cyclic-gmp and Breast-Neoplasms

The cyclic nucleotides, cAMP and cGMP, are ubiquitous second messengers responsible for translating extracellular signals to intracellular biological responses in both normal and tumor cells. When these signals are aberrant or missing, cells may undergo neoplastic transformation or become resistant to chemotherapy. cGMP-hydrolyzing phosphodiesterases (PDEs) are attracting tremendous interest as drug targets for many diseases, including cancer, where they regulate cell growth, apoptosis and sensitization to radio- and chemotherapy. In breast cancer, PDE5 inhibition is associated with increased intracellular cGMP levels, which is responsible for the phosphorylation of PKG and other downstream molecules involved in cell proliferation or apoptosis. In this review, we provide an overview of the most relevant studies regarding the controversial role of PDE inhibitors as off-label adjuvants in cancer therapy.

Topics: Animals; Breast Neoplasms; Clinical Trials as Topic; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Female; Humans; Nitric Oxide; Signal Transduction

One in eight women in the United States will be diagnosed with invasive breast cancer in her lifetime. Advances in therapeutic strategies, diagnosis, and improved awareness have resulted in a significant reduction in breast cancer related mortality. However, there is a continued need for more effective and less toxic drugs for both the prevention and the treatment of breast cancer in order to see a continued decline in the morbidity and mortality associated with this disease. Recent studies suggest that the cGMP signaling pathway may be aberrantly regulated in breast cancer. As such, this pathway may serve as a source of novel targets for future breast cancer drug discovery efforts. This review provides an overview of cGMP signaling in normal physiology and in breast cancer as well as current strategies being investigated for targeting this pathway in breast cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Cyclic GMP; Female; Guanosine Triphosphate; Guanylate Cyclase; Humans; Models, Biological; Signal Transduction

To alter the immunosuppressive tumor microenvironment (TME), we developed an immunostimulatory nanoparticle (NP) to reprogram a tumor's dysfunctional and inhibitory antigen-presenting cells (APCs) into properly activated APCs that stimulate tumor-reactive cytotoxic T cells. Importantly, systemic delivery allowed NPs to efficiently utilize the entire microvasculature and gain access into the majority of the perivascular TME, which coincided with the APC-rich tumor areas leading to uptake of the NPs predominantly by APCs. In this work, a 60 nm NP was loaded with a STING agonist, which triggered robust production of interferon β, resulting in activation of APCs. In addition to untargeted NPs, we employed 'mainstream' ligands targeting fibronectin, α

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Breast Neoplasms; Cell Line, Tumor; Cyclic GMP; Dendritic Cells; Immunity, Innate; Immunologic Factors; Ligands; Macrophages; Mice, Inbred BALB C; Nanoparticles; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; T-Lymphocytes; Tumor Microenvironment

Cardiovascular side effects of broadly used chemotherapeutic drugs such as Tamoxifen citrate (TC), Capecitabine (CP) and Epirubicin (EP) among cancer survivors are well established. Nitric oxide (NO) is known to protect cardiovascular tissues under conditions of stress. NO can act through cyclic guanosine monophosphate (cGMP)-dependent and -independent pathways. Particularly, the S-nitrosylation of SH-groups in a protein by NO falls under cGMP-independent effects of NO. TC, CP, and EP are hypothesized as interfering with cellular protein S-nitrosylation, which, in turn, may lead to endothelial dysfunctions. The results show that all three drugs attenuate nitrosylated proteins in endothelial cells. A significant reduction in endogenous S-nitrosylated proteins was revealed by Saville-Griess assay, immunofluorescence and western blot. Incubation with the drugs causes a reduction in endothelial migration, vasodilation and tube formation, while the addition of S-nitrosoglutathione (GSNO) has a reversal of this effect. In conclusion, results indicate the possibility of decreased cellular nitrosothiols as being one of the reasons for endothelial dysfunctions under TC, CP and EP treatment. Identification of the down-regulated S-nitrosylated proteins so as to correlate their implications on fundamental vascular functions could be an interesting phenomenon.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Movement; Cyclic GMP; Down-Regulation; Endothelial Cells; Endothelium, Vascular; Female; Humans; Nitric Oxide; Nitric Oxide Synthase Type III; Nitrosoguanidines; Protein S; Vasodilation

Metabolic reprogramming to fulfill the biosynthetic and bioenergetic demands of cancer cells has aroused great interest in recent years. However, metabolic reprogramming for cancer metastasis has not been well elucidated. Here, we screened a subpopulation of breast cancer cells with highly metastatic capacity to the lung in mice and investigated the metabolic alternations by analyzing the metabolome and the transcriptome, which were confirmed in breast cancer cells, mouse models, and patients' tissues. The effects and the mechanisms of nucleotide de novo synthesis in cancer metastasis were further evaluated in vitro and in vivo. In our study, we report an increased nucleotide de novo synthesis as a key metabolic hallmark in metastatic breast cancer cells and revealed that enforced nucleotide de novo synthesis was enough to drive the metastasis of breast cancer cells. An increased key metabolite of de novo synthesis, guanosine-5'-triphosphate (GTP), is able to generate more cyclic guanosine monophosphate (cGMP) to activate cGMP-dependent protein kinases PKG and downstream MAPK pathway, resulting in the increased tumor cell stemness and metastasis. Blocking de novo synthesis by silencing phosphoribosylpyrophosphate synthetase 2 (PRPS2) can effectively decrease the stemness of breast cancer cells and reduce the lung metastasis. More interestingly, in breast cancer patients, the level of plasma uric acid (UA), a downstream metabolite of purine, is tightly correlated with patient's survival. Our study uncovered that increased de novo synthesis is a metabolic hallmark of metastatic breast cancer cells and its metabolites can regulate the signaling pathway to promote the stemness and metastasis of breast cancer.

Topics: Adult; Animals; Breast Neoplasms; Cell Line, Tumor; China; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Female; Gene Expression Profiling; Humans; MAP Kinase Signaling System; Metabolomics; Mice; Mice, Inbred BALB C; Neoplastic Stem Cells; Nucleotides; Purines; Ribose-Phosphate Pyrophosphokinase; Signal Transduction

Topics: Breast Neoplasms; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide-Gated Cation Channels; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Guanylate Cyclase; Humans; Lactation; Mammary Glands, Human; Milk, Human; Signal Transduction

Nitric oxide (NO) is an essential signaling molecule in biological systems. Soluble guanylate cyclase (sGC), composing of α1 and β1 subunit, is the receptor for NO. Using radioimmunoassay, we discovered that activation of sGC by treatment with bradykinin or sodium nitroprusside (SNP) is impaired in MCF-7 and MDA-MB-231 breast cancer cells as compared to normal breast epithelial 184A1 cells. The 184A1 cells expressed both sGC α1 and sGCβ1 mRNAs. However, levels of sGCβ1 mRNAs were relatively lower in MCF-7 cells while both mRNA of sGC subunits were absent in MDA-MB-231 cells. Treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) increased mRNA levels of both sGCα1 and sGCβ1 in MDA-MB-231 cells but only sGCβ1 mRNAs in MCF-7 cells. The 5-aza-dC treatment increased the SNP-induced cGMP production in MCF-7 and MDA-MB-231, but not in 184A1 cells. Bisulfite sequencing revealed that the promoter of sGCα1 in MDA-MB-231 cells and promoter of sGCβ1 in MCF-7 cells were methylated. Promoter hypermethylation of sGCα1 and sGCβ1 was found in 1 out of 10 breast cancer patients. Over-expression of both sGC subunits in MDA-MB-231 cells induced apoptosis and growth inhibition in vitro as well as reduced tumor incidence and tumor growth rate of MDA-MB-231 xenografts in nude mice. Elevation of sGC reduced protein abundance of Bcl-2, Bcl-xL, Cdc2, Cdc25A, Cyclin B1, Cyclin D1, Cdk6, c-Myc, and Skp2 while increased protein expression of p53. Our study demonstrated that down-regulation of sGC, partially due to promoter methylation, provides growth and survival advantage in human breast cancer cells.

Topics: Animals; Apoptosis; Azacitidine; Bradykinin; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cell Survival; Cyclic GMP; Decitabine; DNA Methylation; Female; Guanylate Cyclase; Humans; MCF-7 Cells; Mice; Mice, Nude; Nitric Oxide; Nitroprusside; Promoter Regions, Genetic; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase

Cancer vaccination may be our best and most benign option for preventing or treating metastatic cancer. However, breakthroughs are hampered by immune suppression in the tumor microenvironment. In this study, we analyzed whether cyclic diguanylate (c-di-GMP), a ligand for stimulator of interferon genes (STING), could overcome immune suppression and improve vaccination against metastatic breast cancer. Mice with metastatic breast cancer (4T1 model) were therapeutically immunized with an attenuated Listeria monocytogenes (LM)-based vaccine, expressing tumor-associated antigen Mage-b (LM-Mb), followed by multiple low doses of c-di-GMP (0.2 μmol/L). This treatment resulted in a striking and near elimination of all metastases. Experiments revealed that c-di-GMP targets myeloid-derived suppressor cells (MDSC) and tumor cells. Low doses of c-di-GMP significantly increased the production of IL12 by MDSCs, in correlation with improved T-cell responses to Mage-b, whereas a high dose of c-di-GMP (range, 0.3-3 mmol/L) activated caspase-3 in the 4T1 tumor cells and killed the tumor cells directly. On the basis of these results, we tested one administration of high-dose c-di-GMP (3 mmol/L) followed by repeated administrations of low-dose c-di-GMP (0.2 μmol/L) in the 4T1 model, and found equal efficacy compared with the combination of LM-Mb and c-di-GMP. This finding correlated with a mechanism of improved CD8 T-cell responses to tumor-associated antigens (TAA) Mage-b and Survivin, most likely through cross-presentation of these TAAs from c-di-GMP-killed 4T1 tumor cells, and through c-di-GMP-activated TAA-specific T cells. Our results demonstrate that activation of STING-dependent pathways by c-di-GMP is highly attractive for cancer immunotherapy.

Topics: Animals; Antigens, Neoplasm; Breast Neoplasms; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cross-Priming; Cyclic GMP; Female; HEK293 Cells; Humans; Immunotherapy; Listeria monocytogenes; Mammary Neoplasms, Experimental; Membrane Proteins; Mice; Mice, Inbred BALB C; Tumor Microenvironment; Vaccines, Attenuated

Activation of protein kinase G (PKG) by cyclic guanosine 3,5-monophosphate (cGMP) has become of considerable interest as a novel molecular approach for the induction of apoptosis in cancer cells. This study was conducted to investigate the role of PKG isoforms in the regulation of cell growth in human breast cancer cell lines MCF-7 and MDA-MB468. The expression levels of PKG isoforms were also examined using real-time reverse transcriptase polymerase chain reaction. No differences in the gene expression of PKG isoforms were observed between MCF-7 and MDA-MB-468 cells. To investigate the effects of PKG isoforms on the regulation of cell growth, the cGMP analogues 8-APT-cGMP (PKGIα activator), 8-Br-PET-cGMP (PKGIβ activator) and 8-pCPT-cGMP (PKGII activator) were employed. Apoptosis was assessed with the Annexin-V-propidium iodide (PI) staining, cell cycle analysis and caspase-3/9 activity assay. Treatment of MCF-7 and MDA-MB-468 cells with 8-Br-PET-cGMP resulted in a concentration-dependent cell growth inhibition and apoptosis, whereas neither PKGIα nor PKGII activators had any effect on the cell growth. The role of PKGIβ in the inhibition of cell growth was confirmed using PKGI and PKGII inhibitors. The present study is the first to demonstrate the involvement of PKGIβ in the inhibition of cell growth and induction of apoptosis in breast cancer cells.

Topics: Apoptosis; Breast Neoplasms; Caspase 3; Caspase 8; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; Female; Humans

Our previous research data showed that type II cGMP-dependent protein kinase (PKGII) inhibited EGF-induced MAPK/ERK-mediated signal transduction through blocking the phosphorylation of EGFR caused by EGF. Since EGFR also mediates other MAPK-mediated signal transduction pathways, this study was designed to investigate whether PKGII inhibits EGF-induced MAPK/c-Jun N-terminal kinase (JNK) signal transduction. MCF-7 human breast cancer cells were infected with adenoviral constructs encoding the cDNA of PKGII (pAd-PKGII) to increase the expression of PKGII and treated with 8-pCPT-cGMP to activate the enzyme. Western blotting was applied to detect the phosphorylation/activation of EGFR, JNK, MKK7 and c-Jun. The Pull-down method was used to detect the activation of Ras protein. Co-IP was used to analyze the binding between Grb2 and Sos1. TUNEL staining was used to detect the apoptosis of MCF-7 cells. The results showed that EGF treatment increased the phosphorylation of EGFR, the binding between Grb2 and Sos1, the activation of Ras, and the phosphorylation/activation of MKK7, JNK and c-Jun, but decreased the apoptosis of the cells. Increase of PKGII activity through infection with pAd-PKGII and stimulation with 8-pCPT-cGMP efficiently reversed the above changes caused by EGF. The results suggest that PKGII also inhibits EGF-induced MAPK/JNK-mediated signal transduction and further confirmed that PKGII can block the activation of EGFR.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type II; Cyclic GMP-Dependent Protein Kinases; Epidermal Growth Factor; ErbB Receptors; Female; GRB2 Adaptor Protein; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 7; MAP Kinase Signaling System; Phosphorylation; ras Proteins; SOS1 Protein; Thionucleotides

Phosphodiesterase 9 (PDE9) is a major isoform of phosphodiesterase hydrolysing cGMP and plays a key role in proliferation of cells, their differentiation and apoptosis, via intracellular cGMP signalling. The study described here was designed to investigate expression, activity and apoptotic effect of PDE9 on human breast cancer cell lines, MCF-7 and MDA-MB-468.. Activity and expression of PDE9 were examined using colorimetric cyclic nucleotide phosphodiesterase assay and real-time RT-PCR methods respectively; cGMP concentration was also measured. MTT viability test, annexin V-FITC staining, Hoechst 33258 staining and caspase3 activity assay were used to detect apoptosis.. Treatment of both cell lines with BAY 73-6691 lead to reduction in PDE9 mRNA expression, PDE9 cGMP-hydrolytic activity and elevation of the intracellular cGMP response. BAY 73-6691 significantly reduced cell proliferation in a dose- and time-dependent manner and caused marked increase in apoptosis through caspase3 activation.. Our results revealed that BAY 73-6691 induced apoptosis in these breast cancer cell lines through the cGMP pathway. These data suggest that BAY 73-6691 could be utilized as an agent in treatment of breast cancer.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Apoptosis; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Humans; Pyrazoles; Pyrimidines; Signal Transduction

Nonsteroidal anti-inflammatory drugs (NSAID) such as sulindac sulfide (SS) display promising antineoplastic properties, but toxicities resulting from COX inhibition limit their clinical use. Although COX inhibition is responsible for the anti-inflammatory activity of SS, recent studies suggest that phosphodiesterase (PDE) 5 inhibition and activation of cyclic guanosine monophosphate (cGMP) signaling are closely associated with its ability to induce apoptosis of tumor cells. However, the underlying mechanisms responsible for apoptosis induction, factors that influence sensitivity of tumor cells to SS, and the importance of PDE5 for breast tumor cell growth have not been established. Here we show that SS can induce apoptosis of breast tumor cells, which predominantly rely on PDE5 for cGMP hydrolysis but not normal mammary epithelial cells, which rely on PDE isozymes other than PDE5 for cGMP hydrolysis. Inhibition of PDE5 and activation of protein kinase G (PKG) by SS was associated with increased β-catenin phosphorylation, decreased β-catenin mRNA and protein levels, reduced β-catenin nuclear localization, decreased T-cell factor/lymphoid enhancer factor (Tcf/Lef) promoter activity, and decreased expression of Wnt/β-catenin-regulated proteins. Suppression of PDE5 with siRNA or known PDE5 inhibitors was sufficient to selectively induce apoptosis and attenuate β-catenin-mediated transcription in breast tumor cells with minimal effects on normal mammary epithelial cells. These findings provide evidence that SS induces apoptosis of breast tumor cells through a mechanism involving inhibition of PDE5 and attenuation of oncogenic Wnt/β-catenin-mediated transcription. We conclude that PDE5 represents a novel molecular target for the discovery of safer and more efficacious drugs for breast cancer chemoprevention.

Topics: Apoptosis; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Female; Humans; Isoenzymes; Oncogenes; Phosphodiesterase 5 Inhibitors; Signal Transduction; Sulindac; Transcription, Genetic; Wnt Proteins

The activation of protein kinase G (PKG) by cyclic guanosine 3,5-monophosphate (cGMP) has become of considerable interest as a novel molecular approach for the induction of apoptosis in cancer cells. The present study was designed to examine the effects of cGMP and PKG on cell growth and apoptosis in the human breast cancer cell lines, MCF-7 and MDA-MB-468. To achieve this, 1-benzyl-3-(5P-hydroxymethyl-2P-furyl) indazole (YC-1), a soluble guanylyl cyclase activator, and 8-bromo-cGMP (8-br-cGMP), a membrane-permeant and phosphodiesterase-resistant analogue of cGMP, were employed in MCF-7 and MDA-MB-468 cells. Then, the role of PKG in the induction of apoptosis was evaluated using KT5823 and Rp-8-pCPT-cGMP as specific inhibitors of PKG. The expression of PKG isoforms in these cell lines was also investigated. KT5823 and Rp-8-pCPT-cGMP significantly attenuated the loss of cell viability caused by YC-1 and 8-br-cGMP in these cells. This study provides direct evidence that the activation of PKG by cGMP induces growth inhibition and apoptosis in MCF-7 and MDA-MB-468 breast cancer cell lines.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Activators; Female; G1 Phase; Gene Expression Regulation, Neoplastic; Guanylate Cyclase; Humans; Inhibitory Concentration 50; Isoenzymes; Molecular Targeted Therapy; Neoplasm Proteins; Protein Kinase Inhibitors; Receptors, Estrogen; RNA, Messenger

This study investigates angiogenesis and the expression of thrombospondin 1 in invasive ductal carcinoma of the breast and their possible relation to platelet counts and platelet activity. The study included 20 cases of invasive ductal carcinoma. Platelet activity was evaluated by determining thromboxane B2 and cyclic guanosine monophosphate (cGMP) levels by enzyme immunoassay (EIA).Thrombospondin (TSP) 1 and CD34 expression was studied immunohistochemically. Mean platelet count of the patient group was significantly greater than the mean platelet count of the control group (P < 0.05). The platelet counts were positively correlated with tumour size (r=0.609; P < 0.01). Platelet counts were higher in the patients who had grade 3 microvessel density (P < 0.05). The mean basal platelet cGMP level in the patient group was significantly lower than it was in the control group (P < 0.05). Focal TSP immunoreactivity was detectable in 5 (20%) cases in the tumour cells, and in 9 (45%) cases in the stroma. We did not find any correlation between TSP-1 staining and angiogenesis, platelet counts, platelet activity, and the histological prognostic parameters. Our study confirms the essential role of platelets in tumour growth and angiogenesis. Decreased levels of cGMP in the patient group may cause platelet hyperreactivity. Although thrombospondin 1 may be upregulated in malignant breast tissue, this is not sufficient for tumour growth and dissemination according to our results.

Topics: Adult; Aged; Aged, 80 and over; Blood Platelets; Breast Neoplasms; Case-Control Studies; Cyclic GMP; Female; Humans; Immunoenzyme Techniques; Immunohistochemistry; Middle Aged; Neovascularization, Pathologic; Thrombospondin 1

Sulindac displays promising antineoplastic activity, but toxicities from cyclooxygenase inhibition limit its use for chemoprevention. Previous reports suggest that its anticancer properties may be attributed to a cyclooxygenase-independent mechanism, although alternative targets have not been well defined. Here, we show that sulindac sulfide (SS) induces apoptosis and inhibits the growth of human breast tumor cells with IC50 values of 60 to 85 micromol/L. Within the same concentration range, SS inhibited cyclic GMP (cGMP) hydrolysis in tumor cell lysates but did not affect cyclic AMP hydrolysis. SS did not induce apoptosis of normal human mammary epithelial cells (HMEC) nor did it inhibit phosphodiesterase (PDE) activity in HMEC lysates. SS increased intracellular cGMP levels and activated protein kinase G in breast tumor cells but not HMEC. The guanylyl cyclase (GC) activator, NOR-3, and cGMP PDE inhibitors, trequinsin and MY5445, displayed similar growth-inhibitory activity as SS, but the adenylyl cyclase activator, forskolin, and other PDE inhibitors had no effect. Moreover, GC activation increased the sensitivity of tumor cells to SS, whereas GC inhibition reduced sensitivity. By comparing PDE isozyme profiles in breast tumor cells with HMEC and determining the sensitivity of recombinant PDE isozymes to SS, PDE5 was found to be overexpressed in breast tumor cells and selectively inhibited by SS. The mechanism of SS binding to the catalytic domain of PDE5 was revealed by molecular modeling. These data suggest that PDE5 inhibition is responsible for the breast tumor cell growth-inhibitory and apoptosis-inducing activity of SS and may contribute to the chemopreventive properties of sulindac.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Inhibitory Concentration 50; Isoenzymes; Models, Biological; Models, Molecular; Phosphodiesterase 5 Inhibitors; Protein Binding; Sulindac

Protein synthesis is a tightly controlled process, and its deregulation plays an important role in tumorigenesis. Protein synthesis remains poorly understood with very few well-identified validated targets for therapeutic purposes. In this study, we use nitric oxide (NO), which suppresses protein synthesis by inactivating eukaryotic initiation factor 2-alpha (eIF2-alpha), to examine the mechanism by which low and high oxidative stress inhibits protein synthesis. In breast cancer cells, low NO stress induced heme-regulated inhibitor (HRI) activation, which facilitated gradual decline in short half-life proteins. High NO stress induced HRI and protein kinase R (PKR) activation, leading to a sharp decline in protein synthesis as accessed by a decline in short and long half-life proteins and dramatic morphologic changes. In contrast, human mammary epithelial (HME) and Ras transfected untransformed HME (MCF-10A1 neo N) cells were less susceptible to NO-induced inhibition of protein synthesis and cytostasis. Our results suggest that NO-induced cytostasis in breast cancer cells was due to PKR activation and increased phosphorylation of eIF2-alpha, whereas the reduced susceptibility of normal mammary epithelial cells to NO could be due to the inaccessibility of PKR, which is bound to inhibitor p58.

Topics: Apoptosis; Blotting, Western; Breast; Breast Neoplasms; Cell Cycle; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media, Serum-Free; Cyclic GMP; eIF-2 Kinase; Enzyme Activation; Eukaryotic Initiation Factor-2; Female; Free Radical Scavengers; Genes, ras; Heme; Humans; Hydrogen Peroxide; Immunoprecipitation; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitric Oxide; Oxidants; Oxidative Stress; Phosphorylation; Protein Biosynthesis; RNA, Small Interfering

Melatonin, via its MT1 receptor, but not the MT2 receptor, can modulate the transcriptional activity of various nuclear receptors - estrogen receptor alpha (ERalpha) and retinoic acid receptor alpha (RARalpha), but not ERbeta- in MCF-7, T47D, and ZR-75-1 human breast cancer cell lines. The anti-proliferative and nuclear receptor modulatory actions of melatonin are mediated via the MT1 G protein-coupled receptor expressed in human breast cancer cells. However, the specific G proteins and associated pathways involved in the nuclear receptor transcriptional regulation by melatonin are not yet clear. Upon activation, the MT1 receptor specifically couples to the G(alphai2), G(alphai3), G(alphaq), and G(alphall) proteins, and via activation of G(alphai2) proteins, melatonin suppresses forskolin-induced 3',5'-cyclic adenosine monophosphate production, while melatonin activation of G(alphaq), is able to inhibit phospholipid hydrolysis and ATP's induction of inositol triphosphate production in MCF-7 breast cancer cells. Employing dominant-negative and dominant-positive) forms of these G proteins, we demonstrate that G(alphai2) proteins mediate the suppression of estrogen-induced ERalpha transcriptional activity by melatonin, while the G(q) protein mediates the enhancement of retinoid-induced RARalpha transcriptional activity by melatonin. However, the growth-inhibitory actions of melatonin are mediated via both G(alphai2) and G(alphaq) proteins.

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Colforsin; Cyclic AMP; Cyclic GMP; Estrogens; Female; Gene Expression Regulation; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Immunoprecipitation; Luciferases; Melatonin; Phosphorus Radioisotopes; Radioimmunoassay; Receptor, Melatonin, MT1; Receptors, Estrogen; Receptors, Retinoic Acid; Receptors, Thyroid Hormone; Retinoic Acid Receptor alpha; Transcription, Genetic; Transfection

Nitric oxide (NO) in nanomolar (nmol/L) concentrations is consistently detected in tumor microenvironment and has been found to promote tumorigenesis. The mechanism by which NO enhances tumor progression is largely unknown. In this study, we investigated the possible mechanisms and identified cellular targets by which NO increases proliferation of human breast cancer cell lines MDA-MB-231 and MCF-7. DETA-NONOate, a long acting NO donor, with a half-life of 20 h, was used. We found that NO (nmol/L) dramatically increased total protein synthesis in MDA-MB-231 and MCF-7 and also increased cell proliferation. NO specifically increased the translation of cyclin D1 and ornithine decarboxylase (ODC) without altering their mRNA levels or half-lives. Critical components in the translational machinery, such as phosphorylated mammalian target of rapamycin (mTOR) and its downstream targets, phosphorylated eukaryotic translation initiation factor and p70 S6 kinase, were up-regulated following NO treatment, and inhibition of mTOR with rapamycin attenuated NO induced increase of cyclin D1 and ODC. Activation of translational machinery was mediated by NO-induced up-regulation of the Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase/ERK (Raf/MEK/ERK) and phosphatidylinositol 3-kinase (PI-3 kinase)/Akt signaling pathways. Up-regulation of the Raf/MEK/ERK and PI-3 kinase/Akt pathways by NO was found to be mediated by activation of Ras, which was cyclic guanosine 3',5'-monophosphate independent. Furthermore, inactivation of Ras by farnesyl transferase inhibitor or K-Ras small interfering RNA attenuated NO-induced increase in proliferation signaling and cyclin D1 and ODC translation, further confirming the involvement of Ras activation during NO-induced cell proliferation.

Topics: Alkyl and Aryl Transferases; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Cyclic GMP; Dose-Response Relationship, Drug; Eukaryotic Initiation Factor-4E; Humans; MAP Kinase Signaling System; Nitric Oxide; Nitroso Compounds; Phosphatidylinositol 3-Kinases; Protein Biosynthesis; Protein Kinases; Proto-Oncogene Proteins c-akt; raf Kinases; TOR Serine-Threonine Kinases

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

The arginine vasopressin (AVP) gene is expressed in certain cancers such as breast cancer, where it is believed to act as an autocrine growth factor. However, little is known about the regulation of the AVP protein precursor (proAVP) or AVP-mediated signaling in breast cancer and this study was undertaken to address some of the basic issues. The cultured cell lines examined (Mcf7, Skbr3, BT474, ZR75, Mcf10a) and human breast cancer tissue extract were found to express proAVP mRNA. Western analysis revealed multiple forms of proAVP protein were present in cell lysates, corresponding to those detected in human hypothalamus extracts. Monoclonal antibodies directed against different regions of proAVP bound to intact live Mcf7 and Skbr3 cells. Dexamethasone increased the amount of proAVP-associated glycopeptide (VAG) secreted by Skbr3 cells and a combination of dexamethasone, IBMX and 8br-cAMP increased cellular levels of VAG. Exogenous AVP (1, 10, and 100 nM) elevated phospho-ERK1/2 levels, and increased cell proliferation was observed in the presence of 10 nM AVP. Concurrent treatment with the V1a receptor antagonist SR49059 reduced the effects of AVP on proliferation in Mcf7 cells, and abolished it in Skbr3 cells. Results here show that proAVP components are found at the surface of Skbr3 and Mcf7 cells and are also secreted from these cells. In addition, they show that AVP promotes cancer cell growth, apparently through a V1-type receptor-mediated pathway and subsequent ERK1/2 activation. Thus, strategies for targeting proAVP should be examined for their effectiveness in diagnosing and treating breast cancer.

Topics: 1-Methyl-3-isobutylxanthine; Anti-Inflammatory Agents; Antibodies, Monoclonal; Arginine Vasopressin; Blotting, Western; Breast Neoplasms; Cell Proliferation; Cyclic GMP; Dexamethasone; Female; Glycopeptides; Humans; Hypothalamus; Immunoenzyme Techniques; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Ovarian Neoplasms; Phosphodiesterase Inhibitors; Phosphorylation; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction

The synthesis of maspin, a protein with various anti-breast cancer properties has been reported to be produced in normal breast tissue but not in breast cancer cells. We investigated the role of insulin receptors and their upregulation by prostaglandin E(1) in vitro for the stimulation of NO synthesis by the hormone, and its consequence on maspin production in normal neutrophils and malignant cells in breast cancer.. Maspin and NO were determined by ELISA and methemoglobin method, respectively.. The treatment of neutrophils and malignant breast cancer cells with prostaglandin E(1) resulted in partial restoration of the impaired receptor numbers, and resulted in partial normalization of NO and maspin production in these cells compared to normal counterparts. It was not feasible to stimulate NO synthesis to normal ranges by the upregulation of receptor number due to intrinsic decrease of insulin receptors in breast cancer cells. However, aspirin, which was found to stimulate NO synthesis to normal ranges, normalized maspin production in these cells in breast cancer.. The impaired maspin production was found to be the consequence of impaired insulin induced NO production in breast cancer due to the decrease of insulin receptor binding. Restoration of NO production by aspirin can be useful for the restoration of maspin production in breast cancer.

Topics: Aged; Alprostadil; Aspirin; Binding, Competitive; Breast Neoplasms; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Genes, Tumor Suppressor; Humans; Insulin; Middle Aged; Neutrophils; Nitric Oxide; Receptor, Insulin; Sensitivity and Specificity; Serpins; Time Factors; Up-Regulation

Resveratrol (RSVL) is a well-established chemopreventive agent in human breast cancer models. The molecular basis of its action is far less characterized. We investigated the effects of RSVL on activity of adenylate- and guanylate-cyclase (AC, GC) enzymes; two known cytostatic cascades in MCF-7 breast cancer cells. RSVL increased cAMP levels in both time- and concentration-dependent manners (t(1/2), 6.2 min; EC(50) 0.8 micro M). In contrast, it had no effect on cGMP levels. The stimulatory effects for RSVL on AC were not altered either by the protein synthesis inhibitor (actinomycin-D, 5 micro M) or the estrogen-receptor (ER) blockers (tamoxifen and ICI182,780, 1 micro M each). Likewise, cAMP formation by RSVL was insensitive to either the broad-spectrum phosphodiesterase (PDE) inhibitor (IBMX, 0.5 mM) or the cAMP-specific PDE inhibitor (rolipram, 10 micro M). Instead, these PDE inhibitors significantly augmented maximal cAMP formation by RSVL. Parallel experiments showed that either RSVL or rolipram inhibited the proliferation of these cells in a concentration-responsive manner. Further, concurrent treatment with RSVL and rolipram significantly enhanced their individual cytotoxic responses. The antiproliferative effects were appreciably reversed by the kinase-A inhibitors, Rp-cAMPS (100-300 micro M) or KT-5720 (10 micro M). Pretreatment with the cPLA(2) inhibitor arachidonyl trifluoromethyl ketone (10 micro M) markedly antagonized the cytotoxic effects of RSVL, but had no effect on that of rolipram. Altogether, the present study demonstrates, for the first time, that the chemotherapeutic agent RSVL is an agonist for the cAMP/kinase-A system, a documented pro-apoptic and cell-cycle suppressor in breast cancer cells.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-AMP Phosphodiesterases; Adenylyl Cyclases; Antineoplastic Agents, Phytogenic; Arachidonic Acids; Breast Neoplasms; Cell Division; Cyclic AMP; Cyclic GMP; Dactinomycin; Enzyme Activation; Enzyme Inhibitors; Female; Guanylate Cyclase; Humans; Receptors, Estrogen; Resveratrol; Rolipram; Stilbenes; Tamoxifen; Tumor Cells, Cultured

Recently, we demonstrated that relaxin (RLX), a peptide hormone of ovarian origin, inhibits growth and promotes differentiation of MCF-7 breast adenocarcinoma cells. We also showed that RLX stimulates the production of nitric oxide (NO) in several cell types. NO has been reported to have antitumor activity by inhibiting proliferation, promoting differentiation, and reducing the metastatic spread of some tumor cell types. In this study, we aimed at evaluating whether RLX influences the L-arginine-NO pathway in MCF-7 cells. The cells were grown in the absence or presence of RLX at different concentrations, and cell proliferation, constitutive and inducible NO synthase activities, nitrite production, and intracellular levels of cyclic GMP were investigated. The results obtained indicate that RLX increases inducible NO synthase activity and potentiates NO production. This was accompanied by an elevation of intracellular cyclic GMP, which is known to mediate the cell response to NO. The RLX-induced activation of the L-arginine-NO pathway in the MCF-7 cells was inversely related to the rate of cell proliferation. These results suggest that the cytostatic effect of RLX on MCF-7 breast cancer cells may rely on its ability to stimulate endogenous production of NO.

Topics: Arginine; Breast Neoplasms; Cyclic GMP; Female; Humans; Nitric Oxide; Nitric Oxide Synthase; Relaxin; Thymidine; Tumor Cells, Cultured

Topics: Breast; Breast Neoplasms; Cell Count; Cell Division; Cyclic GMP; Female; Humans; Neoplasm Staging; Prognosis

The effects of salmon calcitonin (sCT) and human calcitonin (hCT) and of rat (r) and human (h) calcitonin gene-related peptide (CGRP) on intracellular cAMP accumulation were tested in human breast cancer cells (MCF7). In addition to the well known stimulatory effect, each showed a significant inhibitory effect on cAMP accumulation at low doses. cAMP concentrations in response to sCT, hCT, and rCGRP decreased to 47 +/- 2, 45 +/- 4, and 56 +/- 2% (mean +/- 1 SE) of baseline. The potency ratios for the inhibitory action of sCT, hCT, and rCGRP (1:0.25:0.005, respectively) were similar to the potency ratios for stimulatory action (1:0.3:0.005). The inhibition of cAMP accumulation developed at 300-fold lower peptide concentrations than the stimulation. Preincubation with pertussis toxin or with manganese completely abolished the inhibitory effect of the peptides, suggesting that this is mediated by an inhibitory adenylate cyclase regulatory protein. sCT, hCT, and CGRP each showed unique patterns with regard to time course of inhibition of cAMP accumulation. We conclude that 1) CT can activate an inhibitory adenylate cyclase regulatory protein and a stimulatory adenylate cyclase regulatory protein, and 2) CT effect on an inhibitory adenylate cyclase regulatory protein in MCF 7 cells is evident at far lower hormone concentrations than its effect on a stimulatory adenylate cyclase regulatory protein.

Topics: Adenylate Cyclase Toxin; Breast Neoplasms; Calcitonin; Calcitonin Gene-Related Peptide; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Female; Humans; Manganese; Neuropeptides; Pertussis Toxin; Time Factors; Tumor Cells, Cultured; Virulence Factors, Bordetella

Levels of cAMP and cGMP were higher in breast cancer tissue as compared with benign tumors. Large-fraction irradiation was followed by a decrease in cGMP concentration matched by a rise in cAMP/cGMP ratio in cases of radiosensitive tumors. Small-fraction treatment did not affect cyclic nucleotide levels. Radiotherapy was followed by a decrease in initially raised blood plasma cyclic nucleotide concentration.

Topics: Adenofibroma; Breast Neoplasms; Cyclic AMP; Cyclic GMP; Female; Fibrocystic Breast Disease; Humans

Indices of the T-system of immunity were lowered in breast cancer patients. Small fractional radiotherapy resulted in its greater suppression. A tumor revealed low pathomorphosis accompanied by a sharp increase in the levels of cyclic nucleotides. Large fractional radiotherapy caused no changes in the state of the immune system correlating with high radiation pathomorphosis in a tumor and a decrease in a cGMP level.

Topics: Adult; Breast Neoplasms; Cyclic AMP; Cyclic GMP; Female; Humans; Immunity, Cellular; Middle Aged

The levels of cyclic adenosine 3':5'-monophosphate (cAMP) and cyclic guanosine 3':5'-monophosphate (cGMP) were studied in dimethylbenz(a)anthracene (DMBA)-induced mammary tumors of Sprague-Dawley rats and in human breast cancer. In the rat carcinomas, these levels were significantly lower than in non-malignant tissues when calculated on the basis of DNA content, but higher (cAMP) or equal (cGMP) when calculated on the basis of weight. In human breast cancer the cyclic nucleotide levels were higher than in non-malignant tissues according to both methods of calculation. No correlation was found in human carcinomas between the cyclic nucleotide levels and mitotic index, nuclear grade, tumor size, or lymph node involvement. The rat tumors were subclassified according to state of differentiation, mitotic index, and state of development. Not all the sub-groups had cAMP levels different from normal values. Differences in cAMP levels between the sub-groups could not be correlated with tumor growth rates and/or mitotic index. Thus, cyclic nucleotides may not be useful in prognosis of breast cancer.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Adenofibroma; Animals; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cyclic AMP; Cyclic GMP; DNA, Neoplasm; Female; Fibrocystic Breast Disease; Humans; Mammary Neoplasms, Experimental; Prognosis; Rats; Rats, Inbred Strains

Estradiol binding levels have previously been shown to be increased or decreased by the addition of of cGMP or cAMP respectively to cellular homogenates prepared from human endometrial tissue or from the endometrial cell line HEC 1. Similar cyclic nucleotide dependent effects have now been demonstrated in homogenates prepared from a second endometrial cell line, HEC 50, and from two breast cancer cell lines, CG-5 and T47D. It was previously shown that ATP and Mg2+ are necessary for the generation or inactivation of E2 binding sites. The present study demonstrates that the concentration of K+ and of dithiothreitol can also affect these reactions.

Topics: Breast Neoplasms; Buffers; Cell Line; Cyclic AMP; Cyclic GMP; Dithiothreitol; Endometrium; Estradiol; Female; Humans; Magnesium; Potassium Chloride; Receptors, Estradiol; Receptors, Estrogen; Tromethamine

Cyclic guanosine 3',5' monophosphate (cyclic GMP) and cyclic adenosine 3',5' monophosphate (cyclic AMP) have been determined in random urine specimens from 95 healthy individuals, 60 patients with non-cancerous conditions, 52 patients with benign tumours, and 74 patients with malignant tumours. Concentrations of cyclic GMP have also been determined in a number of other groups, including some undergoing cancer treatment. Ninety-three per cent of cancer patients had raised urinary cyclic GMP concentrations compared to the reference range for healthy subjects. For the non-cancerous and benign groups, 33% and 42% respectively had raised concentrations. The urine cyclic AMP concentrations were similar in all groups. Urine cyclic GMP appeared to rise early in the onset of malignant growth. Successful cancer treatment was accompanied by a dramatic fall in the urine cyclic GMP concentrations, whereas if the treatment was unsuccessful the level did not change. It is concluded that urine cyclic GMP may have important applications in the monitoring of cancer treatment.

Topics: Adolescent; Adult; Aged; Breast Neoplasms; Cyclic AMP; Cyclic GMP; Female; Humans; Male; Middle Aged; Neoplasms; Ovarian Neoplasms; Uterine Cervical Neoplasms; Uterine Prolapse

Topics: Breast Diseases; Breast Neoplasms; Cell Division; Cyclic AMP; Cyclic GMP; Energy Metabolism; Female; Fibrocystic Breast Disease; Humans; Nucleotides, Cyclic; Phosphodiesterase Inhibitors

Topics: Animals; Breast Neoplasms; Cyclic AMP; Cyclic GMP; Female; Humans; Male; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Neoplasm Transplantation; Transplantation, Heterologous

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenofibroma; Adenylyl Cyclases; Breast; Breast Neoplasms; Carcinoma; Cyclic AMP; Cyclic GMP; Cytosol; Female; Humans; Hyperplasia

Intracellular levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate were measured in breast carcinomas. These levels were increased significantly as compared to normal breast tissues. In benign breast tumors, intermediate levels of adenosine 3'5'-cyclic monophosphate were observed, but they did not differ significantly with malignant tissues. No relation has been shown with estradiol or progesterone binding sites.

Topics: Breast Neoplasms; Cyclic AMP; Cyclic GMP; Estradiol; Female; Humans; Receptors, Estrogen; Receptors, Progesterone

Cyclic nucleotide phosphodiesterase activity was studied in 33 malignant neoplastic, 2 benign neoplastic, and 18 nonneoplastic human mammary tissues. Enzyme activity, using both cyclic adenosine 3':5'-monophosphate and cyclic guanosine 3':5'-monosphosphate as substrates, was measured in whole homogenates over a concentration range of 1 to 100 muM. Specific activity was calculated at substrate concentrations of 1 muM (low KM enzyme activity) and 100 muM (high KM activity). Diethylaminoethyl cellulose chromatography was used to separate the different enzyme species. The malignant neoplastic tissues had higher levels of both low-KM cyclic adenosine 3':5'-monophosphate and low-KM cyclic guanosine 3':5'-monophosphate phosphodiesterases. Further, the mean value of the ratio of low-km cyclic adenosine 3':5'-monophosphate to low-KM cyclic guanosine 3':5'-monophosphate activity was higher for the cancer tissues than for the nonneoplastic tissues. Diethylaminoethyl cellulose chromatography indicated the presence of three enzymes in both neoplastic and nonneoplastic mammary tissue. The kinetic as well as regulatory properties of the separated enzymes indicated that they are distinct enzyme activities. The phosphodiesterase properties were similar for neoplastic and nonneoplastic tissues and resembled those described previously in many other mammalian tissues. While both neoplastic and nonneoplastic tissues had detectable levels of the protein activator for phosphodiesterase, the cancer tissues appeared to have a higher level.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Breast; Breast Neoplasms; Calcium; Cyclic AMP; Cyclic GMP; Female; Humans; Kinetics; Phosphoric Diester Hydrolases; Proteins

Topics: Breast Neoplasms; Bucladesine; Cell Migration Inhibition; Colonic Neoplasms; Culture Media; Culture Techniques; Cyclic AMP; Cyclic GMP; Drug Synergism; Humans; Leukocytes; Lung Neoplasms; Neoplasms; Proteins; Theophylline; Tuberculin Test