In Vivo and in Vitro Regulation of Syndecan 1 in Prostate Cells by n-3 Polyunsaturated Fatty Acids
2008; Elsevier BV; Volume: 283; Issue: 26 Linguagem: Inglês
10.1074/jbc.m802107200
ISSN1083-351X
AutoresIris J. Edwards, Haiguo Sun, Yunping Hu, Isabelle M. Berquin, Joseph T. O’Flaherty, J. Mark Cline, Lawrence L. Rudel, Yong Q. Chen,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoSyndecan 1 is the major proteoglycan produced by epithelial cells. It is strategically localized at the plasma membrane to participate in growth factor signaling and cell-cell and cell-matrix interactions. Its expression may modulate the properties of epithelial lineage tumor cells in which it is generally down-regulated compared with nontumor progenitors. The present study examined the regulation of syndecan 1 in prostate epithelial cells by n-3 polyunsaturated fatty acids. In prostate tissue of mice, syndecan 1 immunostaining was demonstrated in epithelial cells throughout each gland. In animals fed an n-3 polyunsaturated fatty acid-enriched diet, syndecan 1 mRNA was increased in all prostate glands. In the human prostate cancer cell line, PC-3, delivery of exogenous n-3 (but not n-6) fatty acids resulted in up-regulation of syndecan 1 expression. This effect was mimicked by a peroxisome proliferator-activated receptor (PPAR) γ agonist, troglitazone, and inhibited in the presence of a PPARγ antagonist and in cells transfected with dominant negative PPARγ cDNA. Using a luciferase gene driven either by a PPAR response element or by a DR-1 site present in the syndecan 1 promoter, reporter activation was increased by n-3 low density lipoprotein, docosahexaenoic acid, and troglitazone, whereas activity of a luciferase gene placed downstream of a mutant DR-1 site was unresponsive. These findings indicate that syndecan 1 is up-regulated by n-3 fatty acids by a transcriptional pathway involving PPARγ. This mechanism may contribute to the chemopreventive properties of n-3 fatty acids in prostate cancer. Syndecan 1 is the major proteoglycan produced by epithelial cells. It is strategically localized at the plasma membrane to participate in growth factor signaling and cell-cell and cell-matrix interactions. Its expression may modulate the properties of epithelial lineage tumor cells in which it is generally down-regulated compared with nontumor progenitors. The present study examined the regulation of syndecan 1 in prostate epithelial cells by n-3 polyunsaturated fatty acids. In prostate tissue of mice, syndecan 1 immunostaining was demonstrated in epithelial cells throughout each gland. In animals fed an n-3 polyunsaturated fatty acid-enriched diet, syndecan 1 mRNA was increased in all prostate glands. In the human prostate cancer cell line, PC-3, delivery of exogenous n-3 (but not n-6) fatty acids resulted in up-regulation of syndecan 1 expression. This effect was mimicked by a peroxisome proliferator-activated receptor (PPAR) γ agonist, troglitazone, and inhibited in the presence of a PPARγ antagonist and in cells transfected with dominant negative PPARγ cDNA. Using a luciferase gene driven either by a PPAR response element or by a DR-1 site present in the syndecan 1 promoter, reporter activation was increased by n-3 low density lipoprotein, docosahexaenoic acid, and troglitazone, whereas activity of a luciferase gene placed downstream of a mutant DR-1 site was unresponsive. These findings indicate that syndecan 1 is up-regulated by n-3 fatty acids by a transcriptional pathway involving PPARγ. This mechanism may contribute to the chemopreventive properties of n-3 fatty acids in prostate cancer. The syndecan family of transmembrane heparan sulfate proteoglycans has roles in cell-cell and cell-matrix interactions, growth factor signaling, and lipoprotein catabolism (1Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2322) Google Scholar). Syndecan 1 (SDC-1) 2The abbreviations used are: SDC-1, syndecan 1; BPH, benign prostrate hyperplasia; BSA, bovine serum albumin; DHA, docosahexaenoic acid; d/n, dominant/negative; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; LA, linoleic acid; LDL, low density lipoproteins; PPARγ, peroxisome proliferator-activated receptor γ; PPRE, PPAR-response elements; PUFA, polyunsaturated fatty acids; FBS, fetal bovine serum; RT, reverse transcription; wt, wild type. is known to play a role in cell adhesion (2Liu W. Litwack E.D. Stanley M.J. Langford J.K. Lander A.D. Sanderson R.D. J. Biol. Chem. 1998; 273: 22825-22832Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 3Woods A. Oh E.S. Couchman J.R. Matrix Biol. 1998; 17: 477-483Crossref PubMed Scopus (78) Google Scholar), inhibit matrix metalloproteinases (4Kaushal G.P. Xiong X. Athota A.B. Rozypal T.L. Sanderson R.D. Kelly T. Br. J. 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High expression of SDC-1, observed in normal prostate epithelial cells, was lost in localized and locally invasive prostate cancer (18Chen D. Adenekan B. Chen L. Vaughan E.D. Gerald W. Feng Z. Knudsen B.S. Urology. 2004; 63: 402-407Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Similarly, compared with benign prostate hyperplasia (BPH) and low grade premalignant intraepithelial neoplasia, SDC-1 expression was reduced in prostate carcinoma and lost entirely in poorly differentiated cases (19Kiviniemi J. Kallajoki M. Kujala I. Matikainen M.T. Alanen K. Jalkanen M. Salmivirta M. APMIS. 2004; 112: 89-97Crossref PubMed Scopus (34) Google Scholar). However, a tissue microarray analysis demonstrated SDC-1 expression associated with high Gleason grade and early recurrence (20Zellweger T. Ninck C. Mirlacher M. Annefeld M. Glass A.G. Gasser T.C. Mihatsch M.J. Gelmann E.P. Bubendorf L. Prostate. 2003; 55: 20-29Crossref PubMed Scopus (113) Google Scholar). Therefore, a clear picture of changes in SDC-1 expression, regulation, and role in tumorigenesis has yet to emerge. Although still controversial, evidence suggests that dietary fat intake may play a role in the development of prostate cancer with diets enriched in fish oil or its n-3 polyunsaturated fatty acids (PUFA) being chemoprotective. A recent review of epidemiological evidence indicated an inverse association between fish oil consumption and advanced/metastatic prostate cancer or prostate cancer mortality (21Terry P.D. Terry J.B. Rohan T.E. J. Nutr. 2004; 134: 3412S-3420SCrossref PubMed Google Scholar). In addition, the Health Professionals' Follow-up study, a large prospective U.S. cohort study, identified significant inverse relationships between intakes of marine fatty acid that were strongest for metastatic cancers (22Davis B.C. Kris-Etherton P.M. Am. J. Clin. Nutr. 2003; 78: 640S-646SCrossref PubMed Google Scholar, 23Leitzmann M.F. Stampfer M.J. Michaud D.S. Augustsson K. Colditz G.C. Willett W.C. Giovannucci E.L. Am. J. Clin. Nutr. 2004; 80: 204-216Crossref PubMed Scopus (232) Google Scholar). Fish oil contains the long-chain PUFA, eicosapentaenoic acid (EPA (20:5, n-3)), and docosahexaenoic acid (DHA (22:6, n-3)). Serum PUFA in patients with prostate cancer and BPH versus cancer-free individuals demonstrated EPA and DHA levels in the order of cancer-free > BPH > prostate cancer (24Yang Y.J. Lee S.H. Hong S.J. Chung B.C. Clin. Biochem. 1999; 32: 405-409Crossref PubMed Scopus (71) Google Scholar). Reduced prostate cancer risk was associated with high erythrocyte EPA and DHA (25Norrish A.E. Skeaff C.M. Arribas G.L. Sharpe S.J. Jackson R.T. Br. J. Cancer. 1999; 81: 1238-1242Crossref PubMed Scopus (171) Google Scholar), and in prostate tissue reduced EPA and DHA levels were associated with cancer compared with BPH (26Mamalakis G. Kafatos A. Kalogeropoulos N. Andrikopoulos N. Daskalopulos G. Kranidis A. Prostaglandins Leukot. Essent. 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Recently, DHA was shown to induce apoptosis in PC-3 cells (30Narayanan N.K. Narayanan B.A. Reddy B.S. Int. J. Oncol. 2005; 26: 785-792PubMed Google Scholar). The opposing effects of n-3 and n-6 PUFA are thought to reflect differences in eicosanoid metabolism (29Rose D.P. Connolly J.M. Prostate. 1991; 18: 243-254Crossref PubMed Scopus (180) Google Scholar, 31Rose D.P. Proc Soc. Exp. Biol. Med. 1997; 216: 224-233Crossref PubMed Scopus (79) Google Scholar), which may influence transcription factor activation, gene expression, and signal transduction (32Larsson S.C. Kumlin M. Ingelman-Sundberg M. Wolk A. Am. J. Clin. Nutr. 2004; 79: 935-945Crossref PubMed Scopus (774) Google Scholar). Our previous studies have shown that, in human breast cancer cells, n-3 PUFA inhibit growth, induce apoptosis, and up-regulate SDC-1(33Edwards I.J. Berquin I.M. Sun H. O'Flaherty J.T. Daniel L.W. Thomas M.J. Rudel L.L. Wykle R.L. Chen Y.Q. Clin. Cancer Res. 2004; 10: 8275-8283Crossref PubMed Scopus (32) Google Scholar, 34Sun H. Berquin I.M. Edwards I.J. Cancer Res. 2005; 65: 4442-4447Crossref PubMed Scopus (40) Google Scholar). In the present study we show that SDC-1 is increased in prostate tissue of mice fed an n-3 PUFA-enriched diet, and that, in human prostate cancer cells, the transcriptional pathway for n-3-PUFA regulation of SDC-1 expression involves the nuclear hormone receptor, peroxisome proliferator-activated receptor (PPAR) γ. Materials—Human prostate cancer PC-3 and DU-145 cells were obtained from the American Type Culture Collection (Rockville, MD). Mouse anti-human syndecan 1 (B-B4) was purchased from Serotec (Oxford, UK), anti-heparan sulfate stub monoclonal antibody (3G10) was from Seikagaku America (Ijamsville, MD), and rabbit polyclonal anti-human syndecan 1 (H-174) was from Santa Cruz Biotechnology (Santa Cruz, CA). Troglitazone and GW259662 were from Cayman Chemical (Ann Arbor, MI), heparinase III was from Sigma, and chondroitin ABC lyase was from Seikagaku America. A Super Signal West Pico Kit was from Pierce, and FuGENE 6 transfection reagent was from Roche Applied Science. Luciferase and β-galactosidase assay kits were from Promega (Madison, WI). Dominant negative (d/n) PPARγ cDNA (L468/E471) was kindly supplied by Dr. V. Chatterjee, University of Cambridge. Preparation of fatty acid-bovine serum albumin (BSA) complexes and n-3 and n-6 PUFA-enriched low density lipoproteins (LDLs) have been previously described (33Edwards I.J. Berquin I.M. Sun H. O'Flaherty J.T. Daniel L.W. Thomas M.J. Rudel L.L. Wykle R.L. Chen Y.Q. Clin. Cancer Res. 2004; 10: 8275-8283Crossref PubMed Scopus (32) Google Scholar). Animals—Male C57BL/6J mice were fed a high n-3 (n-6:n-3 ratio = 1:1) or a high n-6 (n-6:n-3 ratio = 40:1) diet for 8 weeks. Diet composition, body weights, fatty acid ratios in food, blood, and prostate tissues, and dissection of prostate lobes have been described (35Berquin I.M. Min Y. Wu R. Wu J. Perry D. Cline J.M. Thomas M.J. Thornburg T. Kulik G. Smith A. Edwards I.J. D'Agostino R. Zhang H. Wu H. Kang J.X. Chen Y.Q. J. Clin. Invest. 2007; 117: 1866-1875Crossref PubMed Scopus (210) Google Scholar). All animals were maintained in an isolated environment in barrier cages. Animal care was conducted in compliance with the state and federal Animal Welfare Acts and the standards and policies of the Department of Health and Human Services. The protocol was approved by our Institutional Animal Care and Use Committee. Immunostaining of Mouse Prostate Tissue—Prostate glands were dissected from 8-week-old male mice fed a chow diet. Tissues were fixed in Carnoy fluid at 4 °C, dehydrated, embedded in paraffin, sectioned (5 μm), and deparaffinized. Immunostaining was conducted using a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 82-256 of human syndecan 1 (H-174) and the DAKO EnVion + system peroxidase (3,3′-diaminobenzidine) kit according to the manufacturer's instructions. Cell Culture—PC-3 and DU-145 cells were maintained in Advanced Dulbecco's modified Eagle's medium supplemented with 1% fetal bovine serum (FBS) and Eagle's minimal essential medium with 10% FBS, respectively, penicillin/streptomycin, and l-glutamine at 37 °C in 5% CO2. In experiments measuring mRNA, cells were seeded in 6-well plates at a density of 5 × 105 cells/well in growth medium. After 6 h, the medium was changed to Dulbecco's modified Eagle's medium/F-12 with 0.5% FBS for 18 h and then supplemented with 100 μg/ml n-3 or n-6-enriched LDL, 30 μm DHA, 30 μm EPA, 30 μm LA, and 5 μm or 10 μm troglitazone for 8-24 h. Fatty Acid Delivery to Cells—Cells were grown in 100-mm dishes until ∼50% confluent when the growth media was replaced with fresh media containing 0.5% FBS supplemented with LDL or BSA-fatty acid. FBS was used at low levels to minimize competition between its LDL and the n-6 or n-3 LDL added to the cultures. After 24 h, cell monolayers were washed twice with balanced salt solution (137 mm NaCl, 2.7 mm KCl, 1.45 mm KH2PO4, 20.3 mm Na2HPO4), and lipids were extracted with isopropanol for 24 h at 4 °C. Lipid extracts were phased into chloroform and saponified, and fatty acids were methylated and separated by gas-liquid chromatography (36Parks J.S. Gebre A.K. J. Lipid Res. 1991; 32: 305-315Abstract Full Text PDF PubMed Google Scholar). Phospholipids were separated from chloroform extracts by thin layer chromatography prior to fatty acid analysis. Real-time PCR—RNA isolation and cDNA synthesis and real-time RT-PCR were as previously described (34Sun H. Berquin I.M. Edwards I.J. Cancer Res. 2005; 65: 4442-4447Crossref PubMed Scopus (40) Google Scholar). SDC-1 primers were 5′-ggagcaggacttcacctttg (forward) and 5′-ctcccagcacctctttcct (reverse). Versican primers were 5′-cccatgcgctacataaagtca-3′ (forward) and 5′-agaccatttgatgcggagaa-3′ (reverse). Perlecan primers were 5′-cgctggacacattcgtacct-3′ (forward) and 5′-accagggctcggaaataaac-3′ (reverse). Peptidylprolylisomerase B housekeeping gene primers were 5′-gcccaaagtcaccgtcaa (forward) and 5′-tccgaagagaccaaagatcac (reverse). Mouse SDC-1 primers were 5′-tggagaacaagacttcacctttg-3′ (forward) and 5′-ctcccagcacttccttcct-3′ (reverse). Mouse peptidylprolylisomerase B housekeeping gene primers were 5′-aacagcaagttccatcgtgtc-3′ (forward) and 5′-ctttcctcctgtgccatctc-3′ (reverse). Proteoglycan core protein data were normalized to the housekeeping control peptidylprolylisomerase B and are presented relative to control. Enzyme Treatment and Western Blot Analysis—Cells were washed twice with ice-cold phosphate-buffered saline and lysed for 10 min on ice; debris was then removed by centrifugation. Proteins were precipitated by a 2.5-fold volume cold methanol at -20 °C for 2 h. The samples were again centrifuged at 14,000 rpm for 10 min. For membrane proteins, cells were collected in hypotonic buffer (10 mm Tris-HCl, 1 mm EDTA) with protease inhibitors and homogenized, and debris was removed by centrifugation at 8,000 × g. Membranes in the supernatant were pelleted at 100,000 × g, dissolved in lysis buffer, and precipitated with cold methanol as above. Pellets were washed with ice-cold acetone, dried, and resuspended in heparinase buffer (50 mm HEPES, 50 mm NaOAc, 150 mm NaCl, 5 mm CaCl2), and equivalent amounts of protein were treated with heparinase III (2 units/ml) and chondroitinase ABC (1 unit/ml) at 37 °C for 16 h. Proteins were separated by 3.5-15% SDS-PAGE and transferred onto polyvinylidene difluoride membrane. The membranes were blocked with TBST (10 mm Tris-base, 100 mm NaCl, 0.1% Tween 20, pH 7.5) containing 5% nonfat dry milk for 2 h at room temperature, washed with TBST for 5 min × 3, exposed to the primary antibodies in TBST containing 3% BSA at 4 °C overnight, washed three times with TBST, incubated with horseradish peroxidase-conjugated secondary antibody for 1 h, washed with TBST, and developed using the Super Signal West Pico Kit. Wild-type and Mutant SDC-1 DR-1-luciferase Constructs—The one copy reporter construct DR-1 from the SDC-1 promoter (pGL3-DR-1 wt (37Anisfeld A.M. Kast-Woelbern H.R. Meyer M.E. Jones S.A. Zhang Y. Williams K.J. Willson T. Edwards P.A. J. Biol. Chem. 2003; 278: 20420-20428Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar)) was generated by annealing the oligonucleotides 5′-cggttcttcccctttgctctctcggccgtttccgctacacccgagct-3′ and 5′-cgggtgtagcggaaacggccgagagagcaaaggggaagaaccggtac-3′ before ligation into KpnI- and SacI-digested pGL3-luciferase vector. The one copy mutant DR-1 reporter construct (pGL3-DR-1) was generated using the same method and the oligonucleotides 5′-cggttctAGTActGCcCTcactcggccgtttccgctacacccgagct-3′ and 5′-cgggtgtagcggaaacggccgagTgagGGCagTACTagaaccggtac-3′. Mutations are capitalized (37Anisfeld A.M. Kast-Woelbern H.R. Meyer M.E. Jones S.A. Zhang Y. Williams K.J. Willson T. Edwards P.A. J. Biol. Chem. 2003; 278: 20420-20428Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Transfection and Luciferase Assay—PC-3 cells were co-transfected with the PPRE-TK-luciferase reporter plasmid and a control plasmid containing the lacZ gene or with pcDNA3-d/n-PPARγ cDNA and pGL3-luc-DR-1 (wild type or mutant) using FuGENE 6 Transfection Reagent according to the manufacturer's instructions. Transfected cells were incubated with no or 100 μg/ml n-3 LDL, 30 μm DHA, or 10 μm troglitazone for 8 or 24 h. Luciferase activities were measured using a Luciferase Assay Kit in a TD-20e luminometer (Turner Designs Inc., Sunnyvale, CA), and β-galactosidase activity was measured using a β-Galactosidase assay kit in a SpectroMAX plate reader (Molecular Devices Corp., Sunnyvale, CA). Luciferase activities were normalized to β-galactosidase activity and are presented as the percentage of luciferase activity measured in the presence of stimulus, relative to the activity of control cells with no stimulation. Nuclear Extract and Electrophoretic Gel Mobility Shift Assay—PC-3 cells were rinsed three times with phosphate-buffered saline, once with buffer A (20 mm HEPES (pH 7.9), 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride), scraped into buffer A containing 0.2% Nonidet P-40, and spun for 20 s at 15,000 rpm, 4 °C. Pellets were solubilized in 100 μl of buffer B (20 mm HEPES (pH 7.9), 1 mm EDTA, 1 mm EGTA, 420 mm NaCl, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 20% glycerol) and dialyzed against binding buffer (10 mm Tris, 50 mm KCl, 1 mm dithiothreitol, pH 7.5) overnight at 4 °C. DNA binding was performed by mixing 70,000 cpm of a 32P-end-labeled DNA fragment from SDC-1 promoter containing DR-1 (generated by annealing the oligonucleotides 5′-cggttcttcccctttgctctctcggccgtttccgctacacccgagct-3′ and 5′-cgggtgtagcggaaacggccgagagagcaaaggggaagaaccggtac-3′), 50 ng/μl poly(dI·dC) ·poly(dI·dC) (Sigma), 2.5% glycerol, 5 mm MgCl2, 0.05% Nonidet P-40, 20 μg of nuclear protein, 200 ng of unlabeled DNA, and/or 2 μl of PPARγ antibody (Cayman Chemical, Ann Arbor, MI) in a 20-μl reaction volume and incubated for 30 min at room temperature. Samples were loaded on a 5% polyacrylamide gel prerun for 60 min and run in TBE buffer (45 mm Tris base, 45 mm boric acid, 1 mm EDTA, pH 8.3) for 90 min at 200 V. Gels were dried and subjected to autoradiography. Data Analysis—Data were analyzed by analysis of variance and Student's t test. The assays were carried out in triplicate, and the data are shown as means ± S.E. Syndecan 1 Expression in Mouse Prostate Tissue—Anterior, ventral, and dorsolateral prostate lobes were dissected from male mice, embedded in paraffin, and sectioned for immunostaining. An antibody directed against a sequence of the human SDC-1 core protein demonstrated heterogeneous cell surface staining of prostate epithelial cells that was particularly strong at the apical surface of many cells (Fig. 1). Prostate tissues from animals fed an n-3 or n-6 PUFA-enriched diet were similarly dissected and processed for mRNA analysis. Fig. 2 shows that SDC-1 mRNA in the combined prostate lobes was >2 fold higher in animals fed an n-3-enriched diet compared with those fed an n-6 PUFA-enriched diet. Individual lobes from two additional animals in each diet group were analyzed separately and demonstrated that higher SDC-1 expression was achieved in all prostate lobes of the n-3 fed animals.FIGURE 2SDC-1 mRNA in prostate tissues of mice fed n-3 or n-6-enriched diets. Grossly normal prostate glands were dissected from animals at 8 weeks of age, and RNA was isolated and measured by real-time RT-PCR. Shown are SDC-1mRNA in total prostate tissue from animals fed n-3 and n-6 PUFA-enriched diets (n = 3) and in isolated lobes of two additional animals fed n-3 and n-6 PUFA-enriched diets. *, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Syndecan-1 Expression in Human Prostate Cancer Cell Lines—To demonstrate the presence of SDC-1 in human prostate cancer cells, PC-3 cells (Fig. 3A) and DU-145 cells (Fig. 3B) were incubated with a mouse anti-human SDC-1 monoclonal antibody and visualized by a rhodamine-labeled second antibody. Strong immunostaining was observed around the periphery of the cells. A similar staining pattern was observed in a third prostate cancer cell line, LNCaP (not shown). To verify that SDC-1 was the predominant heparan sulfate proteoglycan present on human prostate cancer cells, whole cell lysates of PC-3 cells were either untreated or treated with glycosaminoglycan-degrading enzymes, and Western blotting was performed. Fig. 3C shows the Western analysis using monoclonal antibody 3G10, which recognizes the unsaturated hexuronic acid residue on a heparan sulfate core protein following heparan sulfate chain removal. An 80-kDa core protein was visible in lanes containing heparinase-treated samples. No other core protein bands were detected indicating that one major heparan sulfate species is associated with these cells. In addition, Fig. 3D also shows a Western analysis using a SDC-1 core protein-specific antibody that clearly identified a similarly migrating 80-kDa protein. We conclude that PC-3 cells express SDC-1 as their dominant heparan sulfate-containing proteoglycan. As shown in Fig. 3E, the SDC-1 core protein was markedly elevated in cells incubated with LDL enriched in n-3 PUFA compared with n-6 PUFA. Delivery of PUFA to PC-3 Cells—To further examine in vitro regulation of SDC-1 expression by n-3 PUFA we modeled the two main physiological pathways for delivery of dietary fatty acids to cells: by LDL and by albumin. For this we used LDL isolated from vervet monkeys that had been fed diets enriched in fish oil. Our previous studies had shown that this LDL contained two major species of n-3 PUFA: EPA and DHA (33Edwards I.J. Berquin I.M. Sun H. O'Flaherty J.T. Daniel L.W. Thomas M.J. Rudel L.L. Wykle R.L. Chen Y.Q. Clin. Cancer Res. 2004; 10: 8275-8283Crossref PubMed Scopus (32) Google Scholar). As a control, n-6 PUFA-enriched LDL was isolated from vervet monkeys fed a diet supplemented with linoleic acid (LA (18:2, n-6)), which was the major fatty acid species in the n-6 LDL (33Edwards I.J. Berquin I.M. Sun H. O'Flaherty J.T. Daniel L.W. Thomas M.J. Rudel L.L. Wykle R.L. Chen Y.Q. Clin. Cancer Res. 2004; 10: 8275-8283Crossref PubMed Scopus (32) Google Scholar). Incubation of PC-3 cells with these LDL resulted in delivery of the respective fatty acids to the cells. Table 1 shows the fatty acid percent distribution in total cell lipids following 24-h incubation with either the PUFA-enriched LDL or BSA-bound EPA, DHA, or LA. In cells incubated with no fatty acid supplementation, the n-3 PUFA were ≤1% of total fatty acids. EPA-BSA addition resulted in an increase in EPA content to 15%. These cells effectively converted EPA to docosapentaenoic acid (DPA (22:5, n-3)), which also comprised 15% of total fatty acids. Metabolism of DPA to DHA, however, was not observed during this time period in the EPA-treated cells. DHA-BSA treatment resulted in increased DHA only. The n-3 LDL used for these studies did not contain a detectable level of DPA, but when the PC-3 cells were incubated with this LDL, it was observed that both EPA and DHA were delivered to the cells and that there was formation of DPA. Cells treated with LA-BSA or n-6 LDL showed an increase in LA to 19.4 and 10.1% of total fatty acids, respectively. Cell phospholipids were isolated from the total lipid extracts, and their fatty acid composition is shown in Table 2. The pattern of fatty acid distribution of the cell phospholipids mirrored that of the total cell lipids. Thus both LDL and PUFA-BSA were effective in delivering PUFA to the tumor cell phospholipids, and PC-3 cells were able to metabolize EPA to DPA but unable to metabolize the latter fatty acid further to DHA.TABLE 1Percent fatty acid composition of PC-3 cell total lipids Open table in a new tab TABLE 2Percent fatty acid composition of PC-3 cell phospholipidsView Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab In Vitro Up-regulation of SDC-1 by n-3 PUFA—To demonstrate in vitro regulation of SDC-1 by n-3 PUFA, real-time RT-PCR was used to measure SDC-1 expression in PC-3 cells incubated with n-3 LDL or PUFA-BSA (Fig. 4). The first panel shows that n-3 LDL (100 μg/ml) and DHA-BSA (30 μm) were effective in elevating SDC-1 expression, whereas EPA (30 μm), LA (30 μm), or equimolar concentrations (15 μm each) of EPA plus DHA were ineffective. Higher concentrations of EPA and LA (up to 100 μm) were also ineffective (not shown) as was n-6 LDL at 100 (Fig. 4) or 500 μg/ml (not shown). The effect of n-3 LDL and DHA-BSA was specific for SDC-1 and not a general effect on all cell proteoglycans: neither n-3 LDL nor DHA-BSA stimulated the expression of versican or perlecan, two other proteoglycan species produced by these cells. In addition, n-3 LDL and DHA were also efficient in increasing the expression of SDC-1 in DU-145 prostate cancer cells, but as in PC-3 cells, n-6 LDL and EPA were ineffective. Regulation of SDC-1 by PPARγ—Our previous studies have implicated the nuclear receptor, PPARγ, in the control of SDC-1 expression in human breast cancer cells (34Sun H. Berquin I.M. Edwards I.J. Cancer Res. 2005; 65: 4442-4447Crossref PubMed Scopus (40) Google Scholar). To determine whether a similar pathw
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