WPMY-1 Cells Research Articles

MP19-08 HONOKIOL, A CONSTITUENT OF MAGNOLIA SPEC., INHIBITS ADRENERGIC CONTRACTION OF HUMAN PROSTATE STRIPS AND INDUCES STROMAL CELL DEATH

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 2014MP19-08 HONOKIOL, A CONSTITUENT OF MAGNOLIA SPEC., INHIBITS ADRENERGIC CONTRACTION OF HUMAN PROSTATE STRIPS AND INDUCES STROMAL CELL DEATH Martin Hennenberg, Daniel Herrmann, Andrea Schreiber, Anna Ciotkowska, Frank Strittmatter, Christian G. Stief, and Christian Gratzke Martin HennenbergMartin Hennenberg More articles by this author , Daniel HerrmannDaniel Herrmann More articles by this author , Andrea SchreiberAndrea Schreiber More articles by this author , Anna CiotkowskaAnna Ciotkowska More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Christian G. StiefChristian G. Stief More articles by this author , and Christian GratzkeChristian Gratzke More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.709AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Medical therapy of lower urinary tract symptoms (LUTS) targets smooth muscle contraction in the lower urinary tract, or prostate growth, which may both contribute to bladder outlet obstruction (BOO). Options comprise α1-blockers to induce prostate smooth muscle relaxation, and 5α-reductase inhibitors to reduce prostate growth. As an alternative, phytotherapy is widespread. Honokiol and Magnolol, two lignan constituents of Magnolia spec., are used in traditional Japanese medicine. However, their actions in the lower urinary tract are unknown. Objective: To examine the effects of Honokiol and Magnolol on contraction of human prostate tissue, and on viability of stromal cells. METHODS Prostate tissues were obtained from radical prostatectomy. Contractility was examined in organ bath studies. Contractions were induced by noradrenaline, by the α1-adrenergic agonist, phenylephrine, or by electric field stimulation (EFS). Effects on viability of stromal cells were assessed in cultured WPMY-1 cells (an immortalized line of human prostate stromal cells). Ki-67 mRNA was assessed by RT-PCR, and proliferation by a fluorescence EdU assay. RESULTS Noradrenaline and phenylephrine induced concentration-dependent contractions of prostate strips. Honokiol (100 μM) reduced noradrenaline-induced contractions, which was significant at 10 μM (p<0.02 for Honokiol vs. control), 30 μM (p<0.04), and 100 μM noradrenaline (p<0.04) (n=10 patients) (Figure). Honokiol reduced phenylephrine-induced contractions, which was significant at 3 μM (p<0.02), 10 μM (p<0.005), 30 μM (p<0.003), and 100 μM phenylephrine (p<0.02) (n=8) (Figure). Honokiol reduced EFS-induced contractions very slightly (n=9), while EFS-induced contractions were completely unchanged by solvent. In WPMY-1 cells, Honokiol (24 h) induced cell death (Figure, lower panel, left). Magnolol (100 μM) was without effects on contraction, viability, Ki-67, or proliferation (Figure, lower panel, right). CONCLUSIONS Honokiol may interfer simultaneously with the dynamic component of BOO by inhibition of prostate smooth muscle contraction, and with the static component by killing stromal cells. Whether or not Honokiol induces urodynamic effects or improvement of BOO can only be assessed in clinical studies. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e191-e192 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Martin Hennenberg More articles by this author Daniel Herrmann More articles by this author Andrea Schreiber More articles by this author Anna Ciotkowska More articles by this author Frank Strittmatter More articles by this author Christian G. Stief More articles by this author Christian Gratzke More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Evidence of TGF-β1 mediated epithelial-mesenchymal transition in immortalized benign prostatic hyperplasia cells

Expression of epithelial-mesenchymal transition (EMT) markers has been detected clinically in benign prostatic hyperplasia (BPH) tissues. To understand the molecular basis, we investigated the role of stromal microenvironment in the progression of EMT in BPH cells. First, we used cell culture supernatant from normal prostate stromal WPMY-1 cells to provide supernatant-conditioned medium (WSCM) to culture the BPH-1 cell line. Then, the morphological changes and migratory capacity were detected in BPH-1 cells. The expression of EMT markers was examined in BPH-1 cells by Western blot and immunofluorescent analysis. Finally, to investigate the role of transforming growth factor beta 1 (TGF-β1) in this process, the WSCM-cultured cells were treated with monoclonal antibody against TGF-β1 to study its effect on EMT. We found that the morphology of BPH-1 cells changed to a spindle-like shape after cultured in WSCM, and the levels of E-cadherin and cytokeratin 5/8 (CK5/8) were significantly lower than the cells cultured in ordinary medium. These BPH-1 cells were also tested positive for mesenchymal markers vimentin and a-smooth muscle actin (SMA) as well as Snail. We also found WSCM can increase the migratory capacity of BPH-1 cells. In addition, when they were treated with anti-TGF-β1, upregulation of E-cadherin and CK5/8 levels was observed but no expression of vimentin, alpha-SMA or Snail was detected. Furthermore, phosphorylated-Smad3 expression in WSCM-cultured BPH-1 cells was also suppressed by anti-TGF-β1 treatment. Our results demonstrated that stromal cell supernatant was able to induce EMT in BPH-1 cells, possibly through secreting TGF-β1 to activate Smad signaling. Our results suggest novel molecular targets for clinical treatment of BPH by modification of stromal microenvironment through inhibiting TGF-β1/Smad expression.

Abstract A193: Transmembrane protein TMEM92 as a novel target in prostate cancer.

Abstract TMEM92 is a previously uncharacterized gene predicted to encode a 159 amino acid peptide with a single transmembrane domain and sequence homology to the SHISA group of proteins, some of which have a role in early development. In this study we evaluated the expression and function of TMEM92 in prostate cancer. This gene is expressed in primary prostate tumors and the prostate cancer derived cell lines PC3, DU145 and LNCaP, both at the RNA and protein level. TMEM92 expression appears to be cancer specific, as it is not expressed in normal prostate tissue or in normal adult organs. It is also absent from the WPMY-1 cell line, which is derived from normal prostate epithelium. siRNA-mediated knockdown of TMEM92 in PC3 cells induces apoptosis, as does the treatment of these cells with an anti-TMEM92 antibody, whilst neither treatment is cytotoxic for WPMY-1 cells. Taken together these findings suggest that TMEM92 is a potential therapeutic target in prostate cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A193. Citation Format: Richard Morgan, Gui Feng, Hardev S. Pandha. Transmembrane protein TMEM92 as a novel target in prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A193.

The Effect of LHRH Antagonist Cetrorelix in Crossover Conditioned Media from Epithelial (BPH-1) and Stromal (WPMY-1) Prostate Cells

Stromal cells strictly modulate the differentiation of the normal prostate epithelium. In benign prostatic hyperplasia (BPH) tissue, the ratio of stromal to epithelial cells reaches a 5:1 ratio. In this study, we evaluated the effects of crossover conditioned media (CM) of stromal and epithelial prostate cells before and after treatment with LHRH antagonist Cetrorelix. WPMY-1 human prostate stromal cells and BPH-1 human benign prostatic hyperplasia cells were cultured in vitro and the effects of crossover conditioned media (CM) from those cells were studied. We evaluated the effect of Cetrorelix on the expression of PCNA and p53 in those cells. We then studied the effect of Cetrorelix on BPH-1 cells cultured with the CM from WPMY-1 cells, as well as the mechanisms which govern these interactions. CM from WPMY-1 cells strongly stimulated the proliferation of BPH-1 cells in a dose dependent manner, while CM from BPH-1 cells only slightly increased the proliferation of WPMY-1 cells. Cetrorelix inhibited the proliferation of both cell lines and the expression of PCNA, while the expression of p53 was increased. Cetrorelix also inhibited the proliferation of BPH-1 cells stimulated with the CM from WPMY-1 cells. In the crossover experiment, conditioned media from WPMY-1 and BPH-1 cells increased the expression of phosphorylated ERK1/2 and STAT3. Our results support previous observations on the bidirectional stromal-epithelial interactions in prostate gland and shed more light on the mechanistic action of those effects. Our study strongly supports the hypothesis that LHRH antagonists may be beneficial for BPH prevention and treatment.

Shrinkage of experimental benign prostatic hyperplasia and reduction of prostatic cell volume by a gastrin-releasing peptide antagonist

Gastrin releasing-peptide (GRP) is a potent growth factor in many malignancies. Benign prostatic hyperplasia (BPH) is a progressive age-related proliferation of glandular and stromal tissues; various growth factors and inflammatory processes are involved in its pathogenesis. We have demonstrated that potent antagonists of GRP inhibit growth of experimental human tumors including prostate cancer, but their effect on models of BPH has not been studied. Here, we evaluated the effects of GRP antagonist RC-3940-II on viability and cell volume of BPH-1 human prostate epithelial cells and WPMY-1 prostate stromal cells in vitro, and in testosterone-induced BPH in Wistar rats in vivo. RC-3940-II inhibited the proliferation of BPH-1 and WPMY-1 cells in a dose-dependent manner and reduced prostatic cell volume in vitro. Shrinkage of prostates was observed after 6 wk of treatment with RC-3940-II: a 15.9% decline with 25 μg/d; and a 18.4% reduction with 50 μg/d (P < 0.05 for all). Significant reduction in levels of proliferating cell nuclear antigen, NF-κβ/p50, cyclooxygenase-2, and androgen receptor was also seen. Analysis of transcript levels of genes related to growth, inflammatory processes, and signal transduction showed significant changes in the expression of more than 90 genes (P < 0.05). In conclusion, GRP antagonists reduce volume of human prostatic cells and lower prostate weight in experimental BPH through direct inhibitory effects on prostatic GRP receptors. GRP antagonists should be considered for further development as therapy for BPH.

Mechanisms of synergism between antagonists of growth hormone‐releasing hormone and antagonists of luteinizing hormone‐releasing hormone in shrinking experimental benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) affects aging men. Combined therapy with antagonists of growth hormone-releasing hormone (GHRH) and of luteinizing hormone-releasing hormone (LHRH or GnRH) induces prostate shrinkage in rat models. We investigated the mechanisms of action of this combination on cell cycle traverse and expression of prostatic genes. Effects of GHRH antagonist, JMR-132 (40 µg/day), the LHRH antagonist, cetrorelix (0.625 mg/kg), and their combination were evaluated on testosterone-induced benign prostatic hyperplasia in male Wistar rats. Influence of JMR-132, cetrorelix, and their combinations on cell viability was assessed by MTS assay in BPH-1 human prostate epithelial cells and WPMY-1 normal prostate stromal cells. Cell cycle was analyzed by laser flow cytometry. Real-time PCR arrays were performed. The combination of antagonists caused marked shrinkage of rat prostate (29.5%). In vitro, JMR-132 plus cetrorelix (both 5µM) produced synergistic (57.4%) inhibition of growth of BPH-1 cells, but a lesser inhibition (46%) of WPMY-1 cells. Co-treatment of with JMR-132 plus cetrorelix induced a significant increase of BPH-1 cells blocked in S-phase plus cells with lower G0 /G1 and G2 /M DNA content. Significant changes in expression of >40 gene transcripts related to growth factors, inflammatory cytokines, and signal transduction were identified. GHRH antagonist and LHRH antagonist combination potentiates rat prostate weight reduction and synergistically inhibits of growth of BPH-1 leading to cell cycle arrest in S-phase. These effects were lesser in normal stromal prostate cell line, WPMY-1. Our findings suggest that GHRH antagonists could be useful for BPH therapy, possibly in combination with LHRH antagonists.

Prostaglandin E2 induces stromal cell-derived factor-1 expression in prostate stromal cells by activating protein kinase A and transcription factor Sp1

Recent reports indicate prostaglandin E2 (PGE2) can modulate tumor environment and promote angiogenesis through induction of stromal cell-derived factor 1 (SDF-1) production. We investigated the mechanism of PGE2-induced SDF-1 regulation in human prostate stromal cell and analyzed the effects in a stromal–epithelial interaction model. PGE2 stimulation increased SDF-1 expression in the prostate stromal cell lines WPMY-1 and NAF. We revealed signaling through the PGE2 receptor EP3 and activation of protein kinase A (PKA) are required. The EP3 agonist sulprostone and the cAMP analog forskolin mimicked and the EP3 siRNA, antagonist L798106 and the PKA inhibitor H89 abrogated the effect of PGE2 on SDF-1 expression. SDF-1 promoter truncation experiments demonstrated a 254bp (from nt −219 to nt +34) SDF-1 proximal promoter fragment containing 5 putative transcription factor Sp1 binding motifs is sufficient for PGE2 induction. CHIP assays confirmed binding and PGE2 induced recruitment of Sp1 to the SDF-1 promoter. Sp1 motif mutation identified Sp1 motifs −140/−133 and −9/+1 as the crucial elements responsible for PGE2 induction. Moreover, SDF-1 was up- or down-regulated by Sp1 over-expression or knock-down. We also demonstrate stimulation of migration of prostate cancer cell lines PC3 and DU145 with conditioned media collected from WPMY-1 or NAF cells stimulated with PGE2 and blockade of enhanced migration by a SDF-1 neutralizing antibody. In conclusion, we provide evidence for a paracrine prostate stromal–epithelial interaction induced by upregulation of expression of SDF-1 by PGE2. Our research provides new insights into the mechanism promoting metastasis of prostate carcinoma via stromal–epithelial interaction.

Qianliening capsule (前列宁胶囊) inhibits human prostate cell growth via induction of mitochondrion-dependent cell apoptosis

To investigate the molecular mechanisms by which Qianliening Capsule (, QC) treats benign prostatic hyperplasia (BPH). Human prostate stromal cell line WPMY-1 was treated with 0, 1, 3 and 5 mg/mL of QC for 24, 48 and 72 h, respectively, in the presence of 10 ng/mL basic fibroblast growth factor (bFGF). The viability of WPMY-1 cells was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell morphology was observed by phase-contrast microscopy. 4',6-diamidino-2-phenylindole (DAPI) staining and fluorescence activated cell sorting (FACS) analysis with Annexin-V/propidium iodide (PI) staining were performed to determine cell apoptosis. The loss of mitochondrial membrane potential was examined by FACS analysis with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyarine iodide (JC-1) staining. Activation of caspase-3 and -9 was evaluated by colorimetric assay. The mRNA and protein expression levels of Bcl-2 and Bax were measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. Upon bFGF stimulation, the viability of WPMY-1 cells was increased to 122%-118% compared with the control cells (P <0.05). However, treatment with 1-5 mg/mL of QC for 24, 48 and 72 h decreased the viability of bFGF-stimulated cells to 80%-92%, 59%-82%, 36%-62% compared with the untreated cells (P <0.05). In addition, QC treatment reduced WPMY-1 cell density in a dose-dependent manner. Moreover, QC treatment dose-dependently induced the loss of plasma membrane asymmetry, the nuclear condensation and fragmentation, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase of pro-apoptotic Bax/Bcl-2 ratio. Promoting mitochondrion-dependent apoptosis of prostate stromal cells might be one of the mechanisms by which QC treats BPH.

Hexanic lipidosterolic extract of Serenoa repens inhibits the expression of two key inflammatory mediators, MCP‐1/CCL2 and VCAM‐1, in vitro

What's known on the subject? and What does the study add? Pervasive inflammatory infiltrates, mainly composed of chronically activated T cells and monocytes/macrophages, have been observed in benign prostatic hyperplasia (BPH). Permixon®, a hexanic lipidosterolic extract of Serenoa repens (hexanic LSESr) used to treat urinary dysfunction in BPH patients, has anti-inflammatory activities. This paper provides new insights into the anti-inflammatory properties of Permixon®. We report that hexanic LSESr inhibits early steps of leukocyte infiltration in vitro by downregulating MCP-1/CCL2 and VCAM-1 expression. To investigate the mechanisms by which hexanic lipidosterolic extract of Serenoa repens (hexanic LSESr) may prevent leukocyte infiltration in benign prostatic hyperplasia by studying its impact on monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2) and vascular cell adhesion molecule 1 (VCAM-1) expression in vitro. After pretreatment with hexanic LSESr, human prostate (epithelial and myofibroblastic) cells and vascular endothelial cells were stimulated with proinflammatory cytokines. MCP-1/CCL2 and VCAM-1 mRNA expression was quantified by real-time PCR. ELISA kits were used to determine MCP-1/CCL2 levels in culture supernatants and VCAM-1 expression in living cells. Hexanic LSESr reduced MCP-1/CCL2 mRNA levels in both epithelial (BPH-1) and myofibroblastic (WPMY-1) prostate cell lines. Hexanic LSESr downregulated MCP1/CCL2 secretion by WPMY-1 cells in a concentration-dependent manner, more efficiently than Serenoa repens extracts obtained by supercritical carbon dioxide extraction. Hexanic LSESr inhibited tumour-necrosis-factor-α-induced MCP-1/CCL2 secretion by the human vascular endothelial cell line EAhy.926, as well as surface VCAM-1 protein expression, in a concentration-dependent manner. Hexanic LSESr impedes key steps of monocyte and T cell attraction and adherence by inhibiting MCP-1/CCL2 and VCAM-1 expression by human prostate and vascular cells in an inflammatory environment. These findings provide new insights into the anti-inflammatory effects of the hexanic lipidosterolic extract of Serenoa repens, Permixon®, in benign prostatic hyperplasia.

1578 REGULATION OF PROSTATE STROMAL CELL GENE EXPRESSION BY THE ANDROGEN RECEPTOR AND ITS EFFECTS ON PARACRINE SIGNALING TO PROSTATE CANCER CELLS

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 20111578 REGULATION OF PROSTATE STROMAL CELL GENE EXPRESSION BY THE ANDROGEN RECEPTOR AND ITS EFFECTS ON PARACRINE SIGNALING TO PROSTATE CANCER CELLS Charles Welliver, Matthew Tanner, Mengqian Chen, Badar Mian, Alejandro Godoy, Gary Smith, and Ralph Buttyan Charles WelliverCharles Welliver Albany, NY More articles by this author , Matthew TannerMatthew Tanner Albany, NY More articles by this author , Mengqian ChenMengqian Chen Albany, NY More articles by this author , Badar MianBadar Mian Albany, NY More articles by this author , Alejandro GodoyAlejandro Godoy Buffalo, NY More articles by this author , Gary SmithGary Smith Buffalo, NY More articles by this author , and Ralph ButtyanRalph Buttyan Albany, NY More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1614AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The androgen receptor (AR) is expressed in a subset of prostate stromal cells and functional prostate stromal AR is required for normal prostate developmental. Although we are broadly aware of the specifics of genomic action of AR in prostate cancer cells, we know relatively little regarding the gene targets of functional AR in prostate stromal cells. Here, we describe a novel human prostate stromal cell model that can be used to study gene targets of AR action in stromal cells. METHODS Immortalized human WPMY1 prostate stromal cells were transduced with an AR expression lentivirus and selected for stable AR expression. Control cells (WPMY-Vec) were transduced with empty lentivirus and also selected. Conditional responsiveness to DHT was assessed by measurement of luciferase activity after transfection with a synthetic androgen-responsive promoter reporter vector. RNAs were extracted from the cells (biological duplicate cultures) with or without 10 nM dihydrotestosterone (DHT) treatment. The RNAs were labeled and hybridized to Affymetrix ST1.0 human gene chips and gene expression patterns were compared with Genespring software. Changes in gene expression induced by DHT were confirmed by quantitative RT-PCR (qPCR). The functional ontologies of genes changed by androgen were categorized according to KEGG pathway rules. RESULTS Whereas parental WPMY1 or WPMY-Vec cells express little AR and were unresponsive to DHT, WPMY-AR cells express AR at a level commensurate with LNCaP cells and were highly responsive to DHT. WPMY-AR cells were similar in morphology and growth to WPMY-Vec cells, regardless of the presence of DHT. Treatment of WPMY-AR, but not WPMY-Vec cells, with DHT significantly changed the expression of 141 genes by 2-fold or greater. qPCR testing confirmed these changes for all genes assayed. The predominant functions of the genes affected by androgen include regulation of cancer and cell signaling pathways (TGF-beta, Wnt, Hedgehog and MAP-Kinase). Most gene changes (∼60%) involved gene downregulation by DHT. CONCLUSIONS WPMY-AR provides a cell model to study androgen regulated genes in prostate stromal cells. While DHT did not affect the behavior of these cells in culture, it regulated expression of a number of genes involved in cancer and other cell signaling pathways associated with development. Remarkably, androgens downregulated more genes in these cells than were upregulated. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e633-e634 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Charles Welliver Albany, NY More articles by this author Matthew Tanner Albany, NY More articles by this author Mengqian Chen Albany, NY More articles by this author Badar Mian Albany, NY More articles by this author Alejandro Godoy Buffalo, NY More articles by this author Gary Smith Buffalo, NY More articles by this author Ralph Buttyan Albany, NY More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Estrogens Promote Invasion of Prostate Cancer Cells in a Paracrine Manner through Up-Regulation of Matrix Metalloproteinase 2 in Prostatic Stromal Cells

Accumulating evidence suggests an enhancing effect of estrogens on prostate cancer (PCa) progression. Matrix metalloproteinase 2 (MMP2), which plays an important role in prostate cancer invasion, is mainly expressed in prostatic stromal cells (PrSC). Here we show that estradiol (E(2)) treatment up-regulates MMP2 production in PrSC, which promotes PCa cell invasion in a paracrine manner. Conditioned medium (CM) was collected from E(2)-treated prostatic stromal cell line WPMY-1 and primary PrSC. The CM of E(2)-treated WPMY-1 and PrSC promoted invasion of PCa cells, as measured by Matrigel transwell assays. Treatment with E(2) and 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, an estrogen receptor-alpha (ERα) specific agonist, significantly up-regulated MMP2 expression in WPMY-1 and PrSC cells at both mRNA and protein levels. The CM treated with an anti-MMP2 antibody lost the stimulatory effect on invasion of PCa cells. The ER inhibitor ICI 182,780, as well as a TGFβ1 neutralizing antibody and ERα-specific small interfering RNA effectively suppressed E(2)-induced MMP2 expression in WPMY-1 cells. Mechanistic studies showed that E(2) up-regulated MMP2 in an indirect manner: E(2) induced TGFβ1 expression via ERα; TGFβ1 stimulated MMP2 expression in PrSC; the invasion of PCa cells were stimulated by elevated MMP2 expression induced by E(2) in a paracrine manner. Our data show that E(2) induces MMP2 expression in WPMY-1 and PrSC cells, which was mediated by TGFβ1. The effect of E(2) on invasion of PCa cells is mediated by up-regulation of MMP2 in a paracrine mechanism.

Elevated GnRH receptor expression plus GnRH agonist treatment inhibits the growth of a subset of papillomavirus 18‐immortalized human prostate cells

Human metastatic prostate cancer cell growth can be inhibited by GnRH analogs but effects on virus-immortalized prostate cells have not been investigated. Virus-immortalized prostate cells were stably transfected with rat GnRH receptor cDNA and levels of GnRH binding were correlated with GnRH effects on signaling, cell cycle, growth, exosome production, and apoptosis. High levels of cell surface GnRH receptor occurred in transfected papillomavirus-immortalized WPE-1-NB26 epithelial cells but not in non-tumourigenic RWPE-1, myoepithelial WPMY-1 cells, or SV40-immortalized PNT1A. Endogenous cell surface GnRH receptor was undetectable in non-transfected cells or cancer cell lines LNCaP, PC3, and DU145. GnRH receptor levels correlated with induction of inositol phosphates, elevation of intracellular Ca(2+) , cytoskeletal actin reorganization, modulation of ERK activation and cell growth-inhibition with GnRH agonists. Hoechst 33342 DNA staining-cell sorting indicated accumulation of cells in G2 following agonist treatment. Release of exosomes from transfected WPE-1-NB26 was unaffected by agonists, unlike induction observed in HEK293([SCL60]) cells. Increased PARP cleavage and apoptotic body production were undetectable during growth-inhibition in WPE-1-NB26 cells, contrasting with HEK293([SCL60]) . EGF receptor activation inhibited GnRH-induced ERK activation in WPE-1-NB26 but growth-inhibition was not rescued by EGF or PKC inhibitor Ro320432. Growth of cells expressing low levels of GnRH receptor was not affected by agonists. Engineered high-level GnRH receptor activation inhibits growth of a subset of papillomavirus-immortalized prostate cells. Elucidating mechanisms leading to clone-specific differences in cell surface GnRH receptor levels is a valuable next step in developing strategies to exploit prostate cell anti-proliferation using GnRH agonists.

1408 INDUCTION OF SECRETED WNT FACTOR WISP1/CCN4 BY HUMAN PROSTATE STROMAL CELLS BY MICROENVIORNMENTAL HYPOXIA

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 20101408 INDUCTION OF SECRETED WNT FACTOR WISP1/CCN4 BY HUMAN PROSTATE STROMAL CELLS BY MICROENVIORNMENTAL HYPOXIA Matthew Tanner, Mengqian Chen, Elina Levina, Richard Carkner, and Ralph Buttyan Matthew TannerMatthew Tanner More articles by this author , Mengqian ChenMengqian Chen More articles by this author , Elina LevinaElina Levina More articles by this author , Richard CarknerRichard Carkner More articles by this author , and Ralph ButtyanRalph Buttyan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1100AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Wnt is a ligand-activated cell signaling pathway that plays an important role in normal embryonic development. The effects of Wnt in development are often manifest through a paracrine signaling process wherein Wnt ligands are secreted by one cell type but act on a neighboring cell type. For prostate epithelial cells, Wnt regulates androgen receptor expression and promotes cell growth under low androgen conditions so Wnt activity mediated by age-associated microenvironmental changes has the potential to effect abnormal prostate growth. We sought to determine whether hypoxia, a microenvironmental condition found in aging prostates, affects the expression of Wnt ligands and downstream Wnt targets in human prostate stromal cells as a means to better understand how this condition might contribute to human prostate disease. METHODS Cultured immortalized human prostate stromal cells, WPMY-1 and PS-30, were incubated under normoxic or hypoxic (1% O2) conditions. Hypoxia responsiveness was assessed by the activity of a hypoxia-sensitive luciferase reporter transfected into the cells. The effects of hypoxia on gene expression were determined by comparative expression analysis of RNAs extracted from normoxic or hypoxic cells using the SABiosciences Wnt-targeted gene microarray expression profiling system and the results were confirmed by real-time quantitative reverse-transcriptase PCR. Human WISP1/CCN4 cDNA was cloned into a lentiviral expression vector and used to produce WISP1/CCN4 overexpressing WPMY-1 cells. RESULTS Hypoxia-sensitive luciferase expression peaked at 72 hrs incubation in 1% O2. Several Wnt-related genes were found to be differentially upregulated by 72 hrs of hypoxia, most prominently WISP1/CCN4 (>50 fold), Wnt-1 (>3-fold), Wnt-16 (>2-fold) and WIF1 (>2-fold). Detailed analysis of the entire WISP/CCN gene family showed that expression of all 3 homologues were highly upregulated by hypoxia. Human cDNA encoding WISP1 was cloned and expressed in WPMY-1 cells to provide a cell model to test for the effects of stromal-expressed WISP1 on prostate epithelial cells. CONCLUSIONS Human prostate stromal cells respond to hypoxia by altering the expression of certain genes in the Wnt pathway. This was especially pronounced for WISP1/CCN4, a Wnt-inducible, growth enhancing gene product that is secreted into the extracellular matrix. The availability of a prostate stromal cell model that overexpresses WISP1/CCN4 will enable studies to test how this protein affects the growth of neighboring benign or malignant epithelial cells. Albany, NY© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e543-e544 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Matthew Tanner More articles by this author Mengqian Chen More articles by this author Elina Levina More articles by this author Richard Carkner More articles by this author Ralph Buttyan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Kallikrein‐related peptidase‐4 initiates tumor–stroma interactions in prostate cancer through protease‐activated receptor‐1

In prostate cancer, the mechanism by which the stromal cells surrounding the cancer epithelium become reactive and overproduce growth factors is unclear. Furthermore, the precise process of how these stromal cells stimulate the cancer epithelium is not fully understood. We recently found that protease-activated receptor-1 (PAR-1) in these reactive stromal cells is upregulated. To investigate the role of PAR-1 in the stromal-epithelial interaction, WPMY-1 stromal myofibroblasts were stimulated with PAR-1 agonists including thrombin and PAR-1 activating peptide. We show that WPMY-1 cells have functional PAR-1 by signaling through ERK1/2. Conditioned media (CM) from PAR-1 agonists-treated WPMY-1 cells stimulate the epithelial LNCaP cells leading to ERK1/2 activation and cell proliferation. Cytokine array analysis of the CM demonstrates that PAR-1 induces stromal cells to release numerous cytokines, of which interleukin 6 (IL-6) is the major factor responsible for mitogenic signaling in LNCaP cells. CM further induces expression of prostate-specific kallikrein-related peptidase-3 (KLK3/PSA) and KLK4 in LNCaP cells via the IL-6 pathway. Moreover, KLK4 functions as a potent agonist of PAR-1 by cleaving the receptor at the proper site on cell surface. KLK4 triggers transmembrane signaling and upregulates IL-6 in WPMY-1 cells through PAR-1. Immunohistochemical analysis indicates that PAR-1 is predominantly expressed in peritumoral stroma while KLK4 is produced exclusively by the epithelial cancer cells. These data provide evidence for a novel double-paracrine mechanism whereby cancer epithelium produces KLK4 to activate PAR-1 in the surrounding stroma, which in-turn releases cytokines (IL-6) that stimulate cancer cells to proliferate and increase production of KLKs.

Smad7 is inactivated through a direct physical interaction with the LIM protein Hic-5/ARA55

We recently reported that hydrogen peroxide-inducible clone-5 (Hic-5, also named androgen receptor-associated protein 55) can bind to the transforming growth factor-beta (TGF-beta)-signaling regulator Smad3, thereby inhibiting certain Smad3-dependent TGF-beta responses. We now show that Hic-5 can also control TGF-beta responses through an alternative mechanism involving Smad7, a key negative regulator of TGF-beta signaling. Hic-5 binds directly to Smad7. This interaction requires the LIM3 domain of Hic-5, and enhances TGF-beta signaling through causing loss of Smad7 protein but not mRNA. Enforced expression of Hic-5 reverses the ability of Smad7 to suppress TGF-beta-induced phosphorylation of Smads 2 and 3 and activation of the plasminogen activator inhibitor-1 promoter (in NRP-154 and PC3 prostate carcinoma and WPMY-1 prostate myofibroblast cell lines). Lentiviral-mediated small-hairpin RNA silencing of endogenous Hic-5 reduced TGF-beta responses in PC3 and WPMY-1 cells. Further work suggests that the level of Smad7 is modulated by its physical interaction with Hic-5 and targeted to a degradation pathway not likely to be proteasomal. Our findings support that Hic-5 functions as a cell-type-specific activator of TGF-beta signaling through its ability to physically interact with and neutralize Smad7.

Study on NDRG3 in Mesenchyma of Prostate

Objective To study expression of NDRG3 in prostatic mesenchyma and effect of exogenous NDRG3 on prostatic stromal cells. Methods Immunohistochemical analysis was used to check expression of NDRG3 in prostate mesnenchyma. The WPMY-1 prostate immortalized mesenchyma cell line was stably-transfected with a NDRG3 gene expression vector. The NDRG3-stable transfected WPMY-1 sublines were studied along with parental and empty vector transfected WPMY-1 cells as controls. RT-PCR technology was applied to identity downstream gene expression under regulation of NDRG3 expression. Results Expression of DNRG3 was observed in prostate cancer mesenchyma, over-expression of NDRG3 in WPMY-1 cell up-regulated expression of chemotatic factors-CXCL3 and CXCL5. Conclusion Expression of stromal NDRG3 in prostate cancer specimens is significantly higher than that in benign prostatic hypertrophy (BPH) sample. There is a remarkable difference between the two groups of samples, NDRG3 may be related to angiogenesis in prostatic mesenchyma.

Androgen receptor is a tumor suppressor and proliferator in prostate cancer

Targeting androgens/androgen receptor (AR) functions via androgen deprivation therapy (ADT) remains the standard treatment for prostate cancer. However, most tumors eventually recur despite ADT. Here we demonstrate that the prostate AR may function as both a suppressor and a proliferator to suppress or promote prostate cancer metastasis. Results from orthotopically recombining stromal WPMY1 cells with epithelial PC3 prostate cancer cells in mice demonstrated that restoring AR in epithelial PC3 cells or knockdown of AR in stromal WPMY1 cells suppressed prostate cancer metastasis. Knockdown of the AR in epithelial CWR22rv1 prostate cancer cells also resulted in increased cell invasion in vitro and in vivo. Restoring AR in PC3 cells (PC3-AR9) results in decreased invasion in bone lesion assays and in vivo mouse models. Mice lacking the prostate epithelial AR have increased apoptosis in epithelial luminal cells and increased proliferation in epithelial basal cells. The consequences of these two contrasting results led to the expansion of CK5/CK8-positive intermediate cells, and mice developed larger and more invasive metastatic tumors in lymph nodes and died earlier than wild-type littermates. Mechanistic dissection suggested that androgens/AR might directly or indirectly modulate metastasis-related genes and suppression of TGFbeta1 signals results in the partial inhibition of AR-mediated metastasis. Collectively, our understanding of these opposing roles of prostatic AR may revolutionize the way we combat prostate cancer, and allow the development of new and better therapies by targeting only the proliferative role of AR.

The proliferative effect of estradiol on human prostate stromal cells is mediated through activation of ERK

Estrogen is involved in the development and progression of benign prostatic hyperplasia (BPH). It can stimulate proliferation of prostate stromal cells (PrSCs). However, the exact mechanism remains unclear. We used the primary cultured human PrSCs and a prostate stromal cell line, WPMY-1, to examine the signaling pathways involved in estrogen-mediated proliferation of PrSCs. Cells were treated with 17beta-estradiol (E(2)) or BSA-E(2). Cell proliferation was assessed by the MTT assay and by cell counting. Western blot analysis was used to determine the status of activation of ERK1/2. Results indicated that both E(2) and BSA-E(2) stimulated proliferation of primary PrSCs and WPMY-1 cells. ERK was rapidly activated by E(2) and BSA-E(2). PD98059, which is a selective ERK inhibitor, significantly inhibited estrogen-induced cell proliferation. PrSCs expressed estrogen receptor alpha (ERalpha) and GPR30 but not ERbeta. Small hairpin RNA (shRNA) to ERalpha, but not to GPR30, blocked estrogen-mediated ERK activation and cell proliferation. The results indicated that estrogen could activate ERK pathway through the non-genomic ERalpha pathway, leading to proliferation of PrSCs.

Hic-5/ARA55, a LIM Domain–Containing Nuclear Receptor Coactivator Expressed in Prostate Stromal Cells

Prostate gland development and growth requires both androgen action and epithelial-stromal communications. In fact, androgen signaling through the androgen receptor (AR) may be important in both stromal and epithelial cells of the prostate. Because interaction of AR with the coactivator, Hic-5/ARA55, results in enhanced androgen-induced transcription, we analyzed Hic-5/ARA55 expression in prostate tissue sections from normal human donors and prostate cancer patients. In each sample, Hic-5/ARA55 expression was confined to the stromal compartment of the prostate. Furthermore, a prostate stromal cell line, WPMY-1 cells, expresses Hic-5/ARA55, which is localized both at focal adhesion complexes and within the soluble cytoplasmic compartment. The ability of Hic-5/ARA55 to shuttle between the nuclear and cytoplasmic compartments was revealed on inhibition of nuclear export with leptomycin B. Small interfering RNA ablation experiments established endogenous Hic-5/ARA55 as a coactivator for both viral and endogenous cellular AR-regulated genes. Finally, the mechanism of Hic-5/ARA55 coactivator activity in WPMY-1 cells was revealed by chromatin immunoprecipitation analysis that showed its androgen-dependent recruitment to the promoter of the stromal androgen-responsive keratinocyte growth factor gene. These data provide the first demonstration of a stromal-specific AR coactivator that has an effect on an androgen-regulated growth factor that is essential for stromal/epithelial cell communication in the prostate.

A human prostatic stromal myofibroblast cell line WPMY-1: a model for stromal-epithelial interactions in prostatic neoplasia.

Here we report the characterization of an SV40 large-T antigen-immortalized stromal cell line, WPMY-1, derived from the same prostate as our previously described epithelial cell lines. The WPMY-1 cells were determined to be myofibroblasts on the basis of co-expression of smooth muscle alpha-actin and vimentin. They also show positive staining for androgen receptor, large-T antigen, and positive but heterogeneous staining for p53 and pRb. Their growth is stimulated by the synthetic androgen mibolerone to 145% of control (100%). Platelet-derived growth factor BB, epidermal growth factor and basic fibroblast growth factor, at 10 ng/ml, stimulated growth to 138, 143 and 146% of control, respectively. Transforming growth factor-beta, at 10 ng/ml, inhibited serum-induced growth to 65% of control in the presence of 1% serum, and bFGF-induced growth to 30% of control. A serum-free medium was developed for optimal growth of WPMY-1 cells. They show anchorage-independent growth in soft agar. Studies on paracrine interactions show that myofibroblast-conditioned medium causes a marked inhibition of growth in WPE1-10 cells, while conditioned medium from WPE1-10 prostatic epithelial cells caused only a small increase in the growth of WPMY-1 cells. WPMY-1 cells secrete very low levels of MMP-9 but high levels of MMP-2, markedly higher than the epithelial cells. These epithelial and myofibroblast cell lines, derived from the same prostate, provide novel and useful models for studies on paracrine stromal-epithelial interactions in carcinogenesis, tumor progression, prevention and treatment of prostate cancer and benign prostatic hyperplasia.