WPMY-1 Cells Research Articles

Long noncoding RNA LINC01296 is associated with poor prognosis in prostate cancer and promotes cancer-cell proliferation and metastasis.

Background/purposeLong noncoding RNAs (lncRNAs) have emerged as important regulators and biomarkers of tumor development and progression. This study investigated the clinical significance, biological functions, and underlying mechanisms of long intergenic non-protein-coding RNA 1296 (LINC01296) in prostate cancer.Materials and methodsLINC01296 expression in prostate cancer tissues and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The correlation between LINC01296 expression and clinicopathologic characteristics of prostate cancer was analyzed using Kaplan–Meier analysis and the Cox proportional-hazard model. Small interfering RNA was used to suppress LINC01296, and knockdown efficiency was examined by qRT-PCR. Cell Counting Kit 8 assay, colony-formation assay, migration and invasion assays, and Western blot assay were used to explore the role of LINC01296 in tumor progression further.ResultsLINC01296-expression level was higher in prostate cancer tissues and prostate cancer cells than in adjacent nontumor tissues and immortalized normal prostate stromal WPMY1 cells. LINC01296 expression was correlated with preoperative prostate specific antigen (P=0.002), lymph-node metastasis (P=0.035), Gleason score (P<0.001), and tumor stage (P=0.036). Patients with higher LINC01296 expression displayed advanced clinical features and shorter biochemical recurrence-free survival time than those with lower LINC01296 expression. Multivariate analysis showed that LINC01296 expression was an independent predictor of biochemical recurrence-free survival in prostate cancer. Additionally, LINC01296 knockdown inhibited prostate cancer-cell proliferation, migration, and invasion, demonstrated in an in vitro study involving regulation of PI3K–Akt–mTOR signaling and epithelial–mesenchymal transition.ConclusionThe results demonstrated that LINC01296 is a novel molecule involved in prostate cancer development and progression, and thus is a potential biomarker of prognosis.

Torulene and torularhodin, protects human prostate stromal cells from hydrogen peroxide-induced oxidative stress damage through the regulation of Bcl-2/Bax mediated apoptosis

The current study was designed to elucidate the cytoprotective effects and possible mechanisms of torulene and torularhodin on hydrogen peroxide (H2O2)-induced oxidative stress damage in human prostate stromal cells (WPMY-1). After treated with H2O2, a notable decrease was appeared in cell viability, yet the decrease was attenuated when cells were pretreated with torulene and torularhodin (0.5–10 μM) as evaluated by WST-1 assay. Pretreatment with these two carotenoids significantly attenuated H2O2-induced apoptosis in WPMY-1 cells through the inhibition of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) overproduction, as well as the activation of the activities in catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Finally, pretreatment of cells with carotenoids resulted in the regulation of the mRNA and protein expression of Bcl-2 and Bax in H2O2-exposed prostate stromal cells. The present results indicate that both torulene and torularhodin can protect human prostate stromal cells from oxidative stress damage via Bcl-2/Bax mediated apoptosis.

Smooth muscle contraction and growth of stromal cells in the human prostate are both inhibited by the Src family kinase inhibitors, AZM475271 and PP2.

In benign prostatic hyperplasia, increased prostate smooth muscle tone and prostate volume may contribute alone or together to urethral obstruction and voiding symptoms. Consequently, it is assumed there is a connection between smooth muscle tone and growth in the prostate, but any molecular basis for this is poorly understood. Here, we examined effects of Src family kinase (SFK) inhibitors on prostate contraction and growth of stromal cells. SFK inhibitors, AZM475271 and PP2, were applied to human prostate tissues to assess effects on smooth muscle contraction, and to cultured stromal (WPMY-1) and c-Src-deficient cells to examine effects on proliferation, actin organization and viability. SFKs were detected by real time PCR, western blot and immunofluorescence in human prostate tissues, some being located to smooth muscle cells. AZM475271 (10μM) and PP2 (10μM) inhibited SFK in prostate tissues and WPMY-1 cells. Both inhibitors reduced α1 -adrenoceptor-mediated and neurogenic contraction of prostate strips. This may result from cytoskeletal deorganization, which was observed in response to AZM475271 and PP2 in WPMY-1 cells by staining of actin filaments with phalloidin. This was paralleled by reduced proliferation of wildtype but not of c-Src-deficient cells; cytotoxicity was mainly observed at higher concentrations (>50μM). In human prostate, smooth muscle tone and growth are both controlled by an SFK-dependent process, which may explain their common role in bladder outlet obstruction. Targeting prostate smooth muscle tone and prostate growth simultaneously by a single compound may, in principal, be possible.

The treatment effects of flaxseed-derived secoisolariciresinol diglycoside and its metabolite enterolactone on benign prostatic hyperplasia involve the G protein-coupled estrogen receptor 1.

Secoisolariciresinol diglucoside (SDG), a lignan extracted from flaxseed, has been shown to suppress benign prostatic hyperplasia (BPH). However, little is known about the mechanistic basis for its anti-BPH activity. The present study showed that enterolactone (ENL), the mammalian metabolite of SDG, shared the similar binding site of G1 on a new type of membranous estrogen receptor, G-protein-coupled estrogen eceptor 1 (GPER), by docking simulations method. ENL and G1 (the specific agonist of GPER) inhibited the proliferation of human prostate stromal cell line WPMY-1 as shown by MTT assay and arrested cell cycle at the G0/G1 phase, which was displayed by propidium iodide staining following flow cytometer examination. Silencing GPER by short interfering RNA attenuated the inhibitory effect of ENL on WPMY-1 cells. The therapeutic potential of SDG in the treatment of BPH was confirmed in a testosterone propionate-induced BPH rat model. SDG significantly reduced the enlargement of the rat prostate and the number of papillary projections of prostatic alveolus and thickness of the pseudostratified epithelial and stromal cells when comparing with the model group. Mechanistic studies showed that SDG and ENL increased the expression of GPER both in vitro and in vivo. Furthermore, ENL-induced cell cycle arrest may be mediated by the activation of GPER/ERK pathway and subsequent upregulation of p53 and p21 and downregulation of cyclin D1. This work, in tandem with previous studies, will enhance our knowledge regarding the mechanism(s) of dietary phytochemicals on BPH prevention and ultimately expand the scope of adopting alternative approaches in BPH treatment.

Expression of two WFDC1/ps20 isoforms in prostate stromal cells induces paracrine apoptosis through regulation of PTGS2/COX-2.

Background:WFDC1/Prostate stromal 20 (ps20) is a small secreted protein highly expressed within the prostate stroma. WFDC1/ps20 expression is frequently downregulated or lost in prostate cancer (PCa) and ps20 has demonstrated growth-suppressive functions in numerous tumour model systems, although the mechanisms of this phenomenon are not understood.Methods:Ps20 was cloned and overexpressed in DU145, PC3, LNCaP and WPMY-1 cells. Cellular growth, cell cycle and apoptosis were characterised. WPMY-1 stromal cells expressing ps20 were characterised by transcriptome microarray and the function of WPMY-1 conditioned media on growth of PCa cell lines was assessed.Results:Prostrate stromal 20 expression enhanced the proliferation of LNCaP cells, whereas stromal WPMY-1 cells were inhibited and underwent increased apoptosis. Prostrate stromal 20-expressing WPMY-1 cells secrete a potently proapoptotic conditioned media. Prostrate stromal 20 overexpression upregulates expression of cyclooxygenase-2 (COX-2) in LNCaP and WPMY-1 cells, and induces expression of a growth-suppressive phenotype, which inhibits proliferation of PCa cells by ps20-expressing WPMY-1 conditioned media. This growth suppression was subsequently shown to be dependent on COX-2 function.Conclusions:This work posits that expression of ps20 in the prostate stroma can regulate growth of epithelial and other tissues through the prostaglandin synthase pathway, and thereby restricts development and progression of neoplasms. This provides a rational for selective pressure against ps20 expression in tumour- associated stroma.

P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate.

Prostate smooth muscle tone and hyperplastic growth are involved in the pathophysiology and treatment of male lower urinary tract symptoms (LUTS). Available drugs are characterized by limited efficacy. Patients’ adherence is particularly low to combination therapies of 5α-reductase inhibitors and α1-adrenoceptor antagonists, which are supposed to target contraction and growth simultaneously. Consequently, molecular etiology of benign prostatic hyperplasia (BPH) and new compounds interfering with smooth muscle contraction or growth in the prostate are of high interest. Here, we studied effects of p21-activated kinase (PAK) inhibitors (FRAX486, IPA3) in hyperplastic human prostate tissues, and in stromal cells (WPMY-1). In hyperplastic prostate tissues, PAK1, -2, -4, and -6 may be constitutively expressed in catecholaminergic neurons, while PAK1 was detected in smooth muscle and WPMY-1 cells. Neurogenic contractions of prostate strips by electric field stimulation were significantly inhibited by high concentrations of FRAX486 (30 μM) or IPA3 (300 μM), while noradrenaline- and phenylephrine-induced contractions were not affected. FRAX486 (30 μM) inhibited endothelin-1- and -2-induced contractions. In WPMY-1 cells, FRAX486 or IPA3 (24 h) induced concentration-dependent (1–10 μM) degeneration of actin filaments. This was paralleled by attenuation of proliferation rate, being observed from 1 to 10 μM FRAX486 or IPA3. Cytotoxicity of FRAX486 and IPA3 in WPMY-1 cells was time- and concentration-dependent. Stimulation of WPMY-1 cells with endothelin-1 or dihydrotestosterone, but not noradrenaline induced PAK phosphorylation, indicating PAK activation by endothelin-1. Thus, PAK inhibitors may inhibit neurogenic and endothelin-induced smooth muscle contractions in the hyperplastic human prostate, and growth of stromal cells. Targeting prostate smooth muscle contraction and stromal growth at once by a single compound is principally possible, at least under experimental conditions.

Inflammatory response of a prostate stromal cell line induced by Trichomonas vaginalis.

While Trichomonas vaginalis, a cause of sexually transmitted infection, is known as a surface-dwelling protozoa, trichomonads have been detected in prostatic tissue from benign prostatic hyperplasia and prostatitis by immunoperoxidase assay or PCR. However, the immune response of prostate stromal cells infected with T.vaginalis has not been investigated. Our objective was to investigate whether T.vaginalis could induce an inflammatory response in prostate stromal cells. Incubation of a human prostate stromal myofibroblast cells (WPMY-1) with live T.vaginalis T016 increased expression of the inflammatory chemokines CXCL8 and CCL2. In addition, TLR4, ROS, MAPK and NF-κB expression increased, while inhibitors of TLR4, ROS, MAPKs and NF-κB reduced CXCL8 and CCL2 production. Medium conditioned by incubation of WPMY-1 cells with T.vaginalis stimulated the migration of human neutrophils and monocytes (THP-1 cells). We conclude that T.vaginalis increases CXCL8 and CCL2 production by human prostate stromal cells by activating TLR4, ROS, MAPKs and NF-κB, and this in turn attracts neutrophils and monocytes and leads to an inflammatory response. This study is the first attempt to demonstrate an inflammatory reaction in prostate stromal cells caused by T.vaginalis.

MP44-18 INHIBITION OF SMOOTH MUSCLE CONTRACTION BY THE INHIBITOR FOR CYTOHESIN FAMILIY GUANOSINE NUCLEOTIDE EXCHANGE FACTORS, SECIN H3 IN THE HYPERPLASTIC HUMAN PROSTATE

INTRODUCTION AND OBJECTIVES: In patients with benign prostatic hyperplasia, smooth muscle contraction and stromal growth in the prostate may both contribute to urethral obstruction and voiding symptoms. Although a context between control of smooth muscle tone and prostate growth may be assumed, molecular mechanisms underlying this connection still represent an enigma. Here, we addressed the role of Src family kinases (SFKs) for contraction of human prostate smooth muscle, and for growth of human stromal cells. METHODS: SFK expression was assessed by RT-PCR, Western blot, and immunofluorescence. Effects of two different SFK inhibitors, AZM475271 and PP2, on contraction were assessed in human prostate tissues obtained from radical prostatectomy. Effects on proliferation, actin organization, and cytotoxicity were examined in cultured stromal cells (WPMY-1). RESULTS: C-Src and Lck were detected by RT-PCR and Western blot in human prostate tissues and WPMY1 cells. Colocalization with calponin in immunofluorescence stainings of prostate sections indicated location to smooth muscle cells. AZM475271 (10 mM) and PP2 (10 mM) inhibited SFK activity in prostate tissues and WPMY-1 cell, being assessed as phospho-SFK content. Both inhibitors significantly inhibited contraction of prostate strips by norepinephrine, by the a1-adrenergic agonist phenylephrine, and by electric field stimulation. This may be explained by cytoskeletal deorganization, which was observed by phalloidin staining of actin filaments in response to AZM475271 and PP2 in WPMY-1 cells. This was paralleled by reduced proliferation of WPMY-1 cells, as indicated by EdU assay. Cytotoxicity was mainly observed at higher concentrations (50 mM or more) and longer incubation, but was minor at 10 mM for 24 h. CONCLUSIONS: Synchronized regulation of smooth muscle tone and growth by SFK-dependent control may explain their concomitant contributions to bladder outlet obstruction in the hyperplastic prostate. Targeting smooth muscle contraction and growth in the prostate at once by single agents may be principally possible.

De novo steroid biosynthesis in human prostate cell lines and biopsies

Intratumoral androgen formation may be a factor in the development of prostate cancer (PCa), particularly castration-resistant prostate cancer (CRPC). To evaluate the ability of the human prostate to synthesize de novo steroids, we examined the expression of key enzymes and proteins involved in steroid biosynthesis and metabolism. Using TissueScan™ Cancer qPCR Arrays and quantitative RT-PCR, we performed comparative gene expression analyses between various prostate cell lines and biopsies, including normal, hyperplastic, cancerous, and androgen-deprived prostate cells lines, as well as normal, benign prostate hyperplasia (BPH), PCa, and CRPC human specimens. These studies were complemented with steroid biosynthesis studies in normal and BPH cells. Normal human prostate WPMY-1 and WPE1-NA22, benign prostate hyperplasia BPH-1, and cancer PC-3, LNCaP, and VCaP cell lines, as well as normal, BPH, PCa, and CRPC specimens, were used. Although all cell lines express mRNA encoding for hydroxymethylglutaryl-CoA reductase (HMGCR), the mitochondrial translocator protein TSPO and cholesterol side chain cleavage enzyme CYP11A1 were only observed in WPMY-1, BPH-1, and LNCaP cells. HSD3B1, HSD3B2, and CYP17A1 are involved in androgen formation and were not found in most cell lines. WPE1-NA22 and BPH-1 cells were unable to synthesize de novo steroids from mevalonate. Moreover, androgen-deprived cells did not have alterations in the expression of enzymes that could lead to de novo steroid formation. All prostate specimens expressed TSPO and CYP11A1. HSD3B1/2, CYP17A1, HSD17B5, and CYP19A1 mRNA expression was distinct to the profile observed in cells lines. The majority of BPH (90.9%) and PCa (83.1%) specimens contained CYP17A1, compared to control (normal) specimens (46.7%). BPH (82%), PCa (59%), normal (40%), and CRPC (34%) specimens expressed the four key enzymes that metabolize cholesterol to androgens. These studies question the use of prostate cell lines to study steroid biosynthesis and demonstrate that human prostate samples contain transcripts encoding for key steroidogenic enzymes and proteins indicating that they have the potential to synthesize de novo steroids. We propose CYP17A1 as a candidate enzyme that can be used for patient stratification and treatment in BPH and PCa.

Hypoxia-induced autophagy promotes human prostate stromal cells survival and ER-stress

Benign prostatic hyperplasia (BPH) is emerging as one of the most common diseases seriously threatening the health of elderly men. Accumulating evidences indicate that hypoxia could induce BPH. However, the underlying mechanism of BPH induced by hypoxia is not clear. In the study, hypoxia-induced autophagy could promote cell survival and endoplasmic reticula stress (ER stress) in WPMY-1 cells. Cell viability induced by hypoxia could been decreased by autophagy inhibitors (3-methyladenine, bafilomycin A1) or siRNA interference in two autophagy genes (Beclin1, ATG5) in WPMY-1 cells. Furthermore, ER stress was present in hypoxia-treated WPMY-1 cells, while autophagy and cell survival could been inhibited by C/EBP-homologous protein siRNA (CHOP), which is an important protein of ER stress pathway. Taken together, our data support a novel model that autophagy as a cytoprotective response promotes cell survival via ER stress under hypoxia in human prostate stromal cells.

Inhibition of prostate smooth muscle contraction and prostate stromal cell growth by the inhibitors of Rac, NSC23766 and EHT1864.

Medical therapy of lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH) targets smooth muscle contraction in the prostate, or prostate growth. However, current therapeutic options are insufficient. Here, we investigated the role of Rac in the control of smooth muscle tone in human prostates and growth of prostate stromal cells. Experiments were performed using human prostate tissues from radical prostatectomy and cultured stromal cells (WPMY-1). Expression of Rac was examined by Western blot and fluorescence staining. Effects of Rac inhibitors (NSC23766 and EHT1864) on contractility were assessed in the organ bath. The effects of Rac inhibitors were assessed by pull-down, cytotoxicity using a cell counting kit, cytoskeletal organization by phalloidin staining and cell growth using an 5-ethynyl-2'-deoxyuridine assay. Expression of Rac1-3 was observed in prostate samples from each patient. Immunoreactivity for Rac1-3 was observed in the stroma, where it colocalized with the smooth muscle marker, calponin. NSC23766 and EHT1864 significantly reduced contractions of prostate strips induced by noradrenaline, phenylephrine or electrical field stimulation. NSC23766 and EHT1864 inhibited Rac activity in WPMY-1 cells. Survival of WPMY-1 cells ranged between 64 and 81% after incubation with NSC23766 (50 or 100 μM) or EHT1864 (25 μM) for 24 h. NSC23766 and EHT1864 induced cytoskeletal disorganization in WPMY-1 cells. Both inhibitors impaired the growth of WPMY-1 cells. Rac may be a link connecting the control of prostate smooth muscle tone with proliferation of smooth muscle cells. Improvements in LUTS suggestive of BPH by Rac inhibitors appears possible.

The therapeutic effects of docosahexaenoic acid on oestrogen/androgen-induced benign prostatic hyperplasia in rats

Benign prostatic hyperplasia (BPH) is one of the major disorders of the urinary system in elderly men. Docosahexaenoic acid (DHA) is the main component of n-3 polyunsaturated fatty acids (n-3 PUFAs) and has nerve protective, anti-inflammatory and tumour-growth inhibitory effects. Here, the therapeutic potential of DHA in treating BPH was investigated. Seal oil effectively prevented the development of prostatic hyperplasia induced by oestradiol/testosterone in a rat model by suppressing the increase of the prostatic index (PI), reducing the thickness of the peri-glandular smooth muscle layer, inhibiting the proliferation of both prostate epithelial and stromal cells, and downregulating the expression of androgen receptor (AR) and oestrogen receptor α (ERα). An in vitro study showed that DHA inhibited the growth of the human prostate stromal cell line WPMY-1 and the epithelial cell line RWPE-1 in a dose- and time-dependent manner. In both cell lines, the DHA arrested the cell cycle in the G2/M phase. In addition, DHA also reduced the expression of ERα and AR in the WPMY-1 and RWPE-1 cells. These results indicate that DHA inhibits the multiplication of prostate stromal and epithelial cells through a mechanism that may involve cell cycle arrest and the downregulation of ERα and AR expression.

MP37-04 ANDROGEN RECEPTOR POSITIVE STROMAL CELLS REGULATE PROSTATE CANCER PROLIFERATION THROUGH NON-CANONICAL WNT SIGNALING

You have accessJournal of UrologyProstate Cancer: Basic Research I1 Apr 2015MP37-04 ANDROGEN RECEPTOR POSITIVE STROMAL CELLS REGULATE PROSTATE CANCER PROLIFERATION THROUGH NON-CANONICAL WNT SIGNALING Sayuri Takahashi, Ichiro Takada, Naoki Terada, Yukio Homma, and Robert H. Getzenberg Sayuri TakahashiSayuri Takahashi More articles by this author , Ichiro TakadaIchiro Takada More articles by this author , Naoki TeradaNaoki Terada More articles by this author , Yukio HommaYukio Homma More articles by this author , and Robert H. GetzenbergRobert H. Getzenberg More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1267AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The Wnt signaling pathway (canonical and non-canonical) regulates crucial aspects of embryonic development. Previously, we have reported that non-canonical Wnt members promote proliferation of cancer cells by increasing secretion of growth factors and expression of cancer related genes. We have also demonstrated that Wnt5a is predominantly expressed in prostatic stromal cells and plays a key role in stromal-epithelial communication and in the development of prostate cancer. The purpose of this study is to reveal the relation between the non-canonical Wnt signaling and AR signaling on prostate. METHODS Prostate tissue samples were obtained from patients undergoing radical prostatectomy or cystectomy for prostate and bladder cancer respectively. Cells, representing the cancer, stromal from around the cancer, as well as epithelium and stromal from pathologically normal tissues were isolated by laser capture microdissection Stromal cells (WPMY1 and PrSC) were cultured with DHT ligand and protein. The mRNA levels of Wnt5a, AR, and PSA were determined in both sample types. Wnt5a-knockdown in WPMY1 cells (shWnt5a) along with a vector control (shCTL) was performed and effects on growth evaluated by MTT assay. The cells were co-cultured with PC3 cells, and the mRNA expression of AR was evaluated. A DNA fragment upstream of the Wnt5a gene was inserted into pGL4.17 a vector and the luciferase activity representing Wnt5a inductionby DHT was measured. RESULTS Wnt5a mRNA expression was prediminanly in the stromal cells of the human prostate samples and the expression levels observed were in proportion to PSA mRNA expression levels. DHT increased production of AR as well as PSA and Wnt5a mRNA expression in both WPMY1 and PrSC cells. Wnt5 knockdown decreased the proliferation of WPMY1cells by 50% compared to shCTR. Co-culture of PC3 cells and WPMY1shWnt5a formed fewer and smaller sized colonies than co-culture of PC3 cells and WPMY1shCTR. AR and PSA mRNA expression levels were remarkably lower in WPMY1shWnt5a than in the control. DHT ligand dramatically increased the luciferase activity of Wnt5a promoter in WPMY1 cells. CONCLUSIONS The AR is expressed not only in prostatic epithelium but also prostatic stromal as measured by their response to DHT. In stromal cells, The studies presented here demonstrated an interplay between AR signaling and Wnt5a that appears to modulate the proliferation of prostate cancer cells providing further support for the importance of Wnt5a in the regulation of prostate cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e439 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sayuri Takahashi More articles by this author Ichiro Takada More articles by this author Naoki Terada More articles by this author Yukio Homma More articles by this author Robert H. Getzenberg More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

In search of the molecular mechanisms mediating the inhibitory effect of the GnRH antagonist degarelix on human prostate cell growth.

Degarelix is a gonadrotropin-releasing hormone (GnRH) receptor (GnRHR) antagonist used in patients with prostate cancer who need androgen deprivation therapy. GnRHRs have been found in extra-pituitary tissues, including prostate, which may be affected by the GnRH and GnRH analogues used in therapy. The direct effect of degarelix on human prostate cell growth was evaluated. Normal prostate myofibroblast WPMY-1 and epithelial WPE1-NA22 cells, benign prostatic hyperplasia (BPH)-1 cells, androgen-independent PC-3 and androgen-dependent LNCaP prostate cancer cells, as well as VCaP cells derived from a patient with castration-resistant prostate cancer were used. Discriminatory protein and lipid fingerprints of normal, hyperplastic, and cancer cells were generated by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The investigated cell lines express GNRHR1 and GNRHR2 and their endogenous ligands. Degarelix treatment reduced cell viability in all prostate cell lines tested, with the exception of the PC-3 cells; this can be attributed to increased apoptosis, as indicated by increased caspase 3/7, 8 and 9 levels. WPE1-NA22, BPH-1, LNCaP, and VCaP cell viability was not affected by treatment with the GnRH agonists leuprolide and goserelin. Using MALDI MS, we detected changes in m/z signals that were robust enough to create a complete discriminatory profile induced by degarelix. Transcriptomic analysis of BPH-1 cells provided a global map of genes affected by degarelix and indicated that the biological processes affected were related to cell growth, G-coupled receptors, the mitogen-activated protein kinase (MAPK) pathway, angiogenesis and cell adhesion. Taken together, these data demonstrate that (i) the GnRH antagonist degarelix exerts a direct effect on prostate cell growth through apoptosis; (ii) MALDI MS analysis provided a basis to fingerprint degarelix-treated prostate cells; and (iii) the clusters of genes affected by degarelix suggest that this compound, in addition to its known use in the treatment of prostate cancer, may be efficacious in BPH.

278 Androgen receptor positive stromal cells regulate prostate cancer proliferation through noncanonical Wnt signaling

INTRODUCTION AND OBJECTIVES: The Wnt signaling pathway (canonical and non-canonical) regulates crucial aspects of embryonic development. Previously, we have reported that non-canonical Wnt members promote proliferation of cancer cells by increasing secretion of growth factors and expression of cancer related genes. We have also demonstrated that Wnt5a is predominantly expressed in prostatic stromal cells and plays a key role in stromalepithelial communication and in the development of prostate cancer. The purpose of this study is to reveal the relation between the noncanonical Wnt signaling and AR signaling on prostate. METHODS: Prostate tissue samples were obtained from patients undergoing radical prostatectomy or cystectomy for prostate and bladder cancer respectively. Cells, representing the cancer, stromal from around the cancer, as well as epithelium and stromal from pathologically normal tissues were isolated by laser capture microdissection Stromal cells (WPMY1 and PrSC) were cultured with DHT ligand and protein. The mRNA levels of Wnt5a, AR, and PSA were determined in both sample types. Wnt5a-knockdown in WPMY1 cells (shWnt5a) along with a vector control (shCTL) was performed and effects on growth evaluated by MTT assay. The cells were co-cultured with PC3 cells, and the mRNA expression of AR was evaluated. A DNA fragment upstream of the Wnt5a gene was inserted into pGL4.17 a vector and the luciferase activity representing Wnt5a inductionby DHT was measured. RESULTS: Wnt5a mRNA expression was prediminanly in the stromal cells of the human prostate samples and the expression levels observed were in proportion to PSA mRNA expression levels. DHT increased production of AR as well as PSA and Wnt5a mRNA expression in both WPMY1 and PrSC cells. Wnt5 knockdown decreased the proliferation of WPMY1cells by 50% compared to shCTR. Co-culture of PC3 cells and WPMY1shWnt5a formed fewer and smaller sized colonies than co-culture of PC3 cells and WPMY1shCTR. AR and PSA mRNA expression levels were remarkably lower in WPMY1shWnt5a than in the control. DHT ligand dramatically increased the luciferase activity of Wnt5a promoter in WPMY1 cells. CONCLUSIONS: The AR is expressed not only in prostatic epithelium but also prostatic stromal as measured by their response to DHT. In stromal cells, The studies presented here demonstrated an interplay between AR signaling and Wnt5a that appears to modulate the proliferation of prostate cancer cells providing further support for the importance of Wnt5a in the regulation of prostate cancer.

Cooperative effects of EGF, FGF, and TGF-β1 in prostate stromal cells are different from responses to single growth factors

AimsStromal growth is critical for prostate enlargement during benign prostatic hyperplasia (BPH). While responses of prostate cells to single growth factors have been well characterized, responses to multiple growth factors at once are poorly understood. Here, we examined the effects of combinations between epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor-β1 (TGF-β1) in human prostate stromal cells. Main methodsEGF, FGF, and TGF-β1 were applied to WPMY-1 cells, an immortalized, non-malignant line of stromal cells from the human prostate. Hypertrophic responses were assessed by protein/DNA ratio, and cyclin D1 mRNA by RT-PCR. Expression of EGF, FGF, and TGF-β1 and their receptors in human prostate tissue was analyzed by RT-PCR, Western blot, and fluorescence staining. Key findingsHypertrophic responses to single growth factors and combinations were similar. Combinations showed additive effects on cyclin D1 mRNA. Combination of EGF with TGF-β1, but not EGF or TGF-β1 alone, caused assembly of cells to a new two-dimensional structure, being characterized by dense aggregates connected by branches of few cells. EGF and TGF-β1 were detected together in human prostates. Receptors for EGF and TGF-β colocalized on stromal cells in human prostates. SignificanceResponses of prostate stromal cells to combinations of EGF, FGF, and TGF-β1 may be quantitatively different, qualitatively different, or similar to responses to single growth factors. The combination of EGF and TGF-β1, but not EGF or TGF-β1 alone, induces aggregation of prostate stromal cells, which may be relevant for morphogenesis.

Abstract C39: Docetaxel-induced growth and cell cycle phenotypes in prostate stromal cells

Abstract Background: Docetaxel (D) is a clinically used front-line chemotherapeutic agent for castration resistant advanced prostate cancer. However, its long-term benefits are limited with most patients eventually progressing because of inherent or acquired drug resistance. The treatment of advanced prostate cancer is a significant challenge and there are no effective treatments that stably suppress the disease. The mechanisms behind resistance to Docetaxel are not fully understood. Since in vivo Docetaxel cytotoxicity is directed not only to cancer cells, we hypothesize that the response of prostate stromal cells exposed to chemotherapeutic agents could modify drug response or therapeutic outcome in prostate cancer. One of cellular responses to chemotherapy is NF-kB activation and inflammatory signaling. NF-κB activation has been proposed as an important event in PCa development, castration-resistant progression and resistance to chemotherapy. Objective: In this study, we examined the response of prostate stromal (WPMY- 1) cells to Docetaxel. Current Results: Cellular proliferation was analyzed by MTT assay. The IC50 of Docetaxel for WPMY-1 cells was 10nM. Cell cycle profiles were assessed by flow cytometry. The analysis showed that Docetaxel (10-100nM) induced prominent G2-M arrest in these stromal cells within 24 hours, especially in the higher dose ranges. Interestingly lower doses of Docetaxel (10nM), in contrast to higher doses, consistently resulted in accumulation of sub-G1 population (28% 10nM vs 6% in 100nM) of cells suggestive of fragmented DNA associated with apoptotic process. We show that Docetaxel exhibits strong anti-proliferative effects on prostate stromal WPMY-1 cells. Conclusions: These preliminary results show that stromal cells are sensitive to Docetaxel. Why lower doses of the drug induce sub-G1 (apoptotic) accumulation while higher doses preferentially induce G2-M arrest is currently unknown. Future Steps: We will compare responses to D between prostate cancer cells and stromal cells in individual and co-culture models. NF-kB activity following D treatment or radiation, and the expression of genes in NF-kB pathway in D treated cells will be examined. Citation Format: Dominique Gales, Brionna McMcmeans, Temesgen Samuel. Docetaxel-induced growth and cell cycle phenotypes in prostate stromal cells. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C39. doi:10.1158/1538-7755.DISP13-C39

The prevention and treatment effects of tanshinone IIA on oestrogen/androgen-induced benign prostatic hyperplasia in rats

Benign prostatic hyperplasia (BPH) is one of the major diseases of the urinary system in elderly men. Tanshinone IIA (Tan IIA) is the active ingredient extracted from the traditional Chinese medicine Salvia, and it has effects of anti-oxidation, anti-inflammation, vascular smooth muscle relaxation and tumour growth inhibition. The present study aimed to investigate the therapeutic potential of Tan IIA in the prevention and treatment of BPH. In a rat model of oestradiol/testosterone-induced BPH, Tan IIA inhibited the increase in the thickness of the peri-glandular smooth muscle layer, suppressed the expression of proliferating cell nuclear antigen (PCNA) in both prostate epithelial cells and stromal cells, downregulated the expression of androgen receptor (AR), oestrogen receptor α (ERα), cyclin B1 (CCNB1) and cyclin D1 (CCND1), and effectively prevented the development of the disorder. In vitro, Tan IIA inhibited the proliferation of human prostate stromal cell line WPMY-1 and epithelial cell line RWPE-1 in a dose- and time-dependent manner. In WPMY-1 cells, Tan IIA treatment arrested the cell cycle at the G2/M phase and downregulated the expression of CCNB1. However, in RWPE-1 cells, Tan IIA treatment arrested cell cycle at the G0/G1 phase and reduced the expression of CCND1. Tan IIA also reduced the expression of ERα and AR in WPMY-1 and RWPE-1 cells. These results suggest that Tan IIA can inhibit the growth of prostate stromal and epithelial cells both in vivo and in vitro by a mechanism that may involve arresting the cell cycle and downregulating ERα and AR expression.

Honokiol, a constituent of Magnolia species, inhibits adrenergic contraction of human prostate strips and induces stromal cell death.

PurposeSmooth muscle contraction and prostate growth are important targets for medical therapy of lower urinary tract symptoms (LUTS) in patients with benign prostatic hyperplasia. Honokiol and Magnolol are lignan constituents of Magnolia species, which are used in traditional Asian medicine. Here, we examined effects of honokiol and magnolol on contraction of human prostate tissue and on growth of stromal cells.MethodsProstate tissues were obtained from radical prostatectomy. Contraction of prostate strips was examined in organ bath studies. Effects in stromal cells were assessed in cultured immortalized human prostate stromal cells (WPMY-1). Ki-67 mRNA was assessed by reverse transcription-polymerase chain reaction, and proliferation by a fluorescence 5-ethynyl-2′-deoxyuridine assay.ResultsHonokiol (100μM) reduced noradrenaline-induced contractions, which was significant at 10 to 100μM noradrenaline. Honokiol reduced phenylephrine-induced contractions, which was significant at 3 to 100μM phenylephrine. Honokiol reduced electric field stimulation-induced contractions very slightly. In WPMY-1 cells, honokiol (24 hours) induced cell death. Magnolol (100μM) was without effects on contraction, and cellular viability.ConclusionsHonokiol inhibits smooth muscle contraction in the human prostate, and induces cell death in cultured stromal cells. Because prostate smooth muscle tone and prostate growth may cause LUTS, it appears possible that honokiol improves voiding symptoms.

VDR activity is differentially affected by Hic-5 in prostate cancer and stromal cells.

Patients with prostate cancer treated with androgen deprivation therapy (ADT) eventually develop castrate-resistant prostate cancer (CRPC). 1,25-Dihydroxyvitamin D3 (1,25D3/calcitriol) is a potential adjuvant therapy that confers antiproliferative and pro-differentiation effects in vitro, but has had mixed results in clinical trials. The impact of the tumor microenvironment on 1,25D3 therapy in patients with CRPC has not been assessed. Transforming growth factor β (TGFβ), which is associated with the development of tumorigenic "reactive stroma" in prostate cancer, induced vitamin D3 receptor (VDR) expression in the human WPMY-1 prostate stromal cell line. Similarly, TGFβ enhanced 1,25D3-induced upregulation of CYP24A1, which metabolizes 1,25D3 and thereby limits VDR activity. Ablation of Hic-5, a TGFβ-inducible nuclear receptor coregulator, inhibited basal VDR expression, 1,25D3-induced CYP24A1 expression and metabolism of 1,25D3 and TGFβ-enhanced CYP24A1 expression. A Hic-5-responsive sequence was identified upstream (392-451 bp) of the CYP24A1 transcription start site that is occupied by VDR only in the presence of Hic-5. Ectopic expression of Hic-5 sensitized LNCaP prostate tumor cells to growth-inhibitory effects of 1,25D3 independent of CYP24A1. The sensitivity of Hic-5-expressing LNCaP cells to 1,25D3-induced growth inhibition was accentuated in coculture with Hic-5-ablated WPMY-1 cells. Therefore, these findings indicate that the search for mechanisms to sensitize prostate cancer cells to the antiproliferative effects of VDR ligands needs to account for the impact of VDR activity in the tumor microenvironment. Hic-5 acts as a coregulator with distinct effects on VDR transactivation, in prostate cancer and stromal cells, and may exert diverse effects on adjuvant therapy designed to exploit VDR activity in prostate cancer.