Malignant Human Prostate Tissue Research Articles

Nuclear beta-catenin as a marker of outcome in localized prostate cancer

9570 Background: Beta-catenin, in its role as a nuclear signaling molecule, has been implicated in prostate carcinogenesis through modulation of androgen receptor activity. Mice in whom beta-catenin is overexpressed in the nuclei of prostatic epithelium develop hyperplastic and dysplastic lesions. Hence this study focused on nuclear beta-catenin expression and aimed to define the pattern of beta-catenin protein expression in the nuclei of normal, hyperplastic and malignant human prostate tissue and to determine whether changes in expression were associated with disease progression and/or prognosis. Methods: Five normal prostates, 26 benign prostatic hypertrophy specimens, 232 radical prostatectomy specimens from patients with clinically localized prostate cancer (PC), 186 specimens of hyperplasia adjacent to PC and 20 cases of advanced PC were assessed for beta-catenin expression using immunohistochemistry. Results: There were significantly lower levels of nuclear beta-catenin expression in localized PC compared to benign prostate tissue (p<0.001). Nuclear beta-catenin expression was also lower in advanced compared to localized PC (p<0.001). In addition, lower levels of nuclear beta-catenin expression (immunoreactivity in <10% of malignant cells) predicted for a shorter biochemical relapse-free survival in patients with localized PC (HR 1.9, 95%CI 1.2–3.0; p=0.008) and were inversely correlated with pre-operative prostate-specific antigen (PSA) levels (p=0.01). Analysis of a low-risk subgroup of patients with pre-operative PSA levels <10 ng/ml (n=114), demonstrated that lower levels of nuclear beta-catenin (<10% of malignant cells) again predicted for a poorer prognosis (HR 3.2, 95%CI 1.4–7.3; p=0.006). Conclusions: Lower levels of nuclear beta-catenin expression are found in malignant compared to benign prostate tissue. Lower nuclear beta-catenin expression is also associated with a poorer prognosis in localized PC, in particular in the low-risk subgroup of patients with pre-operative PSA levels <10 ng/ml. Consequently, nuclear beta-catenin expression may be of clinical utility as a pre-operative prognostic marker in patients with low-risk PC. No significant financial relationships to disclose.

Inhibition of Telomerase Activity and Secretion of Prostate Specific Antigen by Silibinin in Prostate Cancer Cells

The androgen sensitive prostate cancer cell line LNCaP is strongly positive for dihydrotestosterone (DHT) dependent telomerase activity, which is an important factor in cellular immortality and carcinogenesis. In this study we determined the potential of silibinin as an anticancer drug that down-regulates telomerase activity and prostate specific antigen (PSA) together with the co-activator of the androgen receptor prostate epithelium specific Ets transcription factor. LNCaP cells were treated with various concentrations of silibinin in the presence or absence of 5alpha-DHT. We used real-time reverse transcriptase-polymerase chain reaction to quantify mRNA expression of PSA, prostate epithelium specific Ets transcription factor and the catalytic subunit of telomerase vs the housekeeping gene porphobilinogen deaminase with gene specific, dual labeled fluorescence probes. PSA secretion from LNCaP cells in conditioned medium was measured with an Elecsys System 2010 (Roche Diagnostics, Mannheim, Germany) and telomerase activity in extracts from LNCaP cells was measured with a TRAP (telomeric repeat amplification protocol) assay. Silibinin down-regulated PSA mRNA expression and PSA secretion in conditioned medium. Simultaneous stimulation with silibinin and 10(-8) M DHT also resulted in PSA down-regulation, whereas DHT alone increased PSA secretion. Telomerase catalytic subunit mRNA decreased significantly after silibinin stimulation. Telomerase activity was down-regulated by silibinin and stimulated by DHT. The 2 agents in combination resulted in telomerase down-regulation. The down-regulation of PSA by silibinin and its counteraction on DHT effects indicate that this compound can interact with the expression of genes that are regulated through the androgen receptor. Silibinin can also inhibit the telomerase activity that mediates cell immortality and carcinogenesis. The 2 effects underline the possible therapeutic use of silibinin as an antiproliferative agent in intervention for prostate cancer.

Expression of PTEN in malignant and non-malignant human prostate tissues: comparison with p27 protein expression.

The role of the putative tumour suppressor PTEN in prostate carcinogenesis is controversial. There are conflicting data regarding the rate of its gene inactivation, the role of transcriptional and post-transcriptional factors, as well as its relationship to tumour progression and to the potential downstream regulator, the cell-cycle inhibitor p27. The present study has assessed the in situ expression of PTEN mRNA and protein in 26 prostate intraepithelial neoplasias (PINs), 58 primary prostate carcinomas, and 15 metastases. Although there was a correlation between PTEN mRNA and protein expression, mRNA detection exceeded detection of protein in 19% of PINs and 30% of all invasive tumours. Using RT-PCR and western blotting on microdissected tissue, this discrepancy was attributed, at least in part, to transcription of the PTEN pseudo-gene, which lacks introns. Total or partial loss of PTEN protein occurred with tumour progression but this association was not statistically significant. Analysing the relationship between PTEN and p27 protein expression on consecutive sections by immunohistochemistry, the results do not support a direct link between the two oncosuppressors, other than an associated loss of expression in advanced tumour stages. However, in the basal cells of prostate glands and in most PINs, an inverse relationship was observed between PTEN and p27. This may reflect the existence of a functional balance that controls the cell cycle in prostatic epithelium and that is probably disturbed in invasive tumour cells.

Membranous expression of secreted frizzled-related protein 4 predicts for good prognosis in localized prostate cancer and inhibits PC3 cellular proliferation in vitro.

Activation of the Wnt-signaling pathway is implicated in aberrant cellular proliferation in a variety of cancers. Secreted frizzled-related protein 4 (sFRP4) is a secreted protein with putative inhibitory activity of the Wnt-signaling cascade through binding and sequestering Wnt ligands. Because sFRP4 mRNA is overexpressed in prostate cancers (PCs), the aim of this study was to define the pattern of sFRP4 protein expression in normal and malignant human prostate tissue and to determine whether changes in expression were associated with disease progression and prognosis, as well as to define the phenotype of sFRP4-overexpression in an in vitro model of PC. Polyclonal antibodies were raised against a COOH-terminal peptide of sFRP4, characterized and used to assess sFRP4 protein expression in benign prostate tissue and 229 patients with clinically localized PC (median follow-up 77 months, range 1-156). In vitro studies of the function of sFRP4 overexpression were performed using PC3 cells transfected with sFRP4. Benign and malignant prostate tissue demonstrated cytoplasmic sFRP4 immunoreactivity, but there was a decrease in the expression of membranous sFRP4 in PCs compared with the hyperplastic lesions (P < 0.0001). Kaplan-Meier analysis revealed that patients whose PC expressed membranous sFRP4 in >20% of cells had improved relapse-free survival compared with those with </=20% membranous expression (P = 0.002). Moreover, membranous sFRP4 expression (P = 0.04) was an independent predictor of relapse when modeled with Gleason score (P = 0.006), pathological stage (P = 0.002), and pre-operative prostate-specific antigen levels (P = 0.004). In addition, in vitro studies demonstrated a decrease in the proliferation rate of PC3 cells transfected with sFRP4 when compared with the control PC3-empty vector cells (P < 0.0001). Decreased levels of phosphorylated glycogen synthase kinase 3beta in PC3-sFRP4 cells suggested that this phenotype is mediated by the "Wnt/beta-catenin" pathway. These data suggest that sFRP4 expression may be prognostic for localized PC, potentially as a consequence of an inhibitory effect on PC cell proliferation.

Cell specific expression of CD10/neutral endopeptidase 24.11 gene in human prostatic tissue and cells.

Neutral endopeptidase (NEP/CD10) is a cell surface zinc metalloproteinase that functions as part of a regulatory loop controlling local concentrations of peptide substrates and associated peptide-mediated signal transduction processes. In contrast to the encouraging data dealing with NEP activity and regulation in prostate epithelial cells, only a few studies are available on the cellular expression and localization of neutral endopeptidase in the prostatic stromal and cancer cells. Here, we describe the cellular localization of NEP in human prostatic tissue and cells using in situ RT-PCR as a novel molecular biological approach. Immunofluorescence and Western blot experiments were performed to control the expression and distribution of the NEP in normal and malignant human prostatic tissues and cell lines. NEP gene expression was monitored by RT-PCR, NEP mRNA was detected in paraffin tissue sections and cultured cells of human prostate by the highly sensitive method of one step-in situ reverse transcriptase-polymerase chain reaction (RT-PCR). NEP mRNA was detected in human prostatic tissue and in cultured cells by means of in situ RT-PCR. Prostatic tissue showed strong signals in the glandular epithelium and weak signals in the stroma, cultured cells displayed strong signals in prostate cancer cells (LNCaP) and weak signals in stromal cells (hPCPs). Western blot experiments were performed using whole cell extracts to proof the presence of NEP protein in LNCaP and hPCPs. The experiments confirm the expression of NEP by both cell types, however, the experiment with hPCPs cells showed two bands. NEP-immunofluorescence was strong in normal prostatic epithelium and confined to the apical plasma membrane. In dedifferentiated prostate cancer specimens, immunofluorescence of apical plasma membranes was lost, and both the cytoplasm and portions of the plasma membrane were immunoreactive for NEP. Prostate cancer cells (LNCaP) showed a strong immunoreaction of the plasma membrane and the cytoplasm. In comparison with LNCaP cells, only a weak cytoplasmic immunofluorescence was found in some stromal cells (hPCPs). In normal prostatic tissue and specimens derived from human prostate cancer, NEP mRNA and protein are expressed mainly by the epithelial cells and to a minor extent by the stromal cells of human prostate glands. In situ RT-PCR is a powerful and straightforward approach for the routine and rapid detection of cellular specific expression of low copy genes.

Immunohistochemical expression of hepatocyte growth factor and c-Met/HGF receptor in benign and malignant human prostate tissue

Hepatocyte growth factor (HGF) has been proposed to be an autocrine/paracrine growth factor for carcinomas of various organs. We recently demonstrated that HGF produced by prostate-derived stromal cells was a paracrine growth factor that stimulated the growth of androgen-independent prostate cancer cells in vitro and in vivo. To assess possible involvement of HGF in prostate cancer, we examined the immunohistochemical expression and localization of HGF and c-Met/HGF receptor in benign and malignant human prostate tissues. In benign glands, columnar cells generally were negative for c-Met, but basal cells were stained uniformly at a high level. In high-grade prostatic intraepithelial neoplasia (PIN) and carcinoma, more than 50% of these foci were stained uniformly. There was no difference in the frequency of positively stained cells by Gleason score. The prostate stroma stained diffusely for HGF, and the staining intensity varied depending upon the amounts of smooth-muscle cells that were stained more intensely than the connective-tissue matrix. A great majority of benign columnar cells were negative for HGF whereas high-grade PIN and carcinoma foci stained focally for HGF. Hormonal ablation therapy prior to prostatectomy did not seem to alter the expression of HGF/c-Met in carcinoma cells. These results indicate that, as the degree of neoplasia progresses, epithelial cells begin to express c-Met protein, that PIN and carcinoma may have developed a c-Met-HGF paracrine loop with the stroma, and that in some carcinoma foci an autocrine loop may operate with HGF expressed by carcinoma cells themselves.

Stat3 activation in prostatic carcinomas.

Activated Stat3 is found in various types of immortal cell lines and cancers. We and others have previously demonstrated that Stat3 is constitutively activated in rat and human prostate cancer cell lines, and that Stat3 activation is involved in IL-6-mediated signaling transduction in prostate cancer cells. The aim of this study is to examine quantitative Stat3 activity in benign and malignant human prostate tissues and analyze the association between Stat3 activity levels and the clinical and pathologic parameters. Stat3 activity levels were analyzed in a total of 104 human primary prostate tissues using electromobility shift assay and immunohistochemical staining for phosphorylated Stat3. The tissue samples used were 42 prostate carcinomas, 42 matched normal prostate tissues from patients with prostatic adenocarcinoma (normal adjacent to tumor), and 20 normal prostate tissues from organ donors. Significantly higher levels of constitutive Stat3 activity were detected in both prostate carcinomas and the matched normal prostate tissues adjacent to tumors compared to the normal prostates from donors without prostate cancer. There was no significant difference of Stat3 activity in foci of tumor and normal prostate tissue adjacent to tumor. No correlation was seen between Stat3 activity and Gleason grade or serum PSA levels in samples from prostate carcinomas. These results indicate that Stat3 is constitutively activated in prostate cancer. The high level of Stat3 activity in both the prostate carcinomas and the normal prostate tissues adjacent to tumors suggests that Stat3 activation may occur before detectable histological alterations of the prostate.

PURIFICATION OF A HUMAN PROSTATE SPECIFIC ANTIGEN

No AccessJournal of UrologyCLINICAL UROLOGY: Milestone in Urology1 Mar 2002PURIFICATION OF A HUMAN PROSTATE SPECIFIC ANTIGEN1 M.C. WANG, L.A. VALENZUELA, G.P. MURPHY, and T.M. CHU M.C. WANGM.C. WANG More articles by this author , L.A. VALENZUELAL.A. VALENZUELA More articles by this author , G.P. MURPHYG.P. MURPHY More articles by this author , and T.M. CHUT.M. CHU Send requests for reprints to Dr. T. M. Chu, Roswell Park Memorial Institute, Buffalo, New York 14263.Submitted for publication March 26, 1979; accepted June 7, 1979. More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(05)65270-6AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Rabbit antiserum raised against the crude extract of normal human prostatic tissue contained antibodies to a prostatic tissue-specific antigen as shown by immunoprecipitation techniques. Using this antiserum a prostate antigen was detected in normal, benign hypertrophic, and malignant prostatic tissues, but not in other human tissues. The prostate antigen was purified to homogeneity from prostatic tissues and showed a single protein band on analytical polyacrylamide gel electrophoresis and isoelectric focusing. This report thus presents the first demonstration of the purification of a prostate-specific antigen that does not represent prostatic acid phosphatase. Department of Diagnostic Immunology Research and Biochemistry, Roswell Park Memorial Institute, Buffalo, New York (Reprinted with permission from Invest. Urol, 17: 159–163, 1979) References 1 : Carcinoma of the prostate in elderly men: incidence, growth characteristics and clinical significance.. J. Urol.1969; 101: 602. Abstract, Google Scholar 2 : An “acid” phosphatase occurring in the serum of patients with metastasizing carcinoma of the prostate gland.. J. Clin. Invest.1938; 17: 473. Google Scholar 3 : Studies on the antigenic properties of prostate tissue I.. J. Urol.1960; 84: 134. Abstract, Google Scholar 4 : Tissue- and species-specific antigens of normal human prostatic tissue.. J. Immunol.1970; 104: 1329. Google Scholar 5 : Immunologic studies of normal, benign and malignant human prostatic tissue.. Cancer1972; 29: 1570. Google Scholar 6 : Investigation of specific antigens in prostate cancer.. Cancer Chemother. Rep.1975; 59: 105. Google Scholar 7 : Tissue specific and tumor specific antigens in human prostate.. Fed. Proc.1977; 36: 1254. Google Scholar 8 : Isolation of Wilm’s tumor antigens by chelation.. Clin. Chim. Acta1975; 61: 411. Google Scholar 9 : Enzyme markers in human prostatic carcinoma.. Cancer Treat. Rep.1977; 61: 193. Google Scholar 10 : Immunochemical detection of serum prostatic acid phosphatase. Methodology and clinical evaluation.. Invest. Urol.1978; 15: 319. Google Scholar 11 : A procedure to increase the sensitivity of staining by Coomassie brilliant blue R250-perchloric acid solution.. Anal. Biochem.1976; 75: 634. Google Scholar 12 : The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis.. J. Biol. Chem.1969; 244: 4406. Google Scholar 13 Weeke B. General remarks on principles, equipment, reagents and procedures. In A Manual of Quantitative Immunoelectrophoresis. Methods and Applications, p. 15, N. H. Axelson, J. Krøll, and B. Weeke (Editors). Universitetsforlaget, Oslo, 1973. Google Scholar 14 : Protein measurement with the Folin phenol reagent.. J. Biol. Chem.1951; 193: 265. Google Scholar 15 : An improved acid phosphatase procedure.. Clin. Chim. Acta1966; 13: 264. Google Scholar 16 : Purification and characterization of a human prostate tissue-specific antigen.. Fed. Proc.1979; 38: 1069. Google Scholar © 2002 by American Urological Association, Inc.FiguresReferencesRelatedDetails Volume 167Issue 3March 2002Page: 1226-1230 Advertisement Copyright & Permissions© 2002 by American Urological Association, Inc.AcknowledgmentThe authors thank J. Ogledzinski for her help in preparing this manuscript.MetricsAuthor Information M.C. WANG More articles by this author L.A. VALENZUELA More articles by this author G.P. MURPHY More articles by this author T.M. CHU Send requests for reprints to Dr. T. M. Chu, Roswell Park Memorial Institute, Buffalo, New York 14263.Submitted for publication March 26, 1979; accepted June 7, 1979. More articles by this author Expand All Advertisement PDF downloadLoading ...

Differential Expression of Estrogen Receptor β (ERβ) and Its C-Terminal Truncated Splice Variant ERβcx as Prognostic Predictors in Human Prostatic Cancer

Estrogens have been widely used for the treatment of advanced prostatic adenocarcinoma. However, their direct effect to prostatic cancer cells via estrogen receptors remains unclear. We investigated expression of ERα, wild-type ERβ (wtERβ), and a C-terminal truncated splice variant of ERβ (ERβcx) in 50 benign and 100 malignant human prostatic tissue samples by immunohistochemistry. While strong immunostaining of ERα was consistently identified in the stromal compartment, wtERβ was expressed in epithelial cells in both the benign and malignant foci. However, wtERβ expression was significantly lower in the cancers than in the benign epithelium and inversely correlated with Gleason tumor grade (P < 0.0001 and P = 0.0099, respectively). In contrast, ERβcx was significantly more expressed in the high-grade cancers (83%) compared with the low-grade tumors (22%) and the benign sites (11%) (P < 0.0001, both). Cancer-specific survival of patients with lower wtERβ expression was significantly worse than those with higher expression of wtERβ (P = 0.0018). Conversely, higher ERβcx expression significantly correlated with poor cancer-specific survival (P = 0.0058). These results suggest that differential expressions of wtERβ and ERβcx may be prognostic predictors for prostatic cancer.

Castration-induced reduction of vascular endothelial growth factor expression in benign human prostate tissue is lost in advanced prostate cancer.

To determine the role of vascular response in the castration-induced regression of benign and malignant human prostate tissue, as recent studies show that castration rapidly decreases blood flow and induces endothelial cell death, which may be important for subsequent epithelial cell death and involution of the glandular tissue of the prostate. The expression of vascular endothelial growth factor (VEGF) and its receptors was analysed using the quantitative reverse transcriptase-polymerase chain reaction, in benign and tumour areas of core biopsies taken before, and approximately 1 week after castration therapy. The castration-induced VEGF response was related to therapy-induced changes in tumour cell apoptotic index and subsequent response in serum prostate-specific antigen (PSA). In another set of patients, serum VEGF was quantified by enzyme-linked immunosorbent assay before, and at 3--6 months after castration therapy. VEGF mRNA was down-regulated after castration in benign prostate tissue (P < or = 0.05), whereas in tumour tissue, VEGF levels were reduced in some of the patients but unchanged or increased in others. In most patients whose tumour tissue responded with VEGF reduction, there was a corresponding increase in tumour cell apoptosis. Serum VEGF levels were not significantly changed after castration. Almost all patients responded with a substantial reduction in serum PSA after castration. Castration reduces VEGF mRNA expression in benign prostate tissue and generally in those prostate tumours where castration also induces tumour cell apoptosis. This suggests that a therapy-induced down-regulation of VEGF could be important for tumour cell death.

Profiling and Verification of Gene Expression Patterns in Normal and Malignant Human Prostate Tissues by cDNA Microarray Analysis

cDNA microarray technology allows the ‘profiling’ of gene expression patterns for virtually any cellular material. In this study, we applied cDNA microarray technology to profile changes in gene expression associated with human prostate tumorigenesis. RNA prepared from normal and malignant prostate tissue was examined for the expression levels of 588 human genes. Four different methods for data normalization were utilized. Of these, normalization to ACTB expression proved to be the most rigorous technique with the least probability of producing spurious results. After normalization to ACTB expression, 15 of 588 (2.6%) genes examined by array analysis were differentially expressed by a factor of 2x or more in malignant compared to normal prostate tissues. The expression patterns for 8 of 15 genes have been reported previously in prostate tissues (TGFβ3, TGFBR3, IGFII, IGFBP2, VEGF, FGF7, ERBB3, MYC), but those of seven genes are reported here for the first time (MLH1, CYP1B1, RFC4, EPHB3, MGST1, BTEB2, MLP). These genes describe at least four metabolic and signaling pathways likely disrupted in human prostate tumorigenesis. Reverse transcriptase polymerase chain reaction (RTPCR) and Northern blot analyses quantitated with reference to ACTB expression levels verified the trends in gene expression levels observed by array analysis for 14/15 and 8/8 genes, respectively. However, RT-PCR and Northern blot analyses accurately verified the ‘fold’ differences in expression levels for only 6/15 (40%) and 7/8 (88%) of genes examined, respectively, demonstrating the need to better validate quantitative differences in gene expression revealed by array-based techniques.

Identification and characterization of neutral endopeptidase (EC 3. 4. 24. 11) from human prostasomes--localization in prostatic tissue and cell lines.

An antibody directed against a 100 kDa protein was immunoselected from a polyvalent antiserum against human prostasomes. The antibody as well as biochemical characteristics of the respective antigen were used to study the structural relationship of the latter with prostate membrane specific antigen (PMSA), another 100 kDa membrane protein of the prostate. The isolated purified 100 kDa protein was characterized by tryptic degradation, aminoacid-sequencing and mass spectroscopy peptide-fingerprinting as well as mono-saccharide analysis and lectin binding and identified as a prostasomal neutral endopeptidase (NEP, EC 3.4.24.11). Immunohistochemistry, immunoelectron microscopy, in situ hybridization, and RT-PCR were performed to analyze the expression and distribution of the protein in normal and malignant human prostatic tissues and cell lines. Prostatic NEP, which has no relationship with PMSA, is a glycosylated, integral membrane protein type II. The prevalent glycosyl residues are NeuNAc, GlcNAc, GalNAc, Gal, Man, Fuc. NEP-mRNA is expressed in human prostatic epithelial and some stromal cells. NEP-immunoreactivity is strong in normal prostatic epithelium and confined to the apical plasma membrane. During apocrine secretion, the enzyme is released from the secretory cells, contributing to the formation of prostasomes. In prostate cancer specimens, immunoreactivity of apical plasma membranes is lost, while generalized cytoplasmic immunoreactivity develops. Prostatic secretory cells contain a membrane-bound, highly glycosylated neutral endopeptidase which is restricted to the apical plasma membrane. The enzyme is released from the cells in an apocrine fashion and contributes to the formation of prostasomes. In prostate cancer cells a preferential cytoplasmic localization is observed, pointing to alterations in intracellular targeting.

Overexpression and regulation of expression of scatter factor/hepatocyte growth factor in prostatic carcinoma

Objectives. Scatter factor (hepatocyte growth factor) (SF/HGF) is a multifunctional polypeptide growth factor that has been implicated in tumor proliferation, angiogenesis, invasiveness, and metastasis. Little is known of the expression of SF/HGF in human prostatic carcinoma. The aims of this investigation were to quantitate the level of SF/HGF expression in benign versus malignant human prostatic tissues and to assess regulation of SF/HGF expression by human prostatic stromal myofibroblasts. Methods. We determined the level of SF/HGF expression in 10 human prostatic tissue samples (5 benign, 5 carcinoma) by Western blot analysis. Five purified growth factors—basic fibroblast growth factor (bFGF), interleukin-1beta (IL-1β), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and endothelial growth factor (EGF)—were tested for their capacity to induce SF/HGF expression by a human prostatic stromal myofibroblastic cell line, as assessed by enzyme-linked immunosorbent assay. Supernatant from the normal PrEC prostatic epithelial cell line and the DU 145 carcinoma cell line were assayed for SF/HGF-inducing activity. Results. SF/HGF exhibited a mean fourfold overexpression in carcinoma tissues compared with benign prostatic tissue. Significant stimulation of SF/HGF expression by prostatic stromal myofibroblasts was detected for IL-1β (8.1-fold), PDGF (6.2-fold), bFGF (4.0-fold), VEGF (3.7-fold), and EGF (2.9-fold). DU 145-conditioned media, but not the PrEC-conditioned media, contained SF/HGF-inducing activity, which was determined to include IL-1β, bFGF, and PDGF by antibody-blocking experiments. Conclusions. SF/HGF is overexpressed in human prostatic carcinoma tissues. Prostatic carcinoma cell stimulation of SF/HGF expression by adjacent benign myofibroblastic cells as a type of epithelial–stromal paracrine interaction could potentially influence prostatic carcinoma cell behaviors.

Rational basis for Trk inhibition therapy for prostate cancer.

Prostatic cancer cells are lethal because they acquire the ability to activate survival pathways that do not require androgenic stimulation. As a rational approach to developing effective therapy for these devastating cells, specific signal transduction pathways uniquely required for the survival of these nonandrogen-dependent prostate cancer cells must be identified. Previous studies suggested that the neurotrophin/trk signal transduction axis may regulate such unique survival pathways. In the present study, the changes in expression of the neurotrophins (NGF, BDNF, and NT-3) and their cognate receptors (i.e., trk and p75NTR) during the progression of normal prostatic epithelial cells to malignancy were documented. Additionally, the consequences of inhibiting these trk signaling pathways on the in vitro survival of prostate cancer cells was tested. Immmunocytochemistry, RT-PCR, and ELISA assays were used to characterize the changes in the neurotrophin ligands (i.e., NGF, BDNF, and NT-3) and their cognate high-affinity (i.e., trk A, B, and C) and low-affinity neurotrophin (i.e., p75 NTR) receptors in normal vs. malignant human prostatic tissues. CEP-751 is an indolocarbazole compound specifically designed to inhibit the initiation of these neurotrophin/trk signal transductions. The consequence of CEP-751 inhibition of trk signaling for in vitro clonogenic survival of a series of human prostatic cancer lines was also tested. These studies demonstrated that normal prostatic tissue from patients without prostate cancer contains substantial levels of nerve growth factor (NGF), which is produced in a paracrine manner by stromal cells. These stromal cells lack both trk and p75NTR receptors. In contrast, normal prostatic epithelial cells from patients without prostate cancer do not secrete detectable levels of neurotrophins, but do express trk A and p75 NTR. While the NGF/trkA/p75 NTR axis is present in the normal prostate, normal prostatic epithelial cells do not depend on this axis for their survival. In contrast, malignant prostate epithelial cells directly secrete a series of neurotrophins (i.e., NGF, BDNF, and/or NT-3) and express at least one if not more of the trk receptor proteins (i.e., trk A, B, and/or C), while no longer expressing the p75NTR receptors. In addition, inhibition of autocrine trk signaling via CEP-751 treatment induces the apoptotic death of these malignant cells. Prostate carcinogenesis involves molecular changes leading to the paracrine and/or autocrine production of a series of neurotrophins. This is coupled to the ectopic expression of trk B and trk C, as well as to the continued expression of trk A, and the loss of expression of p75NTR receptors. These changes result in the acquisition by malignant prostate cells of a unique requirement for trk signaling pathways for survival. Based on these findings, trk inhibition is a novel, rational approach for prostate cancer therapy.

Rational basis for Trk inhibition therapy for prostate cancer

Prostatic cancer cells are lethal because they acquire the ability to activate survival pathways that do not require androgenic stimulation. As a rational approach to developing effective therapy for these devastating cells, specific signal transduction pathways uniquely required for the survival of these nonandrogen-dependent prostate cancer cells must be identified. Previous studies suggested that the neurotrophin/trk signal transduction axis may regulate such unique survival pathways. In the present study, the changes in expression of the neurotrophins (NGF, BDNF, and NT-3) and their cognate receptors (i.e., trk and p75NTR) during the progression of normal prostatic epithelial cells to malignancy were documented. Additionally, the consequences of inhibiting these trk signaling pathways on the in vitro survival of prostate cancer cells was tested.Immmunocytochemistry, RT-PCR, and ELISA assays were used to characterize the changes in the neurotrophin ligands (i.e., NGF, BDNF, and NT-3) and their cognate high-affinity (i.e., trk A, B, and C) and low-affinity neurotrophin (i.e., p75 NTR) receptors in normal vs. malignant human prostatic tissues. CEP-751 is an indolocarbazole compound specifically designed to inhibit the initiation of these neurotrophin/trk signal transductions. The consequence of CEP-751 inhibition of trk signaling for in vitro clonogenic survival of a series of human prostatic cancer lines was also tested.These studies demonstrated that normal prostatic tissue from patients without prostate cancer contains substantial levels of nerve growth factor (NGF), which is produced in a paracrine manner by stromal cells. These stromal cells lack both trk and p75NTR receptors. In contrast, normal prostatic epithelial cells from patients without prostate cancer do not secrete detectable levels of neurotrophins, but do express trk A and p75 NTR. While the NGF/trkA/p75 NTR axis is present in the normal prostate, normal prostatic epithelial cells do not depend on this axis for their survival. In contrast, malignant prostate epithelial cells directly secrete a series of neurotrophins (i.e., NGF, BDNF, and/or NT-3) and express at least one if not more of the trk receptor proteins (i.e., trk A, B, and/or C), while no longer expressing the p75NTR receptors. In addition, inhibition of autocrine trk signaling via CEP-751 treatment induces the apoptotic death of these malignant cells.Prostate carcinogenesis involves molecular changes leading to the paracrine and/or autocrine production of a series of neurotrophins. This is coupled to the ectopic expression of trk B and trk C, as well as to the continued expression of trk A, and the loss of expression of p75NTR receptors. These changes result in the acquisition by malignant prostate cells of a unique requirement for trk signaling pathways for survival. Based on these findings, trk inhibition is a novel, rational approach for prostate cancer therapy.

Expression and localization of aminopeptidase A, aminopeptidase N, and dipeptidyl peptidase IV in benign and malignant human prostate tissue

Cell-surface peptidases are ectoenzymes which regulate the access of bioactive peptides to their receptors on cell membranes. Abnormalities in their expression and function result in altered peptide activity which contribute to neoplastic transformation and/or progression. Expression of aminopeptidase A (APA), aminopeptidase N (APN, CD13), and dipeptidyl peptidase IV (DPP IV, CD26) was immunohistochemically examined in 20 benign and 33 malignant prostate tissues (19 primaries and 14 metastases). Benign prostatic stroma exhibited no APA, APN, or DPP IV immunoreactivity. Stromal cells surrounding prostatic carcinoma cells demonstrated increased APA expression in 24/33 (73%) of tumors. Benign prostatic epithelial cells strongly expressed APN and DPP IV but not APA. In contrast, APN was expressed in > 80% of tumor cells in 5/33 (15%) of specimens, heterogeneously expressed (20-80% of cells positive) in 4/33 (12%) of specimens, and minimally expressed or absent in 24/33 (73%) of tumor specimens, with a similar pattern of expression in primary and metastatic tumors. DPP IV was expressed by > 80% of tumor cells in 18/19 (95%) of primary prostate cancer specimens, but in only 7/14 (50%) of metastases. These data show that cell-surface peptidases are differentially expressed by normal prostatic stromal and epithelial cells, with increased expression of APA in the stroma surrounding prostate cancer cells, absent APN expression in most tumor cells, and a decreased frequency of DPP IV expression in metastatic tumors. Further studies will elucidate the biological effects of the presence or loss of cell-surface peptidases in the benign and malignant prostate.

Expression and localization of aminopeptidase A, aminopeptidase N, and dipeptidyl peptidase IV in benign and malignant human prostate tissue

Expression and localization of aminopeptidase A, aminopeptidase N, and dipeptidyl peptidase IV in benign and malignant human prostate tissue

Vascular Endothelial Growth Factor (VEGF) Expression in Prostate Cancer and Benign Prostatic Hyperplasia

Purpose Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial cell growth and is expressed at elevated levels in several tumor types. In this study immunohistochemical localization and distribution of isoforms of VEGF were examined in malignant and non-malignant human prostatic tissues. Materials and Methods Immunohistochemical localization of VEGF was performed on thirty well, moderately and poorly differentiated stage D 2 prostate cancer specimens and twenty benign prostatic hyperplasia (BPH) specimens. VEGF mRNA was determined by polymerase chain reaction and VEGF protein isoforms were detected by Western blotting of prostate cancer and BPH specimens. Results Cytoplasmic immunoreactivity for VEGF was detected in tumor cells and peritumoral stromal cells of prostate cancer specimens and in non-malignant glandular epithelial cells and interglandular stromal cells in BPH specimens. Staining was focal with areas of strongly to weakly stained cells adjacent to negatively staining areas. mRNA's for VEGF 121, VEGF sub 165 and VEGF 189 were present in all benign and malignant prostate specimens. VEGF protein isoforms of molecular sizes corresponding to VEGF 165 and VEGF 189 were detected in cytosolic extracts of prostate cancers and BPH specimens by Western blotting. In addition, two novel higher molecular weight immunoreactive bands were detected in the prostate specimens. Conclusions Widespread distribution of VEGF in prostate cancers and BPH specimens suggest that the VEGF 165, VEGF 189 isoforms and novel 90 and 110 kD forms detected may contribute to the establishment or progression of these conditions.

Parathyroid hormone-related protein is not an autocrine growth factor for normal prostatic epithelial cells.

Parathyroid hormone-related protein (PTHrP) is the primary factor responsible for humoral hypercalcemia of malignancy. The hypercalcemic actions of PTHrP occur via stimulation of renal distal tubular calcium reabsorption and increased osteoclastic bone resorption. These effects of PTHrP are thought to be mediated through a common parathyroid hormone (PTH)/PTHrP receptor. In addition to the well-established role of PTHrP in bone remodeling, PTHrP is believed to be an important mediator of cellular growth and differentiation in a number of nonbony tissues. We recently demonstrated abundant expression of PTHrP in normal and malignant human prostatic tissues, and in cultured prostatic epithelial cells. In vitro assays were used to test growth-regulatory activity of synthetic and endogenous PTHrP peptides on normal prostatic epithelial cells. No growth-regulatory activity could be demonstrated. PTHrP is not an autocrine growth factor for normal prostatic epithelial cells.

Antioxidant Enzymes In Malignant Prostate Cell Lines and In Primary Cultured Prostatic Cells

The antioxidant enzymes catalase, glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were determined in the androgen-responsive LNCaP and androgen-nonresponsive PC-3 and DU 145 cells as well as in prostatic epithelial cell cultures of benign and malignant human prostatic tissue. There were no differences between the enzyme activities of the human primary cell cultures from cancerous tissue and their normal counterparts. The enzyme activities of the three permanent cell lines were either higher (SOD, catalase, GR) or lower (GST, GPx) than in the primary cell cultures. In LNCaP cells catalase and GR were significantly higher, GST, in contrast, was significantly lower than in PC-3 and DU 145 cells. GST in PC-3 and DU 145 cells, and SOD in all the three cell lines showed no significant differences. Catalase, GPx and GR values were significantly different in the three permanant cell lines. The different enzymatic equipment of the prostate cancer cell lines provides the basis for experimental testing of new concepts of cancer treatment with the help of systematic modulations of the antioxidant defence systems in prostate cancer.