cyclic-gmp and Prostatic-Neoplasms

Androgen deprivation therapy (ADT) is the primary systemic therapy for treating locally advanced or metastatic prostate cancer (PCa). Despite its positive effect on PCa patient survival, ADT causes various adverse effects, including increased cardiovascular risk factors and cardiotoxicity. Lifespans extension, early use of ADT, and second-line treatment with next-generation androgen receptor pathway inhibitors would further extend the duration of ADT and possibly increase the risk of ADT-induced cardiotoxicity. Meanwhile, information on the molecular mechanisms underlying ADT-induced cardiotoxicity and measures to prevent it is limited, mainly due to the lack of specifically designed preclinical studies and clinical trials. This review article compiles up-to-date evidence obtained from observational studies and clinical trials, in order to gain new insights for deciphering the association between ADT use and cardiotoxicity. In addition, potential cardioprotective strategies involving GnRH receptors and second messenger cGMP are discussed.

Topics: Androgen Antagonists; Androgens; Antineoplastic Agents, Hormonal; Cardiotoxicity; Cardiovascular Diseases; Clinical Trials as Topic; Cyclic GMP; Gonadotropin-Releasing Hormone; Humans; Longevity; Male; Observational Studies as Topic; Phosphodiesterase 5 Inhibitors; Prostatic Neoplasms; Receptors, LHRH; Risk Factors; Signal Transduction; Time Factors; Treatment Outcome

Six-transmembrane epithelial antigen of prostate 4 (STEAP4) is involved in the development of human cancers. However, the role of STEAP4 in prostate cancer remains largely unknown. The purpose of this research is to explore the role and action mechanism of STEAP4 in prostate cancer development under lipopolysaccharide (LPS)-induced inflammatory microenvironment. STEAP4 expression was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN and Cancer Cell Line Encyclopedia (CCLE), and its prognostic value was analyzed by LinkedOmics. STEAP4-correlated genes were analyzed by LinkedOmics and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. STEAP4 level was detected by Western blotting or qRT-PCR. Proliferation was investigated by CCK-8 and EdU staining. Inflammatory cytokine levels were detected by ELISA. The cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway was detected by ELISA and Western blotting. STEAP4 level was increased in prostate cancer tissues, and high expression of STEAP4 was associated with the poor overall survival. LPS promoted cell viability and STEAP4 expression. STEAP4 knockdown attenuated LPS-induced inflammation in prostate cancer cells. STEAP4 downregulation mitigated LPS-induced tumorigenesis by decreasing cell proliferation. STEAP4 silencing reversed LPS-induced inactivation of the cGMP-PKG pathway. Inhibition of the cGMP-PKG pathway using inhibitor KT5823 relieved STEAP4 silencing-mediated suppression of cell proliferation and inflammation in LPS-stimulated cells. In conclusion, STEAP4 silencing inhibits LPS-induced proliferation of prostate cancer cells by activating the cGMP-PKG pathway.

Topics: Animals; Cell Proliferation; Cell Survival; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Down-Regulation; Humans; Lipopolysaccharides; Male; Membrane Proteins; Oxidoreductases; Prostate; Prostatic Neoplasms; Signal Transduction; Tumor Microenvironment

A key mechanism mediating cellular adaptive responses to hypoxia involves the activity of hypoxia-inducible factor 1 (HIF-1), a transcription factor composed of HIF-1α, and HIF-1β subunits. The classical mechanism of regulation of HIF-1 activity involves destabilisation of HIF-1α via oxygen-dependent hydroxylation of proline residues and subsequent proteasomal degradation. Studies from our laboratory revealed that nitric oxide (NO)-mediated activation of cyclic guanosine monophosphate (cGMP) signalling inhibits the acquisition of hypoxia-induced malignant phenotypes in tumour cells. The present study aimed to elucidate a mechanism of HIF-1 regulation involving NO/cGMP signalling. Using human DU145 prostate cancer cells, we assessed the effect of the NO mimetic glyceryl trinitrate (GTN) and the cGMP analogue 8-Bromo-cGMP on hypoxic accumulation of HIF-1α. Concentrations of GTN known to primarily activate the NO/cGMP pathway (100 nM-1 µM) inhibited hypoxia-induced HIF-1α protein accumulation in a time-dependent manner. Incubation with 8-Bromo-cGMP (1 nM-10 µM) also attenuated HIF-1α accumulation, while levels of HIF-1α mRNA remained unaltered by exposure to GTN or 8-Bromo-cGMP. Furthermore, treatment of cells with the calpain (Ca2+-activated proteinase) inhibitor calpastatin attenuated the effects of GTN and 8-Bromo-cGMP on HIF-1α accumulation. However, since calpain activity was not affected by incubation of DU145 cells with various concentrations of GTN or 8-Bromo-cGMP (10 nM or 1 µM) under hypoxic or well-oxygenated conditions, it is unlikely that NO/cGMP signalling inhibits HIF-1α accumulation via regulation of calpain activity. These findings provide evidence for a role of NO/cGMP signalling in the regulation of HIF-1α, and hence HIF-1-mediated hypoxic responses, via a mechanism dependent on calpain.

Topics: Calpain; Cell Line, Tumor; Cyclic GMP; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Prostatic Neoplasms; Signal Transduction; Tumor Hypoxia; Tumor Microenvironment

The aberrant activation of the ERG oncogenic pathway due to the TMPRSS2-ERG gene fusion is the major event that contributes to prostate cancer (PCa) development. However, the critical downstream effectors that can be therapeutically targeted remain to be identified. In this study, we have found that the expression of the α1 and β1 subunits of soluble guanylyl cyclase (sGC) was directly and specifically regulated by ERG in vitro and in vivo and was significantly associated with TMPRSS2-ERG fusion in clinical PCa cohorts. sGC is the major mediator of nitric oxide (NO)-cGMP signaling in cells that, upon NO binding, catalyzes the synthesis of cGMP and subsequently activates protein kinase G (PKG). We showed that cGMP synthesis was significantly elevated by ERG in PCa cells, leading to increased PKG activity and cell proliferation. Importantly, we also demonstrated that sGC inhibitor treatment repressed tumor growth in TMPRSS2-ERG-positive PCa xenograft models and can act in synergy with a potent AR antagonist, enzalutamide. This study strongly suggests that targeting NO-cGMP signaling pathways may be a novel therapeutic strategy to treat PCa with TMPRSS2-ERG gene fusion.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, SCID; Nitric Oxide; Oncogene Proteins, Fusion; Prostate; Prostatic Neoplasms; Serine Endopeptidases; Signal Transduction; Soluble Guanylyl Cyclase; Transcriptional Regulator ERG

Cancer stem cells (CSC) are critical for initiation, metastasis, and relapse of cancers, however, the underlying mechanism governing stemness of CSC remains unknown. Herein, we have investigated the roles of phosphodiesterase 5 (PDE5) in stemness of prostate cancer cells. Both PDE5 and WW domain-containing transcription regulator protein-1 (TAZ), a core effector of Hippo pathway, are highly expressed in the PC3-derived cancer stem cells (PCSC). Either TAZ knockdown or inhibition of PDE5 activity attenuated colony formation, altered expression patterns of stem cell markers, and enhanced cisplatin cytotoxicity, resulting in attenuation of stemness in PCSC. In addition, inhibition of PDE5 activity by its specific inhibitors activates cGMP-dependent protein kinase G (PKG), which in turn induces MST/LATS kinases, resulting in cytosolic degradation of TAZ and activation of Hippo pathway. Accordingly, knockdown of TAZ almost completely abolished PDE5 inhibitor-induced attenuation in stemness in cultured PCSC, whereas knockdown of TAZ not only abolished PDE5 inhibitor-induced attenuation in stemness but also facilitated PDE5 inhibitor-induced trans-differentiation in PCSC xenografts. Together, the present study has uncovered that PDE/cGMP/PKG signal targets to Hippo/TAZ pathway in maintaining stemness of PCSC, and suggested that PDE5 inhibitors in combination with chemotherapeutic agents could effectively prevent initiation, metastasis, and relapse of prostate cancer.

Topics: Animals; Antineoplastic Agents; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Cell Transdifferentiation; Cisplatin; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Dose-Response Relationship, Drug; Hippo Signaling Pathway; Humans; Intracellular Signaling Peptides and Proteins; Male; Mice, Nude; Neoplastic Stem Cells; Phenotype; Phosphodiesterase 5 Inhibitors; Prostatic Neoplasms; Protein Serine-Threonine Kinases; RNA Interference; Signal Transduction; Time Factors; Trans-Activators; Transcription Factors; Transcriptional Coactivator with PDZ-Binding Motif Proteins; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

Doxazosin (Doxa) is an α1-selective adrenergic receptor (ADR) antagonist widely used, alone or in combination, to treat high blood pressure, benign prostatic hyperplasia symptoms, and recently has been suggested as a potential drug for prostate cancer prevention/treatment. This study was designed to evaluate the effect of in vivo Doxa po-application, in clinically relevant dose, on: (i) steroidogenic machinery homeostasis; (ii) cAMP/cGMP signalling; (iii) transcription profile of ADR in Leydig cells of adult rats. The results showed that po-application of Doxa for once (1×Doxa), or for two (2×Doxa) or 10 (10×Doxa) consecutive days significantly disturbed steroidogenic machinery homeostasis in Leydig cells. Doxa po-application significantly decreased circulating luteinizing hormone and androgens levels. The level of androgens in testicular interstitial fluid and that extracted from testes obtained from 1×Doxa/2×Doxa rats decreased, although it remained unchanged in 10×Doxa rats. Similarly, the ex vivo basal androgen production followed in testes isolated from 1×Doxa/2×Doxa rats decreased, while remained unchanged in 10×Doxa rats. Differently, ex vivo testosterone production and steroidogenic capacity of Leydig cells isolated from 1×Doxa/2×Doxa rats was stimulated, while 10×Doxa had opposite effect. In the same cells, cAMP content/release showed similar stimulatory effect, but back to control level in Leydig cells of 10×Doxa. 1×Doxa/2×Doxa decreased transcripts for cAMP specific phosphodiesterases Pde7b/Pde8b, whereas 10×Doxa increased Pde4d. All types of treatment reduced the expression of genes encoding protein kinase A (PRKA) regulatory subunit (Prkar2b), whereas only 10×Doxa stimulated catalytic subunit (Prkaca). Doxa application more affected cGMP signalling: stimulated transcription of constitutive nitric oxide synthases (Nos1, Nos3) in time-dependent manner, whereas reduced inducible Nos2. 10×Doxa increased guanylyl cyclase 1 transcript and PRKG1 protein in Leydig cells. Orally applied Doxa significantly disturbed the transcriptional 'signature' of steroidogenic machinery, cAMP/cGMP signalling and ADRs and β-ADRs kinases in Leydig cells, thus giving new molecular insights into the role of cAMP/cGMP/adrenalin signalling in Leydig cells homeostasis.

Topics: Adrenergic alpha-1 Receptor Antagonists; Androgens; Animals; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; Cyclic GMP; Doxazosin; Guanylate Cyclase; Homeostasis; Hypertension; Leydig Cells; Luteinizing Hormone; Male; Organ Culture Techniques; Prostatic Hyperplasia; Prostatic Neoplasms; Rats; Receptors, Adrenergic; Receptors, Adrenergic, alpha-1; Signal Transduction; Steroids; Testosterone; Transcription, Genetic

Vessel dilator and kaliuretic peptide have anticancer effects in human prostate adenocarcinomas.. The effects of vessel dilator, kaliuretic peptide and cyclic GMP on Ras were examined in human prostate adenocarcinoma cells.. Vessel dilator and kaliuretic peptide decreased the activation of Ras -GTP over a concentration range of 0.01 microM to 1 microM. Vessel dilator and kaliuretic peptide (each 1 muM) inhibited the phosphorylation of Ras by 95% (p<0.0001) and 90% (p<0.0001), respectively. At 0.01 microM of kaliuretic peptide, the maximal inhibition was 95% . The inhibition of Ras lasted for 48 to 72 hours secondary to both peptides. Their ability to inhibit Ras was inhibited by cyclic GMP antibody and cyclic GMP itself inhibited Ras phosphorylation (89%; p=0.0015).. Vessel dilator and kaliuretic peptide both inhibit Ras partially mediated via cyclic GMP as part of their anticancer mechanism(s) of action.

Topics: Adenocarcinoma; Aged; Atrial Natriuretic Factor; Cyclic GMP; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Immunoblotting; Male; Natriuresis; Peptide Fragments; Prostatic Neoplasms; Protein Precursors; ras Proteins; Sodium-Potassium-Exchanging ATPase; Tumor Cells, Cultured

Atrial natriuretic peptide and long-acting natriuretic peptide have anticancer effects in human prostate adenocarcinoma.. The effects of atrial natriuretic peptide and long-acting natriuretic peptide and cyclic GMP on Ras were examined in human prostate adenocarcinoma cells.. Atrial natriuretic peptide and long-acting natriuretic peptide reduced the activation of Ras-GTP over a concentration range of 0.01 microM to 1 microM. Atrial natriuretic peptide and long-acting natriuretic peptide (each 0.1 microM) inhibited the phosphorylation of Ras 90% (p<0.0001) and 83% (p<0.0001), respectively. At 0.01 microM of long-acting natriuretic peptide, the maximal inhibition was 89%, which occurred within 5 minutes. Both peptide hormones inhibited Ras for 24 hours. Their ability to inhibit Ras was inhibited by cyclic GMP antibody and cyclic GMP itself inhibited Ras phosphorylation (72%; p=0.009).. Atrial natriuretic peptide and long-acting natriuretic peptide both inhibit Ras partially mediated via cyclic GMP as part of their anticancer mechanism(s) of action.

Topics: Adenocarcinoma; Aged; Atrial Natriuretic Factor; Blotting, Western; Cyclic GMP; Humans; Immunoblotting; Immunoprecipitation; Male; Peptide Fragments; Phosphorylation; Prostatic Neoplasms; ras Proteins

Soluble guanylyl cyclase (sGC) is a receptor for nitric oxide that generates cGMP. This second messenger molecule has established roles in cellular physiology; however, less is known about its effects in tumour cells.. The effects of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS2028), both selective sGC inhibitors on proliferation, death and migration were determined in prostate cancer cell lines.. Western blot analysis confirmed the presence of alpha1 and beta1 subunits of sGC in LNCaP and PC-3 cells. Sodium nitroprusside (SNP) increased cGMP accumulation in LNCaP and PC-3, but not DU-145 cells. SNP-stimulated cGMP production in LNCaP cells was dose-dependently reduced by ODQ, with more than 90% inhibition being observed at 0.1 microM. ODQ activated caspase-3 in all three cell lines, but not in normal prostate epithelial cells, at concentrations over 10 muM. High concentrations of ODQ also promoted DNA fragmentation and nucleosome accumulation in the cytosol of LNCaP cells. Interestingly, the chemically related inhibitor, NS2028 was without effect on caspase-3. In addition, ODQ inhibited LNCaP, Du145 and PC-3 cell growth. Finally, although fibroblast growth factor-2 did not enhance cGMP levels in LNCaP cells, its ability to stimulate LNCaP motility was abolished by ODQ.. These observations taken together suggest that the action of ODQ in LNCaP cells did not reflect sGC inhibition. We conclude that ODQ promotes cell death and inhibits growth and migration of prostate cancer cells and that these actions are independent of its effects on GMP levels.

Topics: Antineoplastic Agents; Caspase 3; Cell Death; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclic GMP; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Guanylate Cyclase; Humans; Male; Nitroprusside; Oxadiazoles; Oxazines; Prostatic Neoplasms; RNA Interference; Solubility

The growth and progression of prostate cancer are dependent on androgens and androgen receptor (AR), which act by modulating gene expression. Utilizing a gene microarray approach, we have identified the alpha1-subunit gene of soluble guanylyl cyclase (sGC) as a novel androgen-regulated gene. A heterodimeric cytoplasmic protein composed of one alpha and one beta subunit, sGC mediates the widespread cellular effects of nitric oxide (NO). We report here that, in prostate cancer cells, androgens stimulate the expression of sGCalpha1. A cloned human sGCalpha1 promoter is activated by androgen in an AR-dependent manner, suggesting that sGCalpha1 may be a direct AR target gene. Disruption of sGCalpha1 expression severely compromises the growth of both androgen-dependent and androgen-independent AR-positive prostate cancer cells. Overexpression of sGCalpha1 alone is sufficient for stimulating prostate cancer cell proliferation. Interestingly, the major growth effect of sGCalpha1 is independent of NO and cyclic guanosine monophosphate, a major mediator of the sGC enzyme. These data strongly suggest that sGCalpha1 acts in prostate cancer via a novel pathway that does not depend on sGCbeta1. Tissue studies show that sGCalpha1 expression is significantly elevated in advanced prostate cancer. Thus, sGCalpha1 may be an important mediator of the procarcinogenic effects of androgens.

Topics: Base Sequence; Cell Line, Tumor; Cell Proliferation; Cyclic GMP; Dihydrotestosterone; DNA Primers; Guanylate Cyclase; Humans; Male; Nitric Oxide; Prostatic Neoplasms; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Soluble Guanylyl Cyclase

Topics: Actins; Adrenergic alpha-Antagonists; Aged; Animals; Blotting, Western; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Doxazosin; Humans; Immunohistochemistry; Male; Middle Aged; Phosphodiesterase Inhibitors; Piperazines; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Purines; Sildenafil Citrate; Sulfones

Phosphodiesterase 5 (PDE5) inhibitors improve smooth muscle relaxation and therefore are considered for pharmacotherapy of benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS). Cyclic guanosine monophosphate (cGMP)-dependent protein kinase-1 (cGKI) has been identified as one of the downstream targets for cGMP. The aim of the present study was to evaluate, by means of immunohistochemistry and Western blot analysis, the expression and localization of cGKI isoforms in relation to smooth muscle alpha-actin and cGMP in the human prostate.. Cryostat sections of tissue segments excised from the transition zone of human prostates from 11 patients (aged 54-68 yr) were incubated with primary antibodies directed against smooth muscle alpha-actin, cGMP, cGKI, cGKIalpha, and cGKIbeta. Visualization of double-labelled immunofluorescent staining was achieved by laser microscopy. Western blot analysis was performed to confirm the expression of cGKI isoforms.. Immunoreactivities specific for cGKI, cGKIalpha, and cGKIbeta were observed in the smooth musculature of the transition zone. Double-staining revealed the colocalization of smooth muscle alpha-actin, cGMP, and cGKI isoforms in smooth muscle cells of the fibromuscular stroma. The expression of cGKI isoforms was confirmed by Western blot analysis.. Our results confirm the presence of cGKI isoforms alpha and beta in the transition zone of human prostate tissue. In addition, the colocalization of alpha-actin, cGMP, and cGKI isoforms provides further evidence for a significant role of the nitric oxide/cGMP pathway in the regulation of smooth muscle contractility in human prostate tissue and therefore could provide additional targets for pharmacotherapy of BPH and LUTS.

Topics: Actins; Aged; Blotting, Western; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Humans; Immunohistochemistry; Male; Middle Aged; Prostate; Prostatic Neoplasms

Hypoxia contributes to drug resistance in solid cancers, and studies have revealed that low concentrations of nitric oxide (NO) mimetics attenuate hypoxia-induced drug resistance in tumor cells in vitro. Classic NO signaling involves activation of soluble guanylyl cyclase, generation of cyclic GMP (cGMP), and activation of cGMP-dependent protein kinase. Here, we determined whether chemosensitization by NO mimetics requires cGMP-dependent signaling and whether low concentrations of NO mimetics can chemosensitize tumors in vivo.. Survival of human prostate and breast cancer cells was assessed by clonogenic assays following exposure to chemotherapeutic agents. The effect of NO mimetics on tumor chemosensitivity in vivo was determined using a mouse xenograft model of human prostate cancer. Drug efflux in vitro was assessed by measuring intracellular doxorubicin-associated fluorescence.. Low concentrations of the NO mimetics glyceryl trinitrate (GTN) and isosorbide dinitrate attenuated hypoxia-induced resistance to doxorubicin and paclitaxel. Similar to hypoxia-induced drug resistance, inhibition of various components of the NO signaling pathway increased resistance to doxorubicin, whereas activation of the pathway with 8-bromo-cGMP attenuated hypoxia-induced resistance. Drug efflux was unaffected by hypoxia and inhibitors of drug efflux did not significantly attenuate hypoxia-induced chemoresistance. Compared with mice treated with doxorubicin alone, tumor growth was decreased in mice treated with doxorubicin and a transdermal GTN patch. The presence of GTN and GTN metabolites in plasma samples was confirmed by gas chromatography.. Tumor hypoxia induces resistance to anticancer drugs by interfering with endogenous NO signaling and reactivation of NO signaling represents a novel approach to enhance chemotherapy.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cyclic GMP; Drug Resistance, Neoplasm; Female; Humans; Isosorbide Dinitrate; Male; Mice; Neoplasms, Experimental; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Prostatic Neoplasms; Signal Transduction

We characterized the effects of sildenafil citrate on the growth and metastasis of human prostate cancer cells in nude mice.. The androgen independent human prostate cancer cell line PC-3 was inoculated into the prostate of nude mice to produce orthotopic primary prostate cancers and metastases. Sildenafil citrate gavage was started on day 31 after tumor cell inoculation and given every other day 15 times (30 days). The 7 mice in the low dose group received 25 mg/kg body weight sildenafil citrate per gavage, while the 7 in the high dose group received 50 mg/kg body weight sildenafil citrate and the 9 in the control group received water. Autopsy was performed on day 75 to evaluate primary tumor growth and metastasis. Plasma cyclic guanosine monophosphate concentrations were measured after the single dose of 50 mg/kg sildenafil citrate in the mice.. Plasma cyclic guanosine monophosphate concentration increased 4-fold 1 hour after sildenafil citrate administration. The plasma concentration decreased rapidly and returned to normal after 8 hours. There was no significant difference in tumor weight between any of the 3 groups. The number of metastatic lymph nodes correlated significantly with primary tumor weight (p = 0.03) with a correlation coefficient of 0.454 but there was no significant correlation between the number of involved lymph nodes and sildenafil administration. Distant metastases were not significantly promoted by sildenafil administration.. Incontinuous oral administration of sildenafil citrate did not promote primary tumor growth and metastasis in an orthotopic prostate cancer model.

Topics: Animals; Cyclic GMP; Disease Models, Animal; Lymphatic Metastasis; Male; Mice; Mice, Inbred BALB C; Phosphodiesterase Inhibitors; Piperazines; Prostatic Neoplasms; Purines; Sildenafil Citrate; Sulfones; Tumor Cells, Cultured; Vasodilator Agents

The intracellular signaling pathways mediating the nuclear exclusion of the androgen receptor (AR) by melatonin were evaluated in PC3 cells stably transfected with the AR. The melatonin-induced nuclear exclusion of the AR by melatonin (100 nM, 3 h) was blocked by LY 83583 (an inhibitor of guanylyl cyclases). 8-Bromo-cGMP (a cell-permeable cGMP analog), mimicked the effect of melatonin, as did ionomycin (a calcium ionophore) and PMA [an activator of protein kinase C (PKC)], and their effects were blocked by GF- 109203X (a selective PKC inhibitor). BAPTA (an intracellular calcium chelator) blocked the effects of melatonin and 8-bromo-cGMP but not of PMA. Inhibition or activation of the protein kinase A pathway did not affect basal or melatonin-mediated AR localization. We conclude that the melatonin-mediated rise in cGMP elicits AR nuclear exclusion via a pathway involving increased intracellular calcium and PKC activation. These results define a novel signaling pathway that regulates AR localization and androgen responses in target cells.

Topics: Active Transport, Cell Nucleus; Aminoquinolines; Antineoplastic Agents; Cell Nucleus; Chelating Agents; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activators; Enzyme Inhibitors; Humans; Ionophores; Male; Melatonin; Prostatic Neoplasms; Protein Kinase C; Receptors, Androgen; Signal Transduction; Transfection; Tumor Cells, Cultured

Melatonin, secreted nocturnally by the pineal gland, can bind to human benign prostate epithelial cells and attenuate their growth and viability. In the present study, melatonin binding and responses were explored in the human steroid-independent PC3 prostatic tumor cells. PC3 cells bound 125I-melatonin with low affinity (Kd ca. 0.9 nM) at high as well as low cell density. Melatonin enhanced cGMP and 3H-thymidine incorporation at low, but attenuated them at high cell density. In addition, melatonin inhibited cAMP at low, but augmented it at high cell density. These effects were associated with an increase in cell count at low- but not high-density cultures. Pertussis toxin treatment suppressed 125I-melatonin binding and ablated all the effects of melatonin on 3H-thymidine incorporation, cAMP, and cGMP at both cell densities. Cholera toxin treatment failed to block the effects of melatonin on 3H-thymidine incorporation, but prevented the modulation by melatonin of cAMP at low and cGMP at high cell density. The cGMP analog 8-Br-cGMP, inhibited melatonin's effects on 3H-thymidine incorporation at both cell densities. H89, a protein kinase A inhibitor, prevented melatonin's effects on 3H-thymidine incorporation at low but not high cell density. These results provide the first demonstration of direct interaction of melatonin with hormone-insensitive prostate tumor cells. The melatonin receptors in the PC3 cells are coupled to pertussis toxin-sensitive G proteins to induce cell density-dependent changes in cGMP, cAMP, and cell growth.

Topics: Binding Sites; Cell Count; Cell Survival; Cholera Toxin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; DNA; Humans; Isoquinolines; Male; Melatonin; Pertussis Toxin; Prostatic Neoplasms; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Melatonin; Serotonin Antagonists; Signal Transduction; Sulfonamides; Thymidine; Tumor Cells, Cultured; Virulence Factors, Bordetella

In this study we sought to determine whether the main components of the nitric oxide (NO) pathway are localized within the Leydig cells of the human testis and whether the soluble guanylyl cyclase (sGC), the enzyme that accounts for NO effects, is functionally active in these cells. Using an amplified immunocytochemical technique, immunoreactivity for nitric oxide synthase (NOS-I), sGC and cyclic guanosine monophosphate (cGMP) was detected within the cytoplasm of human Leydig cells. Distinct differences in staining intensity were found between individual Leydig cells, between cell groups and between Leydig cells of different patients. By means of a specific cGMP-RIA, a concentration-dependent increase in the quantity of cGMP was measured in primary cultures of human Leydig cells following exposure to the NO donor sodium nitroprusside. In addition, NOS-I immunoreactivity was seen in Sertoli cells, whereas cGMP and sGC immunoreactivity was found in Sertoli cells, some apically situated spermatids and residual bodies of seminiferous tubules. Dual-labelling studies and the staining of consecutive sections showed that there are several populations of Leydig cells in the human testis. Most cells were immunoreactive for NOS-I, sGC and cGMP, but smaller numbers of cells were unlabelled by any of the antibodies used, or labelled for NOS-I or cGMP alone, for sGC and cGMP, or for NOS-I and sGC. These results show that the Leydig cells possess both the enzyme by which NO is produced and the active enzyme which mediates the NO effects. There are different Leydig cell populations that probably reflect variations in their functional (steroidogenic) activity.

Topics: Adult; Aged; Aged, 80 and over; Animals; Cells, Cultured; Cyclic GMP; Guanylate Cyclase; Humans; Leydig Cells; Male; Middle Aged; Nitric Oxide Synthase; Prostatic Neoplasms; Rats; Rats, Wistar; Testis

We have recently described the presence of a guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] inhibitor (GCI) in an aqueous extract of the balsam pear (Momordica charantia abbreviata). Because the guanylate cyclase-cyclic GMP system is though to be involved in cell growth, DNA and RNA synthesis, and possible malignant transformation, we examined the effect of the aqueous extract containing GCI on an undifferentiated adenocarcinoma of the rat prostate and concanavalin-A-stimulated [3H]thymidine incorporation into cultured splenic lymphocytes, a process thought to be mediated by cyclic GMP. The results demonstrate that the extract of the balsam pear blocks both the growth of the rat prostatic adencarcinoma in vitro and [3H]thymidine incorporation into DNA. DNA histograms from flow cytometry indicated that the extract containing GCI inhibited in the G2 + M phase of the cell cycle, a presumed locus of cyclic GMP effects. In addition, guanylate cyclase activity was significantly greater in the tumor than normal prostate tissue and was decreased by the extract containing GCI. Cyclic GMP levels in the tumor in culture wer also decreased by addition of the extract. It remains to be determined whether or not the anti-tumor agent and GCI are the same substance.

Topics: Adenocarcinoma; Animals; Cell Cycle; Concanavalin A; Cyclic GMP; Guanylate Cyclase; In Vitro Techniques; Male; Neoplasms, Experimental; Plant Extracts; Prostatic Neoplasms; Thymidine

Adenylate, guanylate cyclase and protein kinases in a fibrous sarcoma originating from rat prostate have been studied. A decrease in levels of adenosine 3', 5'-monophosphate (cyclic AMP) and adenylate cyclase activities and an increase in levels of guanosine 3',5'-monophosphate (cyclic GMP) and guanylate cyclase activities were observed in the tumor tissue when compared with the normal prostatic tissue of rats. Protein kinases from the tumor and the prostate were both responsive to exogenous cyclic AMP, with an apparent Ka of 0.08 muM in the tumor and of 0.11 muM in the prostate. It is of interest that the protein kinases from the tumor responded to cyclic AMP to the same extent as was observed in the enzyme preparation from the prostate. The protein kinase from the tumor was more sensitive to cyclic GMP than that from the prostate, showing an apparent Ka of 0.88 muM in the tumor and of 4.85 muM in the prostate. This tumor has been characterized with an increase in guanylate cyclase activities with a subsequent rise in cellular cyclic GMP and an increased sensitivity of the protein kinase to cyclic GMP.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenylyl Cyclases; Animals; Carrier Proteins; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Guanylate Cyclase; Male; Prostate; Prostatic Neoplasms; Protein Kinases; Rats; Sarcoma