cyclic-gmp and Melanoma

Human epidermal melanocytes as melanin producing skin cells represent a crucial barrier against UV-radiation and oxidative stress. It was shown that the intracellular signaling molecule cyclic guanosine-3',5'-monophosphate (cGMP), generated by the guanylyl cyclases (GCs), e.g., the nitric oxide (NO)-sensitive soluble GC (sGC) and the natriuretic peptide-activated particulate GC (GC-A/GC-B), plays a role in the melanocyte response to environmental stress. Importantly, cGMP is involved in NO-induced perturbation of melanocyte-extracellular matrix interactions and in addition, increased NO production during inflammation may lead to loss of melanocytes and support melanoma metastasis. Further, the NO-sensitive sGC is expressed predominantly in human melanocytes and non-metastatic melanoma cells, whereas absence of functional sGC but up-regulated expression of GC-A/GC-B and inducible NO synthase (iNOS) are detected in metastatic cells. Thus, suppression of sGC expression as well as up-regulated expression of GC-A/GC-B/iNOS appears to correlate with tumor aggressiveness. As the cGMP pathway plays important roles in melanocyte (patho)physiology, we present an overview on the differential effects of altered gravity (hypergravity/simulated microgravity) on the cGMP signaling pathway in melanocytes and melanoma cells with different metastatic potential. We believe that future experiments in real microgravity may benefit from considering cGMP signaling as a possible factor for melanocyte transformation and in medication.

Topics: Animals; Cyclic GMP; Gravity, Altered; Guanylate Cyclase; Humans; Melanocytes; Melanoma; Nitric Oxide; Nitric Oxide Synthase Type II; Signal Transduction

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenylyl Cyclases; Carcinoma, Hepatocellular; Cell Division; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Cyclic AMP; Cyclic GMP; Cytosine Nucleotides; DNA; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Growth Substances; Liver; Liver Neoplasms; Lymphocyte Activation; Lymphocytes; Melanoma; Mitogens; Neoplasms; Skin; Thymus Gland

Drug resistance mechanisms still characterize metastatic melanoma, despite the new treatments that have been recently developed. Targeting of the cGMP/protein kinase G pathway is emerging as a therapeutic approach in cancer research. In this study, we evaluated the anticancer effects of two polymeric-linked dimeric cGMP analogs able to bind and activate protein kinase G, called protein kinase G activators (PAs) 4 and 5. PA5 was identified as the most effective compound on melanoma cell lines as well as on patient-derived metastatic melanoma cells cultured as three-dimensional spheroids and in a zebrafish melanoma model. PA5 was able to significantly reduce cell viability, size, and invasion of melanoma spheroids. Importantly, PA5 showed a tumor-specific outcome because no toxic effect was observed in healthy melanocytes exposed to the cGMP analog. We defined that by triggering protein kinase G, PA5 interfered with the EGF pathway as shown by lower EGFR phosphorylation and reduction of activated, phosphorylated forms of protein kinase B and extracellular signal‒regulated kinase 1/2 in melanoma cells. Finally, PA5 significantly reduced the metastatic process in zebrafish. These studies open future perspectives for the cGMP analog PA5 as a potential therapeutic strategy for melanoma.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Melanocytes; Melanoma; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Skin Neoplasms; Zebrafish

Sildenafil, an inhibitor of the cGMP-degrading phosphodiesterase 5 that is used to treat erectile dysfunction, has been linked to an increased risk of melanoma. Here, we have examined the potential connection between cGMP-dependent signaling cascades and melanoma growth. Using a combination of biochemical assays and real-time monitoring of melanoma cells, we report a cGMP-dependent growth-promoting pathway in murine and human melanoma cells. We document that C-type natriuretic peptide (CNP), a ligand of the membrane-bound guanylate cyclase B, enhances the activity of cGMP-dependent protein kinase I (cGKI) in melanoma cells by increasing the intracellular levels of cGMP. Activation of this cGMP pathway promotes melanoma cell growth and migration in a p44/42 MAPK-dependent manner. Sildenafil treatment further increases intracellular cGMP concentrations, potentiating activation of this pathway. Collectively, our data identify this cGMP-cGKI pathway as the link between sildenafil usage and increased melanoma risk.

Topics: Animals; Butadienes; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic Nucleotide Phosphodiesterases, Type 5; Female; Humans; Melanoma; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Natriuretic Peptide, C-Type; Nitriles; Phosphodiesterase 5 Inhibitors; Protein Isoforms; Signal Transduction; Sildenafil Citrate; Transplantation, Homologous

Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations and effects of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). The role of PDEs in malignant tumor cells is still uncertain. The role of PDEs, especially PDE2, in human malignant melanoma PMP cell line was examined in this study. In PMP cells, 8-bromo-cAMP, a cAMP analog, inhibited cell growth and invasion. However, 8-bromo-cGMP, a cGMP analog, had little or no effect. PDE2 and PDE4, but not PDE3, were expressed in PMP cells. Growth and invasion of PMP cells were inhibited by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a specific PDE2 inhibitor, but not by rolipram, a specific PDE4 inhibitor. Moreover, cell growth and invasion were inhibited by transfection of small interfering RNAs (siRNAs) specific for PDE2A and a catalytically-dead mutant of PDE2A. After treating cells with EHNA or rolipram, intracellular cAMP concentrations were increased. Growth and invasion were stimulated by PKA14-22, a PKA inhibitor, and inhibited by N(6)-benzoyl-c AMP, a PKA specific cAMP analog, whereas 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, an Epac specific cAMP analog, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell line. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenine; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 2; Humans; Melanoma; RNA Interference; RNA, Small Interfering; Rolipram

We show that in melanoma cells oncogenic BRAF, acting through MEK and the transcription factor BRN2, downregulates the cGMP-specific phosphodiesterase PDE5A. Although PDE5A downregulation causes a small decrease in proliferation, its major impact is to stimulate a dramatic increase in melanoma cell invasion. This is because PDE5A downregulation leads to an increase in cGMP, which induces an increase in cytosolic Ca(2+), stimulating increased contractility and inducing invasion. PDE5A downregulation also this leads to an increase in short-term and long-term colonization of the lungs by melanoma cells. We do not observe this pathway in NRAS mutant melanoma or BRAF mutant colorectal cells. Thus, we show that in melanoma cells oncogenic BRAF induces invasion through downregulation of PDE5A.

Topics: Animals; Calcimycin; Calcium; Cardiac Myosins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Down-Regulation; Gene Expression; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 4 or More Rings; Homeodomain Proteins; Humans; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Myosin Light Chains; Neoplasm Invasiveness; Phosphodiesterase 5 Inhibitors; Phosphorylation; POU Domain Factors; Promoter Regions, Genetic; Protein Binding; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; RNA, Small Interfering; Transplantation, Heterologous

Despite increasing use of "targeted therapy," interleukin-2 (IL-2) is unique, because this cytokine can induce long-term remissions in 5% to 7% of patients with metastatic melanoma and renal cancer. Clinical use of IL-2 is limited by severe toxicities, such as hypotension and vascular leak syndrome (VLS). Nitric oxide seems to be involved in the pathogenesis of these toxicities. On the basis of previous studies, we hypothesized that the endothelial nitric oxide synthase (eNOS) is the major source of nitric oxide. Mice with a knockout of the eNOS isoenzyme were treated with IL-2 (800,000 IU twice daily for 5 d). Blood pressure and vascular leak were measured. Inhibitors of superoxide, nitric oxide, and soluble guanylate cyclase were used to probe the mechanism. These experiments showed that IL-2 treatment increased eNOS messenger ribonucleic acid expression and nitric oxide metabolite excretion in eNOS knockout mice. Unlike normal and inducible nitric oxide synthase knockout mice, eNOS knockout mice proved resistant to IL-2-induced hypotension and vascular leak. Although hypotension seems to be mediated by superoxide or peroxynitrite, vascular leak seemed to be mediated by nitric oxide. Inhibition of guanylate cyclase and cyclic guanylate monophosphate formation during IL-2 treatment using methylene blue (MB)-inhibited vascular leak. MB treatment did not interfere with IL-2-induced antitumor mechanisms. Our experiments established that eNOS is a key mediator of IL-2-induced VLS and hypotension. A clinical trial of MB infusion during IL-2 therapy is currently being planned.

Topics: Animals; Blood Pressure; Capillary Leak Syndrome; Carcinoma, Renal Cell; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Humans; Hypotension; Interleukin-2; Manganese; Melanoma; Methylene Blue; Mice; Mice, Inbred Strains; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase Type III; omega-N-Methylarginine; Organometallic Compounds; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Soluble Guanylyl Cyclase; Superoxides

Curcumin inhibits proliferation of many cancer cells. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing intracellular cyclic adenosine-3',5'-monophosphate (cAMP) and/or cyclic guanosine-3',5'-monophosphate (cGMP), play a pivotal role in signalling pathways involved in cell proliferation. Therefore, this study investigated PDE1-5 participations in the anti-proliferative properties of curcumin in B16F10 murine melanoma cells.. We report that curcumin inhibits PDE1-5 activities (IC(50) ≅10(-5)  M), indicating that curcumin acts as a non-selective PDE inhibitor. In melanoma cells, PDE4 and PDE1 represent the major cAMP-PDEs and cGMP-PDEs activities, respectively. Curcumin treatment decreased PDE1 and PDE4 activities and dose dependently increased intracellular cGMP levels, whereas cAMP levels were unchanged. Curcumin inhibited cell proliferation and cell cycle progression by accumulating cells in the S- and G2/M-phases with enhanced expressions of cyclin-dependent kinase inhibitors. In contrast, expressions of PDE1A, cyclin A and the epigenetic integrator ubiquitin-like containing PHD and Ring Finger domains 1 (UHRF1) and DNA methyltransferase 1 (DNMT1) were decreased by curcumin. Interestingly, PDE1A overexpression increased UHRF1 and DNMT1 expressions and rescued the B16F10 cells from curcumin anti-proliferative effects. Nimodipine, a PDE1 inhibitor, mimicked the curcumin effects.. Curcumin exerts its anti-cancer property by targeting PDE1 that inhibits melanoma cell proliferation via UHRF1, DNMT1, cyclin A, p21 and p27 regulations. This suggests that natural PDE1 inhibitors present in food might be effective in preventing cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; CCAAT-Enhancer-Binding Proteins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Curcumin; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 1; Cyclin-Dependent Kinase Inhibitor Proteins; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; Gene Expression Regulation, Neoplastic; Isoenzymes; Melanoma; Mice; Molecular Targeted Therapy; Neoplasm Proteins; Nuclear Proteins; Phosphodiesterase Inhibitors; Recombinant Proteins; RNA, Messenger; Ubiquitin-Protein Ligases

Malignant melanoma is one of the most aggressive human neoplasms which develop from the malignant transformation of normal epithelial melanocytes and share the lineage with retinal cells. cGMP-phosphodiesterase 6 (PDE6) is one of the cancer-retina antigens newly identified in melanoma cells. Normally, PDE6 hydrolyzes the photoreceptor second messenger cGMP allowing the visual signal transduction in photoreceptor cells. cGMP also play an important signaling role in stimulating melanogenesis in human melanocytes. Here, we present evidence that PDE6 is a key enzyme regulating the cGMP metabolism in melanoma cells. Decrease in intracellular cGMP leads to calcium accumulation in melanoma cells. In these cells, cGMP-phosphodiesterase 6 can be activated by another cancer-retina antigen, transducin, through Wnt5a-Frizzled-2 cascade, which leads to a lowering of cGMP and an increase in intracellular calcium mobilization. Thus, the aberrant expression of PDE6 may control cGMP metabolism and calcium homeostasis in melanoma cells.

Topics: Calcium; Cell Line, Tumor; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Frizzled Receptors; Homeostasis; Humans; Melanoma; Models, Biological; Protein Subunits; Proto-Oncogene Proteins; Receptors, G-Protein-Coupled; RNA, Small Interfering; Signal Transduction; Skin Neoplasms; Transducin; Wnt Proteins; Wnt-5a Protein

In our previous works, we have demonstrated that terfenadine (TEF) induces DNA damage and apoptosis in human melanoma cell lines. In this present work, we have studied the effect of histamine on viability of A375 human melanoma cells and the cell-signalling pathways through which TEF may induce its apoptotic effect. We have found that exogenous histamine stimulates A375 melanoma cell proliferation in a dose- and time-dependent manner. Moreover, TEF-induced apoptosis seems to occur via other cellular pathways independent of the histamine-signalling system since co-treatment of histamine with TEF did not protect melanoma cells from the cytotoxic effect of TEF, and alpha fluoromethylhistidine did not induce the same cytotoxic effect of TEF. In addition, we have observed that knocking down the H1 histamine receptor (HRH1) by small interference RNA approach protects melanoma cells only slightly from TEF-induced apoptosis. To explore the molecular mechanisms responsible for histamine and TEF effect on the cell growth, we analysed intracellular cyclic nucleotides and Ca(2+) levels. TEF did not modify intracellular levels of cyclic adenosine 3',5'-monophosphate and cyclic guanine 3',5'-monophosphate; however, TEF induced a very sharp and sustained increase in cytosolic Ca(2+) levels in A375 melanoma cells. On the contrary, histamine did not modulate intracellular Ca(2+). TEF-induced Ca(2+) rise and apoptosis appear to be phospholipase C (PLC) dependent since neomycin and U73122, two inhibitors of PLC, abolished cytosolic Ca(2+) increase and protected the cells completely from cell death. Furthermore, inhibition of tyrosine kinase activity by genistein blocked cytosolic Ca(2+) rise and TEF-induced apoptosis. These results suggest that TEF modulates Ca(2+) homeostasis and induces apoptosis through other cellular pathways involving tyrosine kinase activity, independently of HRH1.

Topics: Apoptosis; Base Sequence; Calcium; Cell Line, Tumor; Cyclic AMP; Cyclic GMP; DNA Primers; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Fluorescent Antibody Technique; Histamine H1 Antagonists, Non-Sedating; Homeostasis; Humans; Inositol Phosphates; Melanoma; Protein-Tyrosine Kinases; Receptors, Histamine H1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Terfenadine; Type C Phospholipases

There will be an estimated 59,940 new cases of melanoma and 8,110 deaths from melanoma in the United States in 2007. There has been no improvement in survival with melanomas in the last 22 years, with current treatment indicating that new treatment(s) of melanoma are drastically needed. Four cardiac hormones ie, atrial natriuretic peptide, vessel dilator, long-acting natriuretic peptide, and kaliuretic peptide, have significant anticancer effects in adenocarcinomas.. Dose-response curves evaluated the effects of these cardiovascular hormones on cell death and DNA synthesis in several melanoma cell lines in culture for 96 hours. Receptors to mediate these peptide hormones effects were examined in the melanoma cells with Western blots. Their intracellular mediator-analog 8-bromo-cyclic GMP was used to determine if it could mimic their effects on decreasing melanoma cell number and DNA synthesis.. The four cardiac hormones caused cell death in up to 71% (P < 0.001) of the melanoma cells within 24 hours. Cardiac hormone receptors (NPR-A, -B, -C) were present on the melanoma cells, and each of the peptide hormones decreased DNA synthesis within the melanoma cells up to 73% (P < 0.001) at their 1-microM concentrations. 8-Bromo-cyclic GMP mimicked their effects, decreasing the number of melanoma cells up to 67% and their DNA synthesis by 58% (both at P < 0.01).. These results indicate that 4 cardiac hormones have potent beneficial effects by increasing cell death in up to 71% of melanoma cells within 24 hours mediated in part by a 73% decrease in their DNA synthesis.

Topics: Adult; Atrial Natriuretic Factor; Cell Death; Cell Line, Tumor; Cyclic GMP; DNA, Neoplasm; Guanylate Cyclase; Humans; Male; Melanoma; Middle Aged; Peptide Fragments; Protein Precursors; Receptors, Atrial Natriuretic Factor; Skin Neoplasms; Time Factors

Gravity alteration (micro- and hypergravity) is known to influence cell functions. As guanosine 3',5'-cyclic monophosphate (cGMP) plays an important role in human melanocyte functions and different guanylyl cyclase isoforms are responsible for cGMP synthesis in human non-metastatic and metastatic melanoma cells, we investigated the effects of hypergravity on the regulation of cGMP levels in cultured human melanocytes and in melanoma cell lines with different metastatic potentials. Hypergravity was produced by horizontal centrifugal acceleration. Here we report that long-term application of hypergravity (up to 5 g for 24 h) stimulated cGMP efflux in cultured melanocytes and in non-metastatic melanoma cells in the presence of 0.1 mM 3-isobutyl-1-methylxanthine (IBMX), a non-selective phosphodiesterase (PDE) inhibitor. Under these conditions, cAMP synthesis and melanin production were up-regulated in pigmented melanocytes and non-metastatic melanoma cells. Hypergravity also stimulated cGMP transport in the presence of 1 microM trequinsin, an inhibitor of cGMP-binding PDE (PDE5) and of transport by multidrug resistance proteins MRP4/5, whereas 50 microM trequinsin partially inhibited cGMP transport. Transport was further inhibited by probenecid, an inhibitor of endogenous non-selective transporters as well as of MRP4/5 and by cycloheximide as an inhibitor of de novo protein synthesis. In contrast, hypergravity did not affect cGMP efflux in metastatic melanoma cells, which might be related to an up-regulated cGMP efflux at 1 g. The results of the present study indicate that hypergravity may stimulate cGMP efflux in melanocytes and in non-metastatic melanoma cells most probably by an enhanced expression of endogenous transporters and/or MRP4/5. Thus, an altered acceleration vector may induce signaling events in melanocytic cells.

Topics: Acceleration; Biological Transport; Centrifugation; Cyclic GMP; Enzyme Inhibitors; Humans; Hypergravity; Melanocytes; Melanoma

Previously, we showed that 5-norbornene-2,2-dimethanol (5-NBene-2,2-DM) is an effective inducer of melanogenesis in cultured cells and guinea-pig skin [Brown et al. (1998) J. Invest. Dermatol., 110:428-437]. This study shows that 2,3-cis/exo-pinanediol (2,3-cs/ex-PinD) is a more effective inducer of melanogenesis than 5-NBene-2,2-DM in S91 mouse melanoma cells. Furthermore, 2,3-cs/ex-PinD appears to penetrate guinea-pig skin better than 5-NBene-2,2-DM and to induce higher levels of pigmentation. Both 5-NBene-2,2-DM and 2,3-cs/ex-PinD induce synthesis of nitric oxide (NO) in S91 cells, and the melanogenic activity of both compounds is reduced by inhibitors of the NO/cyclic guanosine monophosphate (cGMP)/protein kinase(PK) G signaling pathway, but not by inhibitors of the PKC or PKA pathways. Thus, these bicyclic monoterpene diols appear to induce melanogenesis by the same pathway in S91 cells as that shown previously for ultraviolet radiation in melanocytes (Romero-Graillet et al. (1996) J. Biol. Chem., 271:28052-28056). These compounds also induce NO synthesis, neurite outgrowth, and tyrosine hydroxylase activity in PC12 pheochromocytoma cells. Neurite outgrowth in PC12 cells is blocked by the guanylate cyclase inhibitor, LY83583 (6-anilino-2,8-quinolinequinone), indicating that, similar to S91 cells, the induction of morphological differentiation of PC12 cells by bicyclic monoterpene diols is regulated by a cGMP-dependent pathway.

Topics: Animals; Boranes; Cell Cycle; Cell Differentiation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Guanosine Monophosphate; Guinea Pigs; Melanoma; Mice; Monophenol Monooxygenase; Nitric Oxide; Norbornanes; PC12 Cells; Protein Kinases; Rats; Terpenes; Tumor Cells, Cultured

Adhesion of circulating tumor cells to microvascular endothelium plays an important role in tumor metastasis to distant organs. The purpose of this study was to determine whether nitric oxide (NO) would attenuate tumor cell adhesion (TCA) to naive or lipopolysaccharide (LPS)-treated postcapillary venules. A melanoma cell line, RPMI 1846, was shown to be much more adhesive to postcapillary venules isolated from rat mesentery than to corresponding precapillary arterioles. Although venules exposed to LPS for 4 h demonstrated an increased adhesivity for the melanoma cells, TCA to LPS-treated arterioles was not altered. Isolated venules exposed to DETA/NO (1 mM), an NO donor, for 30 min prior to tumor cell perfusion prevented the increment in adhesion induced by LPS and attenuated TCA to naive postcapillary venules. While L-arginine (100 microM), an NO precursor, failed to decrease TCA to naive postcapillary venules, this treatment abolished LPS-stimulated TCA to postcapillary venules. The effect of L-arginine was reversed by administration of N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM), an NO synthase (NOS) inhibitor. These observations indicate that both exogenous and endogenous NO modulate TCA to postcapillary venules. To assess the role of NO-induced activation of cGMP in the reduction in TCA produced by DETA/NO, two additional series of experiments were conducted. In the first series, LY-83583 (10 microM), a guanylyl cyclase inhibitor, was shown to completely reverse the effect of DETA/NO on TCA to both naive and LPS-activated postcapillary venules. On the other hand, administration of 8-bromoguanosine 3',5'-cyclic monophosphate (8-B-cGMP) (1 mM), a cell permeant cGMP analog, mimicked the effect of DETA/NO and reduced TCA to LPS-stimulated postcapillary venules. These data suggest that (a) tumor cells are more likely to adhere to postcapillary venules than to corresponding precapillary arterioles, (b) LPS enhances TCA to postcapillary venules, (c) both exogenously applied (DETA/NO) and endogenously generated (L-arginine) NO attenuate the enhanced adhesion induced by LPS, but only DETA/NO reduced TCA to naive postcapillary venules, and (d) the NO-induced reduction in TCA to LPS-activated postcapillary venules occurs by a cGMP-dependent mechanism.

Topics: Aminoquinolines; Animals; Arterioles; Cell Adhesion; Cell Adhesion Molecules; Cricetinae; Cyclic AMP; Cyclic GMP; DEET; Endothelium, Vascular; Enzyme Inhibitors; Guanylate Cyclase; Lipopolysaccharides; Male; Melanoma; Nitric Oxide; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Tumor Cells, Cultured; Venules

The effect of congeners of nitrogen monoxide (NO) on iron (Fe) uptake from 59Fe-125I-transferrin (Tf) and release of 59Fe from prelabelled cells have been investigated in SK-MEL-28 human melanoma cells, human K562 cells and mouse MDW-4 cells. These studies have been initiated as it has been suggested that the tumoricidal effects of NO may be mediated by its acting to release Fe from cells (Hibbs et al., 1984 Biochem. Biophys. Res. Commun. 123, 716-723; Hibbs et al., 1988 Biochem. Biophys. Res. Commun. 157, 87-94). The nitrosonium ion (NO+) generator, sodium nitroprusside (SNP), decreased 59Fe uptake by melanoma cells to 57% of the control without decreasing 125I-Tf uptake after a 4-h incubation with 59Fe-125-Tf (1.25 microM). Longer incubations up to 24 h decreased 59Fe uptake and also 125I-Tf uptake. Two breakdown products of SNP, ferricyanide and cyanide, had no effect on 59Fe uptake. In addition, photolysis of the SNP solution prevented the inhibition of 59Fe uptake, suggesting that NO was the active agent. Two nitric oxide (NO.) producing agents, 3-morpholinosydnonimine (SIN), and S-nitroso-N-acetylpenicillamine (SNAP), also decreased 59Fe uptake from 59Fe-125I-Tf. Superoxide dismutase increased the efficacy of SIN, and the NO-scavenger, oxyhaemoglobin, prevented the inhibition of 59Fe uptake mediated by SNAP, again suggesting that NO was the active agent. Furthermore, dialysis studies demonstrated that none of the NO-generating agents could remove 59Fe from 59Fe-125I-Tf, suggesting that the decrease in cellular Fe uptake observed was not due to NO releasing Fe from the Fe-binding sites of Tf. Despite the ability of NO-producing agents at inhibiting 59Fe uptake by cells, they could not remove significant amounts of 59Fe from melanoma cells prelabelled with either 59Fe-citrate or 59Fe-125I-Tf. Similar data were obtained using K562 and MDW-4 cells. Interestingly, the NO+ generating agent, SNP, had no effect on [3H]thymidine uptake. However, when SNP was converted to an NO. generator by the addition of 1 mM ascorbate, its effect was similar to the NO. generator, SNAP, markedly reducing [3H]thymidine incorporation to 33% of the control value. The addition of unlabelled diferric Tf (0.625 microM) to SNAP ameliorated its inhibitory effect on cellular [3H]thymidine uptake, suggesting that the interaction of NO. with Fe was of importance in the inhibition observed. The results are discussed in the context of the cytostatic potential of NO via its binding to F

Topics: Animals; Cell Line; Cyclic GMP; Humans; Iodine Radioisotopes; Iron; Iron Radioisotopes; Melanoma; Mice; Molsidomine; Nitric Oxide; Nitrites; Nitroprusside; Thymidine; Transferrin

Nitric oxide (NO) and the NO generating agent nitroprusside (SNP), inhibit the binding of [125I] vasoactive intestinal peptide (VIP) to its receptor at the surface of IGR39 human melanoma cells. Cysteine (10 mM) increases the sensitivity of the system to SNP while N-acetylcysteine (10 mM) decreases it. The NO gas as well as SNP inhibits the [125I]VIP binding capacity. These observations sustain an effect of SNP-generated NO rather than an effect of the SNP molecule per se or the cyanoferrate portion of the molecule. The inhibitory effect of NO is time and concentration dependent and is fully reversible. Affinity constants of high and low affinity VIP receptors of SNP-treated IGR39 cells are not modified while maximal binding capacity (Bmax) of both receptor types are decreased to the same extent. Production of cGMP by SNP-treated cells is time and concentration dependent and the maximum amount of cGMP obtained reaches 13 times the basal level. The cAMP production is not affected by SNP. However, the SNP effects on the [125I]VIP binding are not mimicked by the membrane permeant cGMP analogs dibutyryl cGMP and 8-bromo cGMP even at concentrations as high as 0.5 mM. Taken altogether, these data demonstrate a regulatory action of NO on VIP binding capacity of IGR39 melanoma cells which is not cGMP mediated. They also evidence a new step which could be involved in the NO-VIP interaction.

Topics: Cyclic AMP; Cyclic GMP; Dibutyryl Cyclic GMP; Humans; Melanoma; Neoplasm Proteins; Nitric Oxide; Nitroprusside; Protein Binding; Receptors, Vasoactive Intestinal Peptide; Skin Neoplasms; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Mice with transplantable Harding-Passey melanoma were treated with polysaccharide mannan (50 mg/kg), cyclophosphamide (20 and 100 mg/kg) and their combinations. Mannan proved to possess considerable antitumor properties. It potentiated the therapeutic effect of cyclophosphamide and diminished its toxicity. Mannan treatment resulted in two-phase dynamics of cyclic nucleotide system in mouse liver and melanoma.

Topics: Animals; Antineoplastic Agents; Cyclic AMP; Cyclic GMP; Cyclophosphamide; Drug Therapy, Combination; Liver; Mannans; Melanoma; Mice; Neoplasm Transplantation; Neoplasms, Experimental; Nucleotides, Cyclic; Phosphoric Diester Hydrolases; Polysaccharides

Topics: Agar; Alprostadil; Animals; Cell Adhesion; Cell Division; Cell Line; Cyclic AMP; Cyclic GMP; Melanocyte-Stimulating Hormones; Melanoma; Mice; Prostaglandins A; Prostaglandins E

Topics: Animals; Cells, Cultured; Cyclic AMP; Cyclic GMP; Epinephrine; Fishes; Melanocytes; Melanoma; Melanophores; Pigments, Biological; Theophylline

The acute in vitro actions of two potent melanocytolytic agents, hydroquinone (HQ) and beta-mercaptoethanolamine (MEA), were determined in the B-16, Cloudman S-91 and Harding-Passey (HP) murine melanomas grown in vivo. Drug treated melanoma dice (5--480 min) were analyzed for tyrosinase activity and cyclic nucleotide levels (cAMP, cGMP). HQ and MEA effects on tyrosinase activity are complex and vary with tumor type, duration of treatment and agent tested. MEA or HQ inhibited B-16 tyrosinase activity. With combined drug therapy, low concentrations of MEA plus HQ stimulate B-16 tyrosinase activity while high concentrations of the drugs have little effect on enzymatic activity. MEA depresses tyrosinase activity while HQ elevates enzymatic activity in the S-19 melanoma. Both high and low concentrations of the combined drugs (MEA plus HQ) elicit the same response, stimulation at 10 min followed by continued depression of tyrosinase activity for the remainder of the 4 h study period. MEA initially stimulates HP tyrosinase activity followed by depression of enzymic activity. In contrast, HQ initially depresses HP tyrosinase activity followed by stimulation of enzyme activity. In combination the drugs inhibit HP tyrosinase activity. The effects of MEA and/or HQ on murine melanoma cyclic nucleotide levels are equally complex. MEA or HQ elevate cAMP and cGMP levels in all three tumors with the exception of S-91 cGMP levels which are not altered. In combination the drugs increase cyclic nucleotide levels in each of the three tumor types but at different times. No correlation is present between cyclic nucleotide levels and tyrosinase activity. Thus, the action of increased cyclic nucleotide levels in melanogenesis can not be separated from the direct actions of MEA and HQ upon melanogenesis. The divergent effects of MEA and/or HQ on tyrosinase activity and cyclic nucleotide levels in these melanomas are not correlated with the known in vivo melanocytolytic activity of these drugs. Thus, these parameters appear to be inadequate indicators of melanoma cell viability in chemotherapeutic screening of drugs effective in destroying malignant melanoma.

Topics: Animals; Catechol Oxidase; Cyclic AMP; Cyclic GMP; Hydroquinones; In Vitro Techniques; Melanocytes; Melanoma; Mercaptoethylamines; Mice; Mice, Inbred Strains; Monophenol Monooxygenase; Neoplasms, Experimental; Nucleotides, Cyclic

The acute in vitro action of ACTH and corticosterone individually and in combination were determined in the B-16 melanoma grown in vivo. ACTH elevated levels 10-fold while tyrosinase activity generally was depressed. Corticosterone depressed cAMP levels and tyrosinase activity. ACTH in the presence of corticosterone produced a coincident peak in tyrosinase activity and cAMP levels followed by a depression of enzymatic activity. The results demonstrate that cAMP is not the sole modulator of tyrosinase activity and that ACTH, corticosterone and cAMP interact in the regulation of B-16 melanoma tyrosinase activity.

Topics: Adrenocorticotropic Hormone; Animals; Corticosterone; Cyclic AMP; Cyclic GMP; In Vitro Techniques; Melanins; Melanoma; Mice; Monophenol Monooxygenase; Neoplasms, Experimental

Topics: Adult; Carcinoma, Basal Cell; Cyclic AMP; Cyclic GMP; Eczema; Humans; Leukocytes; Melanoma; Middle Aged; Nevus; Nucleotides, Cyclic; Skin Neoplasms; T-Lymphocytes

Changes in the concentration of cyclic AMP as well as cyclic GMP were measured in different murine tumors and in human tumors of varying malignancy. The quotient of cAMP and cGMP seems to be an important parameter for the molecular-biological derangement. Because of the recently much discussed importance of cAMP and cGMP in the immune defence the changes in the concentration of both nucleotides were measured in the T-lymphocytes of tumor patients. Significant changes occurred in patients with malignant melanoma. Investigations of the stimulatibility of the cAMP and cGMP levels revealed a diminished activatibility of the cAMP level and a higher stimulatibility of the cGMP level in the T-lymphocytes of patients with malignant melanoma as compared with those of the controls. On the basis of the working hypothesis that there is a causal relationship between the deranged dualism of cAMP and cGMP in the T-lymphocytes and the failure of the immunological tumor cell defence, an increase in the cAMP level is offered as a possible therapy. Therapeutic results in tumor-bearing mice and first results in melanoma patients are discussed.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cyclic AMP; Cyclic GMP; Drug Evaluation; Drug Evaluation, Preclinical; Humans; Melanoma; Mice; Neoplasm Transplantation; Neoplasms; Neoplasms, Experimental; Nevus; Skin; Skin Neoplasms; T-Lymphocytes; Time Factors

The portion of B- and T-lymphocytes at the whole white blood count have been estimated in patients with malignant melanoma. An elevation of the B-lymphocytes and a decrease of the T-lymphocytes could be observed in comparison to controls. The CAMP and CGMP level in lymphocytes of patients suffering from melanoma shows a correlation to the degree of metastasing changes. The cyclic nucleotides by mealanoma patients are influenced differently than by normal persons, after addition of physiologic stimulators (isoproterenole, PGE, PGF, acetylcholine). The beta-adrenergic system shows a decreased stimulability and the cholinergic system an increased one depending upon the disease stage in comparison to controls.

Topics: B-Lymphocytes; Cyclic AMP; Cyclic GMP; Humans; Immunity, Cellular; Leukocyte Count; Melanoma; Neoplasm Metastasis; Neoplasm Staging; Nucleotides, Cyclic; Reference Values; T-Lymphocytes

The acute in vitro action of adrenocorticotropin (ACTH) and corticosterone alone and in combination were determined in the Cloudman S-91 melanoma grown in vivo. Hormone-treated melanoma dice (5-240 min) were analyzed for tyrosinase activity (EC 1.14.18.1), cyclic AMP (cAMP) and cyclic GMP (cGMP). ACTH elevated cAMP levels in the S-91 melanoma. However, these increases in cAMP were not accompanied by increased tyrosinase activity. Corticosterone depressed cAMP levels while stimulating tyrosinase activity. ACTH plus corticosterone produced an early cAMP peak followed by depression. ACTH plus corticosterone stimulated tyrosine activity coincident with the early cAMP peak followed by a drop in tyrosinase activity which was subsequently elevated. cGMP levels were not altered by any hormone treatment. The results indicate that cAMP is not the sole modulator of tyrosinase activity and suggest the interaction of ACTH, corticosterone and cAMP in the regulation of melanoma tyrosinase activity.

Topics: Adrenocorticotropic Hormone; Animals; Corticosterone; Cyclic AMP; Cyclic GMP; Drug Interactions; In Vitro Techniques; Melanins; Melanoma; Mice; Mice, Inbred DBA; Monophenol Monooxygenase; Neoplasms, Experimental

The acute in vitro action of adrenocorticotropin (ACTH) and corticosterone alone and in combination were determined in the Harding-Passey (HP) melanoma grown in vivo. Hormone treated melanoma dice (5--240 min) were analyzed for tyrosinase activity, cyclic AMP (cAMP) and cyclic GMP (cGMP). ACTH elevated cAMP and cGMP levels 20- and 13-fold, respectively, in the HP melanoma. However, these large increases in cyclic nucleotide levels were accompanied by only a 49% increase in tyrosinase activity. Corticosterone elicited a similar response. ACTH plus corticosterone produced an early cAMP and cGMP peak followed by depression. ACTH plus corticosterone stimulated tyrosinase activity coincident with the early cyclic nucleotide peak followed by a drop in tyrosinase activity which was subsequently elevated. The results indicate that neither cAMP nor cGMP are the sole modulators of tyrosinase activity and suggest the interaction of ACTH, corticosterone and cyclic nucleotides in the regulation of melanoma tyrosinase activity.

Topics: Adrenocorticotropic Hormone; Animals; Corticosterone; Cyclic AMP; Cyclic GMP; Drug Interactions; In Vitro Techniques; Melanoma; Mice; Monophenol Monooxygenase; Neoplasms, Experimental; Nucleotides, Cyclic

In human tumor tissues of different degrees of differentiation--nevus-cell-nevus, basalioma, malign melanoma--the cAMP and cGMP content was determined and compared with the corresponding normal values. It is demonstrated that the quotient of the cAMP to the cGMP values is of importance rather than the latter values for themselves. For the benign tumor, this quotient differs only slightly from that of the adjacent normal, sound tissue. On the other hand, for the two malign tumors a drastic decrease of the quotient as compared to that of the normal tissue was found to occur.

Topics: Carcinoma, Basal Cell; Cyclic AMP; Cyclic GMP; Humans; Melanoma; Neoplasms; Nevus; Reference Values

The effect of systemic administration of 16,16-dimethyl prostaglandin E2-methyl ester (di-M-PGE2) on the growth of B-16 melanoma tumors has been studied in C57BL/6J mice. Daily i.p. injection of 5 mu of di-M-PGE2 commencing on the day of tumor inoculation with 10(5) and 10(6) viable cells delayed appearance of tumors; for the smaller tumor inoculum, it also increased median survival among treated mice from 23 to 33 days. Di-M-PGE2 treatment of mice with established tumors caused significant inhibition of tumor growth, as measured by a number of parameters including tumor diameters and volumes. At the time of sacrifice, di-M-PGE2-treated mice had tumors that were an average of 32% smaller (by weight), contained 60% fewer melanoma cells, and had higher concentrations of cyclic adenosine 3':5'-monophosphate and cyclic guanosine 3':5'-monophosphate (+225% and +100%, respectively).

Topics: Animals; Cell Count; Cell Division; Cyclic AMP; Cyclic GMP; Female; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms, Experimental; Prostaglandins E, Synthetic; Transplantation, Isogeneic