adrenomedullin and Prostatic-Neoplasms

Adrenomedullin (ADM) is a 52-amino acid peptide with structural homology to calcitonin gene-related peptide (CGRP) initially isolated from human pheochromocytoma. ADM is synthesized and is secreted from many mammalian tissues, including the adrenal medulla, endothelial and vascular smooth muscle cells, as well as the myocardium and central nervous system. ADM has been implicated as a mediator of several diseases such as cardiovascular and renal disorders, sepsis, inflammation, diabetes and cancer. ADM is also expressed in a variety of tumors, including breast, endometrial and prostate cancer. ADM has been shown to be a mitogenic factor capable of stimulating growth of several cancer cell types. In addition, ADM is a survival factor for certain cancer cells and an indirect suppressor of the immune response. ADM plays an important role in environments subjected to low oxygen tension, which is a typical feature of solid tumors. Under these conditions, ADM is up regulated and acts as a potent angiogenic factor promoting neovascularization. The major focus of this review will be on the role of ADM in cancer, with emphasis on its utility in diagnostic and prognostic terms, along with its relevance as a therapeutic target.

Topics: Adrenomedullin; Animals; Disease Progression; Endometrial Neoplasms; Female; Humans; Male; Neoplasms; Neovascularization, Physiologic; Ovarian Neoplasms; Pancreatic Neoplasms; Prognosis; Prostatic Neoplasms; Protein Biosynthesis; Receptors, Adrenomedullin; Receptors, Peptide; Signal Transduction

There is increasing evidence for the important role played by regulatory peptides in the physiology of the normal and neoplastic prostate. Adrenomedullin (AM) and pro-adrenomedullin N-terminal 20 peptide (PAMP) are recently discovered regulatory peptides widely expressed in the normal prostate and in prostate carcinoma. AM is produced in secretory, stroma, and endothelial cells and in neurons of the prostate ganglia. PAMP is only produced by neuroendocrine cells. The expression of AM mRNA is regulated by androgens in the rat prostate. The number of neuroendocrine cells expressing PAMP is increased in prostate carcinoma after androgen deprivation, which shows that this peptide could regulate androgen-independent prostate tumor growth. However, the roles of AM and PAMP in the normal prostate and in prostate carcinoma are yet to be elucidated.

Topics: Adrenomedullin; Animals; Carcinoma; Humans; Immunohistochemistry; Male; Peptide Fragments; Peptides; Prostate; Prostatic Neoplasms; Proteins; Rats

Extra-cellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK) signaling pathway is widely involved in cell proliferation, apoptosis, and drug resistance. MAPK kinase 1 (MEK1) is the upstream protein kinase of ERK that can activate ERK/MAPK signaling pathway. microRNA 203 (MiR-203) down-regulation is found to be associated with prostate cancer pathogenesis. Bioinformatics analysis showed the complementary targeted relationship between miR-203 and the 3'-UTR of MEK1 mRNA. This study explored the role of miR-203 in regulating prostate cancer cell proliferation, apoptosis, and ADM resistance through affecting MEK1 expression.. Dual luciferase assay confirmed the targeted relationship between miR-203 and MEK1. MiR-203, MEK1, p-ERK1/2, and B cell lymphoma 2 (Bcl-2) expressions were compared in normal prostate epithelial cells PrEC, prostate cancer cells PC-3M, and drug resistance cells PC-3M/ADM. PC-3M, PC-3M/ADM cell apoptosis and proliferation were detected by using flow cytometry under ADM treatment at IC50 concentration of PC-3M cells. PC-3M cells were cultured in vitro and divided into four groups, including microRNA-normal control (miR-NC), miR-203 mimic, small interfere NC (si-NC), and si-MEK1.. MiR-203 targeted and inhibited MEK1 expression. MiR-203 levels and cell apoptosis were significantly lower, while MEK1, p-ERK1/2, Bcl-2, and cell proliferation were significantly higher in PC-3M/ADM cells compared to the PC-3M cells. MiR-203 mimic and/or si-MEK1 transfection significantly reduced MEK1, p-ERK1/2, and Bcl-2 levels, attenuated cell proliferation, induced cell apoptosis, and decreased drug resistance.. MiR-203 elevation suppressed prostate cancer PC-3M cell proliferation, promoted apoptosis, and weakened ADM resistance through targeted inhibiting MEK1 expression to alleviate ERK/MAPK signaling pathway and Bcl-2 expression.

Topics: Adrenomedullin; Apoptosis; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Male; MAP Kinase Signaling System; MicroRNAs; Prostatic Neoplasms

Adrenomedullin (AM) is a 52-amino acid peptide initially isolated from human pheochromocytoma. AM is expressed in a variety of malignant tissues and cancer cell lines and was shown to be a mitogenic factor capable of stimulating growth of several cancer cell types. In addition, AM is a survival factor for certain cancer cells. Some data suggest that AM might be involved in the progression cancer metastasis via angiogenesis and cell migration and invasion control. The Transient Receptor Potential channel TRPV2 is known to promote in prostate cancer cell migration and invasive phenotype and is correlated with the stage and grade of bladder cancer. In this work we show that AM induces prostate and urothelial cancer cell migration and invasion through TRPV2 translocation to plasma membrane and the subsequent increase in resting calcium level.

Topics: Adrenomedullin; Cell Adhesion; Cell Line, Tumor; Cell Membrane; Cell Movement; Humans; Male; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Prostatic Neoplasms; Protein Transport; Signal Transduction; TRPV Cation Channels; Urethral Neoplasms

Mutations in the androgen receptor (AR) have been detected in experimental and clinical prostate tumors. Mice with enforced prostate-specific expression of one such receptor variant, AR-E231G, invariably develop prostatic intraepithelial neoplasia by 12 weeks and metastatic prostate cancer by 52 weeks. The aim of this study was to identify genes with altered expression in the prostates of AR-E231G mice at an early stage of disease that may act as drivers of AR-mediated tumorigenesis. The gene expression profile of AR-E231G prostate tissue from 12-week-old mice was compared to an equivalent profile from mice expressing the AR-T857A receptor variant (analogous to the AR-T877A variant in LNCaP cells), which do not develop prostate tumors. One hundred and thirty-two genes were differentially expressed in AR-E231G prostates. Classification of these genes revealed enrichment for cellular pathways known to be involved in prostate cancer, including cell cycle and lipid metabolism. Suppression of two genes upregulated in the AR-E231G model, ADM and CITED1, increased cell death and reduced proliferation of human prostate cancer cells. Many genes differentially expressed in AR-E231G prostates are also deregulated in human tumors. Three of these genes, ID4, NR2F1 and PTGDS, which were expressed at consistently lower levels in clinical prostate cancer compared to nonmalignant tissues, formed a signature that predicted biochemical relapse (hazard ratio 2.2, p = 0.038). We believe that our findings support the value of this novel mouse model of prostate cancer to identify candidate therapeutic targets and/or biomarkers of human disease.

Topics: Adrenomedullin; Animals; Apoptosis Regulatory Proteins; Biomarkers, Tumor; Cell Line, Tumor; COUP Transcription Factor I; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Differentiation Proteins; Intramolecular Oxidoreductases; Lipocalins; Male; Mice; Mice, Transgenic; Mutation; Nuclear Proteins; Prognosis; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Receptors, Androgen; RNA Interference; RNA, Small Interfering; Trans-Activators

Interleukin-13 (IL-13) receptor alpha2 (IL-13Ralpha2), a high-affinity IL-13 binding subunit and a tumor antigen, is amplified in a variety of human tumor cell lines and tumors in vivo. By cDNA microarray, we have shown that gene transfer of human and rat adrenomedullin (AM) up-regulates IL-13Ralpha2 in a human prostate tumor cell line. Here, we show that IL-13Ralpha2 mRNA and protein are also up-regulated in PC-3 prostate tumor cells by recombinant AM (rAM) and human synthetic AM peptide in a dose-dependent manner in vitro and in vivo in mouse prostate tumor model. The 8- to 10-fold up-regulation of IL-13Ralpha2 by rAM or AM peptide in prostate tumor cells in vitro and in vivo increased their sensitivity to IL-13PE cytotoxin consisting of IL-13 and a truncated form of Pseudomonas exotoxin. Immunodeficient mice with established prostate tumors transfected with AM or treated with AM peptide showed reduction in tumor size by intratumoral administration of IL-13PE in a dose-dependent manner. At the highest dose (three 100 mug/kg/d every alternate day), >70% reduction of tumor size was observed compared with controls (P

Topics: ADP Ribose Transferases; Adrenomedullin; Animals; Bacterial Toxins; Cell Line, Tumor; Exotoxins; Gene Expression Regulation, Neoplastic; Humans; Immunotoxins; Interleukin-13; Interleukin-13 Receptor alpha2 Subunit; Male; Mice; Prostatic Neoplasms; Pseudomonas aeruginosa Exotoxin A; RNA, Messenger; Up-Regulation; Virulence Factors

Neuroendocrine (NE) differentiation in prostate cancer (CaP) has been reported to be an early marker associated with the development of androgen independence. The mechanisms by which CaP acquires NE properties are poorly understood. In this study, a putative role of adrenomedullin (AM) in the NE differentiation was investigated. The expression of AM and AM receptors (calcitonin receptor-like receptor (CRLR)/receptor activity modifying protein-2 and -3 (RAMP2 and RAMP3) was evaluated after experimental manipulation of androgen status. Levels of AM mRNA and immunoreactive AM (ir-AM) increased four- to sevenfold in androgen-sensitive LNCaP cells after androgen withdrawal in vitro and in LNCaP xenografts in animals after castration. Treatment of LNCaP cells with androgen analogue (dihydrotestosterone; 10(-9) M) prevented the increase in AM mRNA and ir-AM levels. Interestingly, the expression of CRLR, RAMP2 and RAMP3 is not regulated by androgen status. We demonstrate that in the presence of serum, AM is able to induce an NE phenotype in LNCaP cells via CRLR/RAMP2 and RAMP3, which includes extension of neuritic processes and expression of the neuron-specific enolase (NSE), producing cGMP in a dose-dependent manner, which is mediated by a pertussis toxin-sensitive GTP-binding protein. 8-Bromo-cGMP mimicked the effects of AM on cell differentiation. We demonstrate that AM induces a G-kinase Ialpha translocation to the nucleus. The protein kinase G inhibitor KT-5823 inhibited the neurite outgrowth induced by both AM and 8-bromo-cGMP. In noncastrated animals, administration of AM enhanced expression of NSE and chromogranin A in LNCaP xenografts with a significant increase of NSE levels in serum and no changes in tumor growth. In castrated animals, intraperitoneal injection of AM resulted in a 240+/-18% (P<0.001) increase in tumor volume 36 days after treatment, indicating that the nature of effect of AM in CaP depends on the presence or absence of endogenous androgen. Together, these results demonstrate that AM may function as a mediator of NE-like differentiation in culture as well as in vivo and indicate that its production may be important for tumor resurgence following androgen ablation.

Topics: Adrenomedullin; Androgens; Animals; Antineoplastic Agents, Hormonal; Autocrine Communication; Biomarkers, Tumor; Carcinoma, Neuroendocrine; Cell Differentiation; Cell Proliferation; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; Neoplasms, Hormone-Dependent; Paracrine Communication; Phenotype; Prostatic Neoplasms; Receptors, Adrenomedullin; Receptors, Peptide; Tumor Cells, Cultured; Withholding Treatment; Xenograft Model Antitumor Assays

The 52-aminoacid peptide adrenomedullin (AM) is expressed in the normal and malignant prostate. We have previously shown that prostate cancer cells produce and secrete AM, which acts as an autocrine growth inhibitory factor. We have evaluated in the present study the role of AM in prostate cancer cell apoptosis, induced either by serum deprivation or treatment with the chemotherapeutic agent etoposide (which acts as an inhibitor of topoisomerase II). For this purpose we over-expressed AM in PC-3, DU 145 and LNCaP cells, which were transfected with an expression vector carrying AM. We also treated the parental cell lines with synthetic AM in normal culture conditions and in conditions of induced-apoptosis. After serum removal, AM prevented apoptosis in DU 145 and PC-3 cells, but not in LNCaP cells. When treated with etoposide, AM prevented apoptosis in PC-3 and LNCaP cells, but not in DU 145 cells. Cell cycle analysis demonstrated a significant decrease in the percentage of AM-overexpressing PC-3 cells in the subG0/G1 phase after treatment with etoposide, as compared to the percentage of mock-transfected PC-3 treated cells. Western blot showed that protein levels of phosphorylated ERK1/2 increased in parental PC-3 cells after treatment with etoposide. In PC-3 cells overexpressing AM, phosphorylated ERK1/2 basal levels were lower than basal levels of parental PC-3 cells, and treatment with etoposide did not result in such an increase. Etoposide produced a significant increase in cleaved PARP in parental PC-3 cells. However, PC-3 clones overexpressing AM that were treated with etoposide only showed a mild increase in fragmented PARP. The ratio Bcl-2/Bax was reduced in parental or mock-transfected PC-3 cells after treatment with etoposide. On the contrary, this ratio was not reduced in PC-3 clones with AM overexpression that were treated with etoposide. All these data demonstrate that AM plays a protective role against induced apoptosis in prostate cancer cells. These results may have important implications in prostate cancer resistance to chemotherapeutic agents.

Topics: Adrenomedullin; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Cell Proliferation; Drug Resistance, Neoplasm; Etoposide; Humans; Male; Mitogen-Activated Protein Kinase 3; Peptides; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Transfection; Tumor Cells, Cultured

Human adrenomedullin (AM) is a 52 amino acid peptide, which shares homology with the calcitonin gene-related peptide. Overexpression of AM in the prostate carcinoma cell line PC-3 results in growth inhibition with a 20% (for human AM) and 35% (for rat AM) increase in doubling time compared to parental or mock-transfected cells. We demonstrate by gene expression profiling that AM overexpression results in the dysregulation of approximately 100 genes. Examples of such genes include many involved in the formation of the cytoskeleton, cell adhesion and the extracellular matrix, as well as regulators of the cell cycle and apoptosis, cytokines and transcription factors. Several genes related to cell growth arrest, such as GADD45, IGF-BP6 and RUNX-3, are upregulated by AM. Interestingly, interleukin-13 receptor alpha 2 (IL-13R alpha 2) transcripts were significantly increased in clones overexpressing AM, which was confirmed by semiquantitative RT-PCR analysis. In addition, PC-3 cells treated with AM showed an overexpression of IL-13R alpha 2, which was abolished when cells were preincubated with an anti-AM blocking antibody. When PC-3 cells overexpressing AM and the IL-13R alpha 2 were treated with the highly specific IL13-PE38 cytotoxin, which binds to this receptor, a concentration-dependent inhibition of protein synthesis was observed. The IC(50) (concentration of cytotoxin inhibiting protein synthesis by 50%) ranged from 1 to 4 ng/ml. This cytotoxicity was specific as it was neutralized by the excess of IL-13 and confirmed by clonogenic assays. This study describes a novel AM-induced mechanism of tumor sensitization through the upregulation of functional IL-13R alpha 2 chain, an ideal target for the highly specific recombinant chimeric cytotoxin IL13-PE38.

Topics: Adrenomedullin; Apoptosis; Cell Cycle; Gene Expression Profiling; Humans; Interleukin-13 Receptor alpha1 Subunit; Male; Oligonucleotide Array Sequence Analysis; Peptides; Prostatic Neoplasms; Receptors, Interleukin; Receptors, Interleukin-13; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured; Up-Regulation

After castration or therapeutic hormone deprivation, most cancer of the prostate (CaP) cells develop androgen-independent (AI) growth. In this work, we studied the effect of androgen depletion (castration) on the growth of experimental model LuCaP 23.1 xenograft. A total of 101 nude mice were implanted and analysed for their growth profile before experimental period 1 (11 weeks) and after castration experimental period 2 (15 weeks). For specific periods, tumors were harvested and assessed for molecular marker expression specific for CaP. Taking into account tumor dynamic growth, prior to castration we found 37 fast growing (FG) tumors (948.9+/-76.9 mm3) and 63 slow growing (SG) tumors (229.6+/-18.4 mm3). Real-time quantitative RT-PCR showed that in comparison to SGs, FGs contained elevated expression of epidermal growth factor receptor type 1 (HER1), urokinase plasminogen activator (uPA), thymidine phosphorylase (TP) and thymidilate synthase (TS) mRNAs expression and low levels of 5alpha-reductase 2 (5alpha-R2) mRNA. After castration all FG tumors progressed rapidly (by 5 weeks) to AI growth (FG-P). In SG castrated tumors, 66% of tumors showed retarded progression (by 12 weeks) to AI (SG-P), whereas 34% responded to castration (SG-R). Molecular analysis demonstrated distinct molecular profiles integrating different pathways associated with AI progression. The progressive tumors FG-P, and some tumors of SG-P subgroup, presented significantly high levels of HER1, epidermal growth factor receptor type 2 (HER2), TS, uPA, TP, tumor necrosis factor superfamily member 6 (FAS) and peptidylglycine alpha-amidating mono-oxygenase (PAM) mRNA all of which correlated with androgen receptor (AR) mRNA. The second subgroup of SG-P tumors showed a high expression of the anti-apoptotic gene Bcl-2. A third subgroup of SG-P tumors showed significant expression of hypoxia-related genes such as adrenomedullin (AM) after castration. LuCaP 23.1 xenograft represent a useful dynamic model to study pre-clinically new therapeutic molecules and evaluate non-randomized therapeutics protocols combining different target inhibition specific to each AI pathways.

Topics: Adrenomedullin; Androgens; Animals; Antimicrobial Cationic Peptides; Castration; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Peptides; Prostate-Specific Antigen; Prostatic Neoplasms; Thymidine Phosphorylase; Transplantation, Heterologous; Tumor Necrosis Factors; Vascular Endothelial Growth Factor A

Adrenomedullin (AM) is a multifunctional peptide expressed in the normal and malignant prostate, and in prostate cancer cells. To elucidate the potential role of AM in prostate cancer, we have transfected the human AM gene into PC-3, DU 145, and LNCaP prostate cancer cells. Northern blot, Western blot, and radioimmunoassay techniques confirmed an increase in the synthesis and secretion of the 6kDa mature peptide, in the AM-transfected clones. Proliferation and cell cycle assays demonstrated that AM overexpression inhibited cell proliferation in PC-3 and LNCaP cells through a G0/G1 cell cycle arrest, but not in DU 145 cells. In vivo growth assays also confirmed that, at least in PC-3, AM produced a very significant reduction of tumor volume. In addition, the three cell lines expressed the CL/RCP/RAMP-2 receptor complex by RT-PCR, which suggests that AM peptide acts through an autocrine loop in prostate cancer cells. Although cAMP elevation is the most common pathway involved in AM signalling, stimulation of PC-3, DU 145, and LNCaP with synthetic AM did not increase intracellular cAMP. However, short-term stimulation of PC-3 cells with synthetic AM increased ERK1/2 activation. On the contrary, long-term stimulation, or AM overexpression, caused a reduction in the basal activation of ERK1/2. In summary, our results demonstrate that AM (either overexpressed or exogenously added) causes an inhibition of prostate cancer cell growth. This inhibition does not depend on changes in intracellular cAMP levels, but may be related to ERK1/2 activation.

Topics: Adrenomedullin; Base Sequence; Cell Division; Cell Line, Tumor; Cyclic AMP; DNA Primers; Humans; Kinetics; Male; Peptides; Polymerase Chain Reaction; Prostatic Neoplasms; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transfection

Adrenomedullin (AM) is a hypotensive peptide, that acts via the calcitonin receptor-like receptor (CRLR), whose interaction with the subtypes 2 and 3 of a family of receptor activity-modifying proteins (RAMP) gives rise to two distinct AM receptors, named AM1 and AM2 receptors. AM derives from the post-translational proteolytic cleavage of pro(p)AM, the last step of which involves the conversion of the inactive AM to active AM by the peptidyl-glycine alpha-amidating monooxigenase (PAM). Compelling evidence suggests that AM, in addition to exerting its well-known regulatory action on blood pressure and water and electrolyte balance, also possesses a growth promoting effect in several normal and neoplastic tissues, including human prostate. Conventional reverse transcription (RT)-polymerase chain reaction (PCR) demonstrated the expression of pAM, PAM, CRLR and RAMP(1-3) mRNAs in both prostate hyperplasias (PH) and carcinomas (PC), and semiquantitative PCR showed that pAM, PAM and RAMP3 mRNA expression was higher in PCs than PHs. Radioimmunoassay measured higher concentrations of immunoreactive AM in PCs than PHs. The expression of pAM, CRLR and RAMP1,2 mRNAs was also detected in the PC-derived cell lines PC-3 and DU-145, RAMP3 expression being restricted to the latter line. AM did not affect the growth rate (duplication time) of PC-3 cells, but it did significantly increase that of DU-145 cells. The growth promoting effect of AM was found to ensue from both the rise in the proliferation rate and the lowering in the apoptosis rate of DU-145 cells. These effects of AM were counteracted by the AM receptor antagonists CGRP(8-37) and AM(22-52), the former antagonist, which is more selective for AM2 than AM1 receptors, being more effective than the latter one. Both antagonists were per se able to induce a slow, but significant decrease in the basal growth rate of DU-145 cells by inhibiting proliferation and enhancing apoptosis, again CGRP(8-37) being more effective than AM(22-52). Taken together, our findings allow us to suggest that: i) endogenous AM system plays an important autocrine-paracrine growth promoting action in the human prostate, being possibly involved in the development of the malignant phenotype of epithelial cells; and ii) the tumor promoting effect of AM in the human prostate is mainly mediated by the AM2 receptor (CRLR/RAMP3) subtype.

Topics: Adrenomedullin; Apoptosis; Cell Proliferation; Cell Transformation, Neoplastic; Gene Expression Profiling; Humans; Male; Peptides; Phenotype; Prostatic Neoplasms; Radioimmunoassay; Receptors, Adrenomedullin; Receptors, Peptide; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation

The expression of the gene encoding adrenomedullin (AM), a multifunctional peptide hormone, in the prostate is localized to the epithelial cells. Prostate cancer cells are derived from prostatic epithelial cells. To elucidate the potential role of the AM gene in prostate cancer progression, we have stably-transfected the PC3 human prostate cancer cell line with an AM gene expression vector. The AM-transfected PC3 sublines were studied along with parental and empty vector transfected PC3 cells as controls. The average level of AM in the conditioned media of AM-transfected cells was 0.959+/-0.113 nM, a physiologically relevant concentration. The ectopic expression of AM gene inhibited the proliferation of PC3 cells in culture dishes. In addition, anchorage-independent growth of the transfected sublines was virtually abolished in soft agar assays. Flow cytometry studies showed that overexpression of AM gene caused a very significant G(1)/G(0) cell cycle arrest. In vivo experiments demonstrated that AM gene expression markedly inhibited the growth of xenograft tumors in nude mice. Our in vivo and in vitro studies suggest that AM could strongly suppress the malignancy of prostate cancer cells, via autocrine and/or paracrine mechanisms.

Topics: Adrenomedullin; Animals; Cell Division; Cell Line, Tumor; Humans; Interphase; Male; Mice; Mice, Nude; Neoplasms, Experimental; Peptides; Prostatic Neoplasms; Resting Phase, Cell Cycle; Transfection; Transplantation, Heterologous

Most of the locally advanced and metastatic prostate carcinomas (PCs) treated with antiandrogenic therapy eventually become refractory to this treatment. Locally produced factors may control prostate tumor biology after androgen withdrawal. Adrenomedullin (AM) is expressed in the prostate and could control cell growth in androgen-independent conditions. AM needs to be amidated by the enzyme peptidylglycine alpha-amidating monooxygenase (PAM) to become fully active. The objective of the present study was to analyze whether the expression of preproadrenomedullin (preproAM) and PAM in PC is regulated by androgens. For this purpose, human in vitro and in vivo PC models were grown in the presence or absence of androgens, and the expression of AM and PAM was examined by immunohistochemistry, Western blotting, RT-PCR, and Northern blotting. Furthermore, immunohistochemical analysis of AM in clinical specimens was performed to test if its expression is related to Gleason score and antiandrogenic therapy. In PC cell lines and xenografts, mRNA and protein AM levels were similar in the presence or absence of androgens. PAM expression seemed to be induced by androgen-withdrawal. Our results in clinical samples showed no relationship between AM expression and Gleason score or antiandrogenic treatment. In conclusion, our results demonstrate that preproAM and PAM expression in the human prostate is androgen-independent. In addition, we also report for the first time the expression of a novel PAM transcript in PC, which has not been previously described in other tissues.

Topics: Adolescent; Adrenomedullin; Adult; Androgens; Base Sequence; Blotting, Western; Humans; Immunohistochemistry; Male; Mixed Function Oxygenases; Molecular Sequence Data; Multienzyme Complexes; Peptides; Prostate; Prostatic Neoplasms

After therapeutic hormone deprivation, prostate cancer (CaP) cells often develop androgen-independent growth through not-well-defined mechanisms. The presence of neuroendocrine (NE) cells is often greater in prostate carcinoma than in normal prostate, and the frequency of NE cells correlates with tumor malignancy, loss of androgen sensitivity, increase of autocrine-paracrine activity, and poor prognosis. In some CaPs, neuropeptides have been previously implicated as growth factors. Peptidylglycine alpha-amidating monooxygenase (PAM) is the enzyme producing alpha-amidated bioactive peptides from their inactive glycine-extended precursors. In the present work, we demonstrate that androgen-independent PC-3 and DU145 cell lines, derived from human CaP, express PAM in vitro and in xenografts implanted in athymic nude mice, indicating that they are able to produce alpha-amidated peptides. Contrarily, barely detectable levels of PAM were found in the androgen-sensitive LNCaP cell line. We also show that whereas PC-3 and DU145 cells produce and secrete adrenomedullin (AM), a multifunctional amidated peptide, no expression was found in LNCaP cells. We further demonstrate that AM acts as a growth factor for DU145 cells, which suggests the existence of an autocrine loop mechanism that could potentially drive neoplastic growth. PAM mRNA levels were found to be 3-fold higher in prostate adenocarcinomas compared with that of human benign prostate hyperplasia (BPH) as demonstrated by real-time quantitative reverse transcription-PCR. The analysis of AM message expression in BPH and CaP (Gleason's score, 6-9) shows a clear distinction between benign and CaP. The expression was detected only in adenocarcinomas tissues with a marked increase in samples with a high Gleason's score. Immunocytochemically, AM was localized in the carcinomatous epithelial compartment. NE phenotype, assessed after the immunocytochemical localization of neuron-specific enolase (NSE), was found in both the epithelial and the stromal compartments of cancers; in BPH, only some spare basal cells were NSE-labeled. Cancer progression could be accelerated by peptides secreted by a population of cells capable of inducing androgen-independent tumoral growth via autocrine-paracrine mechanisms.

Topics: Adenocarcinoma; Adrenomedullin; Animals; Cell Division; Humans; Immunohistochemistry; In Situ Hybridization; Male; Mice; Mice, Nude; Mixed Function Oxygenases; Multienzyme Complexes; Neoplasm Proteins; Neoplasm Transplantation; Neoplasms, Hormone-Dependent; Peptides; Phosphopyruvate Hydratase; Prostatic Hyperplasia; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transplantation, Heterologous; Tumor Cells, Cultured

Most PCs show NE differentiation. Several studies have tried to correlate NE expression with disease status, but the reported findings have been contradictory. Prostatic NE cells synthesize peptides with a wide spectrum of potential functions. Some of these active peptides, such as PAMP, are amidated. PAM is the only carboxy-terminal peptide-amidating enzyme identified. We studied expression of PAMP and PAM in normal prostate and prostatic tumors (clinical specimens and human xenograft models) with or without prior androgen-deprivation therapy and found a wide distribution of both molecules in NE subpopulations of all kinds. Although the correlation of either marker to tumor grade, clinical progression or disease prognosis did not reach statistical significance, PAMP- or PAM-immunoreactive cells were induced after androgen-blockade therapy. In the PC-310 and PC-295 androgen-dependent models, PAMP or PAM NE differentiation was induced after castration in different ways, being higher in PC-310, which might explain its long-term survival after androgen deprivation. We show induction of expression of 2 new NE markers in clinical specimens and xenografted PC after endocrine therapy.

Topics: Adrenomedullin; Androgen Antagonists; Animals; Cell Differentiation; Humans; Immunohistochemistry; Male; Mice; Mixed Function Oxygenases; Multienzyme Complexes; Neoplasm Transplantation; Neurosecretory Systems; Peptide Fragments; Peptides; Prostate; Prostatic Neoplasms; Proteins; Transplantation, Heterologous