (Coulter Corp., Miami, FL), and 200-μl duplicates were counted in a model Z1 Coulter particle counter and averaged. [3H]Thymidine Incorporation—LNCaP cells were plated onto 24-well plates (Corning) with 2 × 104 cells/well and treated with varying concentrations of DIM with and without 1 nm DHT for 24–48 h. [3H]Thymidine (3 μCi) was then added to each well and incubated at 37 °C for 2–3 h. Medium was removed, and the cells were washed 3 times with 2 ml of ice-cold 10% trichloroacetic acid followed by the addition of 300 μl of 0.3 n NaOH to each well and then incubated at room temperature for 30 min. Aliquots (150 μl) were transferred into the scintillation vials with 4 ml of ScintiVerse BD scintillation fluid (Fisher) and counted for radioactivity by a Beckman liquid scintillation counter. Plasmid Reporters and Expression Vectors—The ARE-responsive luciferase reporter plasmid, pPSA-630 luciferase (pPSA-Luc), was a gift from Dr. M. D. Sadar (29Sato N. Sadar M.D. Bruchovsky N. Saatcioglu F. Rennie P.S. Sato S. Lange P.H. Gleave M.E. J. Biol. Chem. 1997; 272: 17485-17494Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). pPSA-Luc contains the PSA promoter region (-630 to 12) with three AREs, all of which are critical to the activity of the pPSA-Luc promoter. The MMTV-Luc, containing one consensus ARE, and the expression vector, pCMV-hAR, which constitutively expresses a fully functional human androgen receptor, were also generously provided by Dr. M. D. Sadar. The pCMV-GFP-rAR was a gift from Dr. A. K. Roy (30Roy A.K. Tyagi R.K. Song C.S. Lavrovsky Y. Ahn S.C. Oh T.S. Chatterjee B. Ann. N. Y. Acad. Sci. 2001; 949: 44-57Crossref PubMed Scopus (104) Google Scholar). RNA Extraction, mRNA Purification, and Northern Hybridization— mRNA isolation and Northern blot analyses were conducted as described previously (20Hong C. Kim H.A. Firestone G.L. Bjeldanes L.F. Carcinogenesis. 2002; 23: 1297-1305Crossref PubMed Scopus (169) Google Scholar, 31Riby J.E. Feng C. Chang Y.C. Schaldach C.M. Firestone G.L. Bjeldanes L.F. Biochemistry. 2000; 39: 910-918Crossref PubMed Scopus (63) Google Scholar). PSA cDNA was generously provided by Dr. M. D. Sadar, and the cDNA probes were biotinylated using NEBlot Phototope kit (New England Biolabs, Beverly, MA), purified via precipitation with 3 m sodium acetate, pH 5.2, and washed with 70% ethanol. After hybridization with cDNA probes, the membrane was incubated with streptavidin then biotinylated with alkaline phosphatase followed by the Phototope-CDP-Star assay (New England Biolabs) and autoradiographed. The amount of mRNA was quantified by Gel Densitometer (Bio-Rad) and normalized with β-actin as an internal control. Analysis of Intracellular and Secreted PSA—LNCaP cells growing on 100-mm plates were treated as indicated for 24 h. Cells were lysed as previously described (20Hong C. Kim H.A. Firestone G.L. Bjeldanes L.F. Carcinogenesis. 2002; 23: 1297-1305Crossref PubMed Scopus (169) Google Scholar) for intracellular PSA analysis. For secreted proteins, spent medium was collected and concentrated 18-fold using Millipore Centriprep YM-10 following the manufacturer's protocol (Bedford, MA). Protease inhibitors (10 μg/ml aprotinin, 10 μg/ml leupeptin, 5 μg/ml pepstatin, 50 μg/ml phenylmethylsulfonyl fluoride) were added, and the proteins were immunoprecipitated with 3 μg/ml monoclonal free PSA antibody (Biodesign International) for 2 h and co-immunoprecipitated overnight at 4 °C with protein A/G-agarose (Santa Cruz Biotechnology) on a rotator. The samples were then subjected to Western blot analysis as described previously (20Hong C. Kim H.A. Firestone G.L. Bjeldanes L.F. Carcinogenesis. 2002; 23: 1297-1305Crossref PubMed Scopus (169) Google Scholar) using a monoclonal PSA (sc-7316) primary antibody and a goat anti-mouse-IgG-AP secondary antibody (sc-2008) from Santa Cruz Biotechnology. Transient Transfections with Reporters and Luciferase Assay—LNCaP and PC-3 cells were transfected with some modifications, as previously described (20Hong C. Kim H.A. Firestone G.L. Bjeldanes L.F. Carcinogenesis. 2002; 23: 1297-1305Crossref PubMed Scopus (169) Google Scholar). For AR transactivation, cells were transfected with 0.1 μg of MMTV-Luc or pPSA-Luc per plate. Co-transfection experiments with pCMV-hAR or pCMV-GFP-rAR, 0.1 μg/plate, was also used. For experiments involving GFP fluorescence imaging, treatments were not added until 30 h after transfection. Hormone Binding Assay—LNCaP cells were grown in 5% DCC-FBS-DMEM medium supplemented with 4.0 g/liter glucose and 3.7 g/liter sodium bicarbonate and harvested in Hepes-buffered saline containing 1.5 mm EDTA by scraping with a rubber policeman. The cells were placed on ice, collected by centrifugation, washed with ice-cold TKEG buffer (20 mm Tris-HCl, pH 7.4, 50 mm KCl, 1 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride, and 10% glycerol) and resuspended in 250 μl/plate homogenization buffer (50 mm Tris-HCl, pH 7.4, 1.5 mm EDTA, 10 mm sodium molybdate, 2.5 mm β-mercaptoethanol, 50 mm KCl, 0.1 mm phenylmethylsulfonyl fluoride, and 10% glycerol). Cells were homogenized using a Polytron apparatus at medium speed for 1 min on ice. The homogenates were centrifuged at 50,000 rpm in 4 °C for 60 min. The supernatant solution was divided into 1.0-ml aliquots, quickly frozen in a dry-ice/ethanol bath, and stored at -80 °C. Protein concentration was determined by the Bradford assay using bovine serum albumin as the standard. For each competitive binding assay, 5 μl of 20 nm [3H]DHT in 50% ethanol, 10 mm Tris, pH 7.5, 10% glycerol, 1 mg/ml BSA, and 1 mm dithiothreitol was placed in a 1.5-ml microcentrifuge tube. Competitive ligands were added as 1.0 μl of 100× solution in Me2SO. After mixing, 95 μl of either LNCaP cell extracts or recombinant AR protein (PanVera, Madison, WI) was added, and the solutions were vortexed and incubated at room temperature for 2–3 h. Proteins were precipitated by the addition of 100 μl of 50% hydroxylapatite slurry equilibrated in TE (50 mm Tris, pH 7.4, 1 mm EDTA) and incubated on ice for 15 min with vortexing every 5 min to resuspend the hydroxylapatite. The pellet was washed with 1.0 ml of ice-cold wash buffer (40 mm Tris, pH 7.4, 100 mm KCl) and centrifuged for 5 min at 10,000 × g at 4 °C. The supernatant was carefully aspirated, and the pellet was washed 2 more times with 1.0 ml of wash buffer. The final pellet was resuspended in 200 μl of ethanol and transferred to a scintillation vial. The tube was washed with another 200 μl of ethanol, which was then added to the same counting vial. A negative control contained no protein, and nonspecific binding was determined using 100-fold (0.1 μm) excess unlabeled DHT. Subcellular Fractionation—Three near confluent (80–90%) cultures of LNCaP cells in 100-mm Petri dishes were used for each treatment. Treatments were added as 1 μl of a 1,000-fold concentrated solution of DIM in Me2SO/ml of medium for the indicated time. After incubation with treatments at 37 °C, cytosolic and nuclear proteins were prepared as described (32Hong C. Firestone G.L. Bjeldanes L.F. Biochem. Pharmacol. 2002; 63: 1085-1097Crossref PubMed Scopus (249) Google Scholar, 33Riby J.E. Chang G.H. Firestone G.L. Bjeldanes L.F. Biochem. Pharmacol. 2000; 60: 167-177Crossref PubMed Scopus (79) Google Scholar) with modifications. Briefly, cells were lysed in hypotonic buffer (10 mm Hepes, pH 7.5) and harvested in MDH buffer (3 mm MgCl2, 1 mm dithiothreitol, 25 mm Hepes, pH 7.5). After homogenization, supernatant was saved for cytosolic proteins, and nuclear proteins were extracted from the pellets using MDHK buffer (3 mm MgCl2,1mm dithiothreitol, 0.1 m KCl, 25 mm Hepes, pH 7.5) followed by HDK buffer (25 mm Hepes, pH 7.5, 1 mm dithiothreitol, 0.4 m KCl). Cytosolic and nuclear extracts were subsequently analyzed by Western blot analysis. Western Blot Analysis—After the indicated treatment, Western immunoblot analyses of androgen receptor from LNCaP cells were performed as described previously (20Hong C. Kim H.A. Firestone G.L. Bjeldanes L.F. Carcinogenesis. 2002; 23: 1297-1305Crossref PubMed Scopus (169) Google Scholar). In short, polyclonal AR antibodies, sc-816 and sc-815, from Santa Cruz Biotechnology were used as primary antibodies with a chemiluminescence protein detection method. Blotted membranes were stained with Coomassie Blue to determine protein loading, or β-actin (sc-8432, Santa Cruz Biotechnology) was used as an internal control. The amount of protein was quantified by Gel Densitometer (Bio-Rad) and normalized with β-actin when used as an internal control. Fluorescence Imaging—PC-3 cells were plated on cover slips in 6-well culture plates at 1.5 × 105 cells/well in 5% DCC-FBS-DMEM medium. Cells were co-transfected with pCMV-GFP-rAR and pPSA-Luc or MMTV-Luc as indicated above. Cover slips were placed on microscope slides, and images were taken at 1000×. Fluorescence imaging of GFP was performed using a Zeiss Axiophot 381 and Q-imaging MicroPublisher at the College of Natural Resources Biological Imaging Facility of the University of California, Berkeley, CA. Modeling of DIM Binding to the AR Ligand Binding Domain— Quantum mechanical geometry optimizations were performed at a high level of theory, 6–31G**/MP2, for DHT, DIM, Casodex, and R1881. Using these molecular coordinates, a solvent-accessible surface was constructed surrounding each molecule; such a surface enables coupling of the ab initio electronic structure calculations to the solution of the Poisson-Boltzmann equation (34Wilson W.D. Schaldach C.M. Bourcier W.L. Chem. Phys. Lett. 1997; 267: 431-437Crossref Scopus (12) Google Scholar). The coupling was accomplished through the single and double layers of charge at the boundary and allowed for relaxation of the quantum electronic charge distribution in response to these surrounding layers. This first principles approach eliminated the need to assign fractional charges to the atoms. The induced polarization charge at the interface was then mapped onto the nodes (“dots”) of the elements of the solvent accessible surface. A comparison was then made between these molecules. The atomic configuration of DHT determined experimentally, i.e. obtained from the crystal structure of the molecule in the androgen receptor, provided a template for comparison of the feasibility of the androgen receptor binding a different ligand (35Sack J.S. Kish K.F. Wang C. Attar R.M. Kiefer S.E. An Y. Wu G.Y. Scheffler J.E. Salvati M.E. Krystek S.R.J. Weinmann R. Enspahr H.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4904-4909Crossref PubMed Scopus (391) Google Scholar). We, therefore, again constructed a solvent-accessible surface, SR, surrounding the DHT molecule (crystal structure) and used this surface as a reference “standard” or template for the androgen receptor's ligand binding site. The center-of-mass of each androgen receptor ligand, DHT, Casodex, R1881 (optimized coordinates), and DIM, was translated to the center-of-mass of the template (crystal structure coordinates) and then rotated about the x, y, and z axes through the center-of-mass. We calculated the fractional surface area of the ligand, which did not fit into the binding site template, as follows. For each element, i, of the ligand surface, SL, we then found its nearest neighbor element j on SR, allowing us to form the vector rij = ri-rj from element j (nearest neighbor to i of SL) on SR to element i. By forming the dot product of rij with the normal to the element at j on SR, rij × nj, we could determine whether element i of SL is inside or outside of SR. In this way we calculated a ΔSL, the fractional surface area of each ligand that lies outside the template DHT surface SR. This method was repeated using the crystal structure of R1881 as the binding site template (36Matias P.M. Donner P. Coelho R. Thomaz M. Peixoto C. Macedo S. Otto N. Joschko S. Scholz P. Wegg A. Basler S. Schafer M. Egner U. Carrondo M.A. J. Biol. Chem. 2000; 275: 26164-26171Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). DIM Inhibits the Proliferation and DNA Synthesis of Uninduced and DHT-induced LNCaP Cells—The effects of DIM on human prostate cancer cell growth were examined using LNCaP and PC-3 cells. After a 96-h treatment, DIM produced a concentration-dependent inhibition of LNCaP cell proliferation with maximal inhibition of 70% at 50 μm. At these concentrations, DIM had no observable effects on the growth of PC-3 cells (Fig. 1A). In addition, we examined the effects of varying concentrations of DIM with and without 1 nm DHT on DNA synthesis in LNCaP cells (Fig. 1B). Our results showed a concentration-dependent inhibition of DNA synthesis of these cells of up to 90% under both uninduced and androgen-induced growth conditions. Inhibition of Endogenous PSA Expression by DIM—Northern blot analysis was used to examine the effect of DIM on endogenous PSA gene expression. Fig. 2 shows concentration-dependent (A) and time-dependent (B) decreases of up to 70% in PSA mRNA levels after DIM treatments. In addition, PSA mRNA induction by DHT with increasing time of treatment was inhibited by up to 80% by 24 h of co-treatment with DIM (Fig. 2C). Furthermore, Western immunoblot analysis showed that DIM reduced levels of intracellular and secreted PSA protein to background concentrations (Figs. 3, A and B) after DHT co-treatments. The reduction of PSA expression was comparable with the reduction in DHT-induced mRNA expression determined by Northern blot analysis. These results are consistent with DIM regulation of PSA expression occurring at the transcriptional level and consistent with the antiandrogenic activity of DIM observed in the cell proliferation experiments.Fig. 3DIM inhibits expression of secreted and intracellular PSA protein.A, Western blot analysis of intracellular PSA protein level in cells treated with Me2SO (DMSO) control and 50 μm DIM in the presence and absence of 1 nm DHT. B, DHT (1 nm) induced the expression of secreted PSA, and co-treatment with 50 μm DIM inhibited the expression of the secreted protein. Coomassie Blue staining was used to verify equal protein loading.View Large Image Figure ViewerDownload Hi-res image Download (PPT) DIM Down-regulates the Activities of DHT-induced Reporter Genes—The antiandrogenic effects of DIM were further examined with reporter assays using a MMTV-Luc promoter construct that contains one ARE and a pPSA-Luc promoter construct containing three AREs. These plasmids were transiently transfected into LNCaP cells and, by luciferase analysis, showed that DIM strongly inhibited DHT induction of androgen-responsive genes by more than 50% at 1 μm and more than 90% at 10 μm in both promoter constructs (Fig. 4, A and B). Treatment with DIM alone failed to induce transactivation of these reporter genes. These results further confirm that DIM inhibition of AR-responsive gene expression occurs at the transcriptional level. The AR Is the Central Modulator of DIM Inhibitory Effects on Androgen-regulated Gene Expression—To confirm the importance of the AR in the transcriptional activation of the ARE promoters, we employed PC-3 cells, which exhibit little or no AR expression. We transfected these cells with the pPSA-Luc promoter and performed luciferase analysis to show that without co-transfection of an AR expression vector, DIM has no effect (data not shown). In contrast, co-transfection of an AR expression vector with the pPSA-Luc reporter construct led to a concentration-dependent inhibition of DHT-induced transactivation by DIM that was similar to the effect we had observed in LNCaP cells (Fig. 5). The same results were seen with the MMTV-Luc promoter (data not shown). Moreover, DIM by itself did not induce transactivation of these reporter genes in either cell line with or without co-transfection of the wild-type androgen receptor. DIM Competes with Androgen for Binding to the AR in LNCaP Cells and in Recombinant AR Protein—Because our results strongly implicate the AR as the focus of the DIM mode of action in prostate cells, we assessed directly the ability of DIM to bind to this receptor. Our results of competitive binding assays with both the mutant AR of LNCaP cells and a wild-type recombinant human AR demonstrate that DIM, in the micro-molar concentration range, competes with labeled DHT for binding to the AR (Fig. 6). Cyproterone acetate and Casodex, two well known antiandrogens, were used as positive controls. DIM and Casodex exhibited similar binding affinity for the AR. Biochemical Ana" @default.
• W2032131631 created "2016-06-24" @default.
• W2032131631 creator A5008109270 @default.
• W2032131631 creator A5023224521 @default.
• W2032131631 creator A5023598018 @default.
• W2032131631 creator A5054324775 @default.
• W2032131631 date "2003-06-01" @default.
• W2032131631 modified "2023-10-06" @default.
• W2032131631 title "Plant-derived 3,3′-Diindolylmethane Is a Strong Androgen Antagonist in Human Prostate Cancer Cells" @default.
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