vasoactive-intestinal-peptide and Melanoma

Topics: Administration, Inhalation; Aged; Antibodies, Monoclonal, Humanized; Antineoplastic Agents, Immunological; Drug Combinations; Humans; Immunotherapy; Lung; Male; Melanoma; Phentolamine; Pneumonia; Programmed Cell Death 1 Receptor; Tomography, X-Ray Computed; Vasoactive Intestinal Peptide

Aggressive melanoma cells can engage in a process termed vasculogenic mimicry (VM) that reflects the ability of tumor cells to express a multipotent, stem cell-like phenotype. Melanoma cell plasticity contributes to the lack of efficient therapeutic strategies targeting metastatic tumors. This study reveals cyclic AMP as a mediator of VM in vitro. In uveal and cutaneous metastatic aggressive human melanoma cells, an increase in cyclic AMP by forskolin, dibutyryl cyclic AMP, or G protein-coupled receptor (GPCR) ligands such as adrenaline and vasoactive intestinal peptide inhibited VM to different extents. Although chemical modulators of protein kinase A (PKA) had no effect, a specific pharmacologic activator of Exchange protein directly activated by cyclic AMP (Epac) impaired VM. Ras-associated protein-1 (Rap1) activation assays revealed that cyclic AMP-elevating agents induce a PKA-independent activation of Epac/Rap1. Pharmacologic inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activity abolished VM. Phosphorylation of ERK1/2 was PKA-independently inhibited by forskolin but not inhibited by Epac/Rap1 signaling, PKA modulation, or GPCR ligands. Furthermore, the forskolin also inhibited phosphatidyl inositol-3-kinase (PI3K)-mediated activation of protein kinase Akt, as monitored by Ser473 phosphorylation. The pharmacologic activation of Epac and GPCR ligands slightly stimulated Akt, a likely concomitant process of VM modulation. Collectively, these data show that forskolin strongly inhibits VM through PKA-independent activation of Epac/Rap1, PKA-, and Epac-independent inactivation of ERK1/2 and inhibition of PI3K/Akt. The data also show that VM inhibition by GPCR ligands involves mainly the Epac/Rap1-activated signal. Thus cyclic AMP inhibits VM through multiple signaling pathways.

Topics: 1-Methyl-3-isobutylxanthine; alpha-MSH; Cell Line, Tumor; Colforsin; Cyclic AMP; Guanine Nucleotide Exchange Factors; Humans; Ligands; MAP Kinase Signaling System; Melanoma; Microvessels; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neovascularization, Pathologic; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptors, G-Protein-Coupled; Shelterin Complex; Signal Transduction; Telomere-Binding Proteins; Vasoactive Intestinal Peptide

A receptor capable of recognizing VIP-related peptides with unusual functional characteristics and selectivity profile was characterized on human IGR37 melanoma cells. When using either [125I]VIP or [125I]N-AcPACAP27 as a tracer, PACAP38 had the highest affinity, while PACAP27, VIP, helodermin, GRF and the VIP fragment VIP10-28 showed the same low affinity. Moreover, this receptor did not recognize PHM, PHV, helospectin, secretin, GIP, glucagon and glucagon-like peptide-1(7-36). Surprisingly, none of the peptides significantly stimulated the cAMP production. By covalent crosslinking, the receptor was shown to have a M(r) = 60,500.

Topics: Binding, Competitive; Cyclic AMP; Humans; Iodine Radioisotopes; Melanoma; Neuropeptides; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

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

Using mono[125I]iodinated vasoactive intestinal peptide (125I-VIP), a very high number of specific binding sites for VIP were identified at the surface of the human melanoma cell line IGR39. The Scatchard analysis of competitive displacement experiments between native VIP and 125I-VIP was consistent with the existence of two classes of VIP-binding sites. IGR39 cells possess 0.54 x 10(6) high-affinity sites with a dissociation constant (Kd) of 0.66 nM and 1.3 x 10(6) sites of moderate affinity with a Kd of 4.7 nM. Pharmacological studies indicated that the order of potency in inhibiting 125I-VIP binding of the VIP/secretin family peptides was VIP much greater than peptide histidine methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin. Glucagon has no effect on the binding of the labelled peptide. By means of photoaffinity labelling a polypeptide of Mr 63,000 was characterized. The labelling of this species was completely abolished by native VIP. The order of potency of VIP-related peptides in inhibiting 125I-VIP cross-linking to its receptor was the same as in the competition experiments. The glycoprotein nature of the VIP-binding sites of IGR39 cells has been investigated by affinity chromatography on wheat-germ-agglutinin-Sepharose.

Topics: Binding, Competitive; Cell Line; Chromatography, Affinity; Humans; Kinetics; Melanoma; Molecular Weight; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) stimulated in a dose-dependent manner the accumulation of cAMP in human melanoma-derived cell line IGR39. The maximal effect (about 100 times the basal level) was observed with 10 nM VIP. Half-maximum cAMP production was obtained at 0.78 nM VIP. VIP-related peptides were also potent in stimulating the cAMP production in IGR39 cells. The order of potency was VIP much greater than peptide histidine-methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin greater than glucagon. Using the same conditions, IGR37 cells, a metastasic counterpart of IGR39 cells, displayed a weak stimulation of cAMP production. After exposure of IGR39 cells to 10 nM VIP, the cAMP response to a new stimulation by VIP was strongly reduced. This desensitization of IGR39 cells to VIP was rapid (t1/2 less than 2 min) and homologous. Preincubation of IGR39 cells in the presence of native VIP induced disappearance of the VIP-binding sites at the cell surface. This phenomenon was dependent on time and VIP concentration. Maximum effect (loss of 80% of binding capacity) was obtained after exposure of the cells at 37 degrees C with a VIP concentration of 1 microM. The t1/2 of maximum disappearance was less than 2 min and the concentration of VIP giving half-maximum decrease in binding of mono[125I]iodinated VIP (125I-VIP) was 8 nM. This phenomenon was also reversible since 85% of the VIP-binding capacity could be restored in less than 1 h by incubating IGR39 cells in a VIP-free medium. The IGR39 cell line should be a useful model for further study of the structure and function of the human VIP receptor.

Topics: Cell Line; Cyclic AMP; Humans; Kinetics; Melanoma; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

By the peroxidase-antiperoxidase technique, the authors studied 7 malignant choroidal melanomas, 7 conjunctival nevi and 1 malignant conjunctival melanoma with the aim to detect the presence of vasoactive intestinal polypeptide (VIP), adrenocorticotropic hormone (ACTH), gastrin, estradiol and testosterone. Positive staining reaction for VIP, estradiol and testosterone was observed in both malignant melanomas of the choroid and conjunctival nevi. The case of conjunctival melanoma was positive for VIP and ACTH but not for estradiol and gastrin.

Topics: Adolescent; Adrenocorticotropic Hormone; Adult; Aged; Child; Child, Preschool; Choroid; Choroid Neoplasms; Conjunctiva; Conjunctival Neoplasms; Estradiol; Female; Gastrins; Hormones, Ectopic; Humans; Immunoenzyme Techniques; Male; Melanoma; Middle Aged; Nevus, Pigmented; Testosterone; Vasoactive Intestinal Peptide