msh--4-nle-7-phe-alpha- and Melanoma

The incidence of malignant melanoma is rising faster than that of any other cancer in the United States. Because of its high expression on the surface of melanomas, MC1R has been investigated as a target for selective imaging and therapeutic agents against melanoma. Eight ligands were screened against cell lines engineered to overexpress MC1R, MC4R, or MC5R. Of these, compound 1 (4-phenylbutyryl-His-dPhe-Arg-Trp-NH(2)) exhibited high (0.2 nM) binding affinity for MC1R and low (high nanomolar) affinities for MC4R and MC5R. Functionalization of the ligand at the C-terminus with an alkyne for use in Cu-catalyzed click chemistry was shown not to affect the binding affinity. Finally, formation of the targeted polymer, as well as the targeted micelle formulation, also resulted in constructs with low nanomolar binding affinity.

Topics: Alkynes; Antineoplastic Agents; Azides; Binding, Competitive; Click Chemistry; Drug Screening Assays, Antitumor; HCT116 Cells; HEK293 Cells; Humans; Ligands; Melanoma; Micelles; Oligopeptides; Polyethylene Glycols; Proteins; Receptor, Melanocortin, Type 1; Receptor, Melanocortin, Type 4; Receptors, Melanocortin; Structure-Activity Relationship

Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. Upon stimulation by αMSH, MC1R triggers the cAMP and ERK1/ERK2 MAPK pathways. In mouse melanocytes, ERK activation by αMSH binding to Mc1r depends on cAMP, and melanocytes are considered a paradigm for cAMP-dependent ERK activation. However, human MC1R variants associated with red hair, fair skin [red hair color (RHC) phenotype], and increased skin cancer risk display reduced cAMP signaling but activate ERKs as efficiently as wild type in heterologous cells, suggesting independent signaling to ERKs and cAMP in human melanocytes. We show that MC1R signaling activated the ERK pathway in normal human melanocytes and melanoma cells expressing physiological levels of endogenous RHC variants. ERK activation was comparable for wild-type and mutant MC1R and was independent on cAMP because it was neither triggered by stimulation of cAMP synthesis with forskolin nor blocked by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine. Stimulation of MC1R with αMSH did not lead to protein kinase C activation and ERK activation was unaffected by protein kinase C inhibitors. Conversely, pharmacological interference, small interfering RNA studies, expression profiles, and functional reconstitution experiments showed that αMSH-induced ERK activation resulted from Src tyrosine kinase-mediated transactivation of the stem cell factor receptor, a receptor tyrosine kinase essential for proliferation, differentiation, and survival of melanocyte precursors, thus demonstrating a functional link between the stem cell factor receptor and MC1R. Moreover, this transactivation phenomenon is unique because it is unaffected by natural mutations impairing canonical MC1R signaling through the cAMP pathway.

Topics: alpha-MSH; Animals; Cell Line, Tumor; Cyclic AMP; Enzyme Activation; HEK293 Cells; Humans; MAP Kinase Signaling System; Melanocortins; Melanocytes; Melanoma; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; PC12 Cells; Phosphorylation; Proto-Oncogene Proteins c-kit; Rats; Receptor, Melanocortin, Type 1; Signal Transduction; src-Family Kinases; Transcriptional Activation

The melanocortin 1 receptor, a G protein-coupled receptor positively coupled to adenylyl cyclase, is a key regulator of epidermal melanocyte proliferation and differentiation and a determinant of human skin phototype and skin cancer risk. Despite its potential importance for regulation of pigmentation, no information is available on homologous desensitization of this receptor. We found that the human melanocortin 1 receptor (MC1R) and its mouse ortholog (Mc1r) undergo homologous desensitization in melanoma cells. Desensitization is not dependent on protein kinase A, protein kinase C, calcium mobilization, or MAPKs, but is agonist dose-dependent. Both melanoma cells and normal melanocytes express two members of the G protein-coupled receptor kinase (GRK) family, GRK2 and GRK6. Cotransfection of the receptor and GRK2 or GRK6 genes in heterologous cells demonstrated that GRK2 and GRK6 impair agonist-dependent signaling by MC1R or Mc1r. However, GRK6, but not GRK2, was able to inhibit MC1R agonist-independent constitutive signaling. Expression of a dominant negative GRK2 mutant in melanoma cells increased their cAMP response to agonists. Agonist-stimulated cAMP production decreased in melanoma cells enriched with GRK6 after stable transfection. Therefore, GRK2 and GRK6 seem to be key regulators of melanocortin 1 receptor signaling and may be important determinants of skin pigmentation.

Topics: Adrenocorticotropic Hormone; alpha-MSH; Animals; beta-Adrenergic Receptor Kinases; Cell Line, Tumor; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; G-Protein-Coupled Receptor Kinases; Humans; Melanocytes; Melanoma; Mice; Mutation; Protein Serine-Threonine Kinases; Receptor, Melanocortin, Type 1; Receptors, G-Protein-Coupled; Signal Transduction; Skin Pigmentation; Transfection

The melanocortin-4 receptor (MC4) modulates physiological functions such as feeding behavior, nerve regeneration, and drug addiction. Using a high throughput screen based on (125)I-NDP-MSH binding to the human MC4 receptor, we discovered 2,3-diaryl-5-anilino[1,2,4]thiadiazoles 3 as potent and selective MC4 receptor agonists. Through SAR development on the three attached aryl rings, we improved the binding affinity from 174 nM to 4.4 nM IC(50). When delivered intraperitoneally, compounds 3a, 3b, and 3c induced significant inhibition of food intake in a fasting-induced feeding model in rats. When delivered orally, these compounds lost activity, mainly due to rapid metabolism to inactive imidoylthiourea reduction products.

Topics: alpha-MSH; Animals; Disease Models, Animal; Fasting; Feeding Behavior; Humans; Infusions, Parenteral; Inhibitory Concentration 50; Iodine Radioisotopes; Male; Melanoma; Rats; Rats, Long-Evans; Receptor, Melanocortin, Type 4; Receptors, Corticotropin; Structure-Activity Relationship; Thiadiazoles; Tumor Cells, Cultured

The localisation of melanocortin 1 receptor (MC1R) in WM266-4 human melanoma cells was investigated by applying an antipeptide antiserum specific for the cloned human MC1R (MSH receptor). In enzyme-linked immunosorbent assay (ELISA), the immunoreactivity was detected in the membrane fraction of WM266-4 cells. The ELISA reactivity could be inhibited by an antiserum pre-absorbed with its specific synthetic peptide. In immunocytochemistry, the specific immunoreactivity was demonstrated on the surface of the cells by using either biotin-avidin immunoalkaline phosphatase- or TRITC-staining method. These results indicate that the MC1R is prominently present on the plasma membrane of WM266-4 human melanoma cells.

Topics: Affinity Labels; alpha-MSH; Cell Membrane; Humans; Immunohistochemistry; Iodine Radioisotopes; Kinetics; Melanocyte-Stimulating Hormones; Melanoma; Radioligand Assay; Receptors, Pituitary Hormone; Tumor Cells, Cultured

Seventeen human melanoma cell (HMC) lines, both melanotic and amelanotic, were incubated in the continuous presence of a potent melanotropic peptide hormone analog, [Nle4,D-Phe7] alpha-MSH, for 72 hr with daily changes of medium. Only one cell line (HD, melanotic) consistently responded to the hormone analog by increased tyrosinase activity. Three (one melanotic, two amelanotic) of the HMC lines also failed to respond to the peptide by either increased or decreased enzyme activity when incubated continuously in the presence of the peptide for longer periods of time (6,15,27,43 days). The HD cell line, however, again responded with increasingly enhanced basal enzyme activity the longer the cells were incubated in the presence of the melanotropin. One amelanotic cell line (C8161) responded with enhanced enzyme activity when grown to confluency in the continuous presence of the peptide. Basal tyrosinase activity of the C8161 cell line may have increased as cell density in the flasks increased. These results suggest that under conditions of increased cell number, phenotypic expression of tyrosinase activity in so called "amelanotic" (tyrosinase-negative) cells is increased and can be enhanced further by stimulation with a melanotropic peptide. Under conditions of increased cell number, the presence of [Nle4,D-Phe7] alpha-MSH caused morphological differentiation (shape change); the cells became enlarged and very dendritic. The number of cells in monolayer (surface of the flask) and in the medium were drastically reduced in both melanotic and "amelanotic" cell lines incubated with [Nle4,D-Phe7] alpha-MSH. The data support other published reports that melanotropic peptides inhibit human melanoma cell growth (proliferation) in vitro, most likely through a cytostatic mechanism. [Nle4,D-Phe7] alpha-MSH also exhibited a prolonged (residual) inhibitory action on HD cell proliferation. In other words, inhibition of cell growth (proliferation) of the HMCs was evident even several days after removal of the melanotropic peptide from the incubation medium.

Topics: alpha-MSH; Animals; Cell Division; Cyclic AMP; Humans; Melanoma; Mice; Monophenol Monooxygenase; Phosphodiesterase Inhibitors; Receptors, Pituitary Hormone; Theophylline; Tumor Cells, Cultured

Investigation of physiological effects of alpha-MSH and its analog (NLe4, D-Phe7)-alpha-MSH on human melanoma cells with different phenotypes has shown that these peptides have a growth-modulating activity. The effect of inhibition or activation of the growth of melanoma cells depended on their phenotypes. (NLe4, D-Phe7)-alpha-MSH activated 1.5-2.5-fold the growth of amelanotic BRO cells at concentrations of 10(-6)-10(-12) M, but inhibited the growth of melanin-producing MS cells under the same conditions not affecting the growth of human lung fibroblasts.

Topics: alpha-MSH; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Fibroblasts; Humans; Lung; Melanoma; Phenotype; Tumor Cells, Cultured

Melanocyte stimulating hormone (alpha-MSH, alpha-melanotropin),Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Ly-Pro-Va l-NH2, regulates melanogenesis within epidermal melanocytes of many animals. An MSH analogue ([Nle4,D-Phe7]alpha-MSH) that exhibits superpotency and prolonged biological activity has been synthesized, biologically characterized, and is presently in clinical trials to determine its possible clinical use in tanning of the skin. It also has potential for the diagnosis, localization, and chemotherapy of melanoma. The effects of this analogue on the growth, metastatic behavior, and invasive potential of a melanotic variant of Cloudman S-91 murine melanoma are reported here. In an intracutaneous murine model of melanoma cell tumor growth, the analogue did not increase primary tumor growth (size) after the period of administration of the peptide hormone analogue and did not affect spontaneous lung metastases. Survival times for the control and melanotropin-treated groups were similar, suggesting that overall tumor burden was not affected by treatment with the hormone analogue. Last, melanoma cell invasion through a human amniotic basement membrane in vitro was not enhanced compared to untreated cells.

Topics: alpha-MSH; Amino Acid Sequence; Animals; Cell Division; Female; Humans; Melanocyte-Stimulating Hormones; Melanoma; Mice; Mice, Inbred DBA; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Tumor Cells, Cultured

Although alpha-MSH increases skin darkening in humans, there are several reports that it fails to have melanogenic effects on human melanocytes in vitro. The purpose of this study was to see whether cultured human melanocytes express MSH receptors. Human melanocytes were grown in the absence of artificial mitogens such as 12-O-tetradecanoyl phorbol-13-acetate (TPA) and cholera toxin (CT) and incubated for 2 h at room temperature with increasing amounts of 125I-labelled Nle4DPhe7-alpha-MSH with and without excess cold peptide. Binding was saturable and specific: Scatchard analysis gave a Kd of 4.9 x 10(-11) M and approximately 700 binding sites/cell. Human keratinocytes and fibroblasts showed no specific binding. The addition of 1 mM dibutyryl cAMP to the culture medium caused a 62% increase in MSH binding to human melanocytes. A smaller increase (25%) was seen with 10(-9) M CT while 25 mM TPA caused a 24% decrease. These results show that human melanocytes in culture express MSH receptors and that this expression can be modulated by mitogens.

Topics: alpha-MSH; Animals; Cells, Cultured; Cholera Toxin; Cyclic AMP; Fibroblasts; Humans; Keratinocytes; Melanocytes; Melanoma; Mice; Receptors, Pituitary Hormone; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Three different monoiodinated radioligands of alpha-MSH (alpha-melanocyte-stimulating hormone) were compared in a binding assay with human D10 melanoma cells: [Tyr(125I)2]-alpha-MSH, [Tyr(125I)2,NIe4]-alpha-MSH, and [Tyr(125I)2,NIe4,D-Phe7]-alpha-MSH. They were prepared either by the classical chloramine T method or by the Enzymobead method. A simple and rapid purification scheme was developed consisting of a primary separation on reversed-phase C18 silica cartridges immediately after the iodination, followed by HPLC purification before each binding experiment. Biological testing of the three radioligands showed that they all retained high melanotropic activity in the B16 melanin assay and the Anolis melanophore assay. However, in human D10 melanoma cells, [Tyr(125I)2,NIe4]-alpha-MSH led to a high degree of non-specific binding to the cells which could not be displaced by excess alpha-MSH and only partially by [NIe4]-alpha-MSH. The [Tyr(125I)2,NIe4,D-Phe7]-alpha-MSH tracer gave similar results but with a much lower proportion of non-specific binding. On the other hand, [Tyr(125I)2]-alpha-MSH proved to be an excellent radioligand whose non-specific binding to the D10 cells was not higher than 20% of the total binding.

Topics: alpha-MSH; Biological Assay; Chloramines; Chromatography, High Pressure Liquid; Chromatography, Liquid; Humans; Iodine Radioisotopes; Isotope Labeling; Melanoma; Radioligand Assay; Receptors, Pituitary Hormone; Tosyl Compounds; Tumor Cells, Cultured

We show that Cloudman melanoma cells undergo rapid arborization in response to [Nle4,D-Phe7]alpha-melanocyte-stimulating hormone, a potent analogue of alpha-melanocyte stimulating hormone (alpha-MSH). The arbors were established by extension of processes and resembled dendrites. We used this system to study the regulation of cell shape. alpha-MSH is known to induce increases in cAMP levels, and agents such as forskolin and isobutylmethylxanthine that led to increased cAMP also caused arborization. However, equally dramatic arbors were formed after incubation with the protein kinase C inhibitor H-7 [1-(5-isoquinolinesulfonyl)-alpha-methyl-piperazine]. Phorbol diesters that activate protein kinase C led to cell rounding and antagonized alpha-MSH. The actions of protein kinase C cannot be rationalized in terms of indirect effects on cAMP: neither H-7 nor phorbol diesters alone altered cAMP levels, nor did they affect the increase in cAMP induced by MSH. We show also that MSH produced longer-term effects that cannot be mimicked by cAMP. Specifically, even in the continued presence of alpha-MSH, arborization was followed by morphological reversal to the unstimulated flattened configuration within 2 hr. (This did not occur with other agents that increase cAMP or with H-7.) Most importantly, whereas MSH-induced arborization occurred in the presence of cycloheximide, actinomycin D, or in enucleated cells, the reversal of arborization did not. Thus, MSH induced a program of rapid shape change that was dependent on new protein synthesis and gene transcription.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; alpha-MSH; Animals; Axons; Cell Line; Cyclic AMP; Cycloheximide; Dactinomycin; Fluorescent Antibody Technique; Isoquinolines; Melanocyte-Stimulating Hormones; Melanoma; Mice; Microscopy, Electron; Piperazines; Protein Kinase C; Sulfonamides; Tetradecanoylphorbol Acetate; Time Factors; Transcription, Genetic

alpha-Melanotropin (alpha-melanocyte stimulating hormone, alpha-MSH) stimulates tyrosinase activity in Cloudman S91 murine melanoma cells. Three [Nle4, D-Phe7]-substituted alpha-melanotropin analogues, [Nle4, D-Phe7]-alpha-MSH, Ac-[Nle4, D-Phe7]-alpha-MSH4-11-NH2, and Ac-[Nle4, D-Phe7]-alpha-MSH4-10-NH2, are at least 100-fold more effective than alpha-MSH in stimulating melanoma tyrosinase, the rate-limiting enzyme in melanin biosynthesis. These [Nle4, D-Phe7]-substituted melanotropin analogues induce tyrosinase activity in melanoma cells with shorter contact times than required by the native hormone, alpha-MSH. [Nle4, D-Phe7]-substituted melanotropins also induce a prolonged (residual) stimulation of melanoma tyrosinase. Following incubation of melanoma cells in the presence of [Nle4, D-Phe7]-alpha-MSH for 24 h, tyrosinase activity is maintained for up to 6 days in the absence of the melanotropin. The shorter 4-10 and 4-11 fragment analogues also exhibit residual melanotropic activity. The prolonged stimulation of tyrosinase in the absence of the analogues is maintained even though melanoma cells continue to divide about every 24 h. These results suggest that melanoma cells possess spare melanotropin receptors and that [Nle4, D-Phe7]-substituted analogues bind almost irreversibly to these receptors or to some other component of the adenylate cyclase enzyme complex responsible for enhancing tyrosinase activity and melanin production.

Topics: alpha-MSH; Animals; Catechol Oxidase; Cell Division; Enzyme Induction; Melanocyte-Stimulating Hormones; Melanoma; Mice; Mice, Inbred DBA; Monophenol Monooxygenase; Peptide Fragments; Receptors, Cell Surface; Receptors, Pituitary Hormone; Structure-Activity Relationship

The superpotent and ultraprolonged melanotropic properties of an alpha-melanotropin analog, [Nle4, D-Phe7]-alpha-MSH, were investigated in a Cloudman S91 (CCL 53.1) melanoma cell line. [Nle4, D-Phe7]-alpha-MSH is 100-fold more effective than the native hormone, alpha-melanocyte stimulating hormone (alpha-MSH), in stimulating melanoma cell tyrosinase activity, as determined from their minimum effective doses (10(-11)M and 10(-9)M, respectively). [Nle4, D-Phe7]-alpha-MSH also exhibits a more sustained effect than alpha-MSH on tyrosinase after removal of the melanotropins from the incubation medium. When cells were exposed to alpha-MSH (10(-7)M) for 24 h, residual activity after removal of the hormone was minimally significant. In contrast, under the same experimental conditions [Nle4, D-Phe7]-alpha-MSH treatment induced tyrosinase activity 2-3 fold above basal level, and maintained remarkable stimulatory effects up to 72 h following melanotropin removal. When the exposure time to melanotropins was reduced to 4 h, alpha-MSH failed to elicit significant tyrosinase activity, whereas [Nle4, D-Phe7]-alpha-MSH stimulated significant tyrosinase activity during the first 24 h subsequent to melanotropin removal. Interestingly, this stimulation by the analog increased at 48 h, reached a maximum at 72 h following removal of the melanotropin analog, and remained significantly stimulated for 6 consecutive days in the absence of the analog.

Topics: Adenylyl Cyclases; alpha-MSH; Animals; Catechol Oxidase; Cell Division; Cell Line; Enzyme Activation; Melanocyte-Stimulating Hormones; Melanoma; Mice; Monophenol Monooxygenase; Receptors, Cell Surface; Receptors, Pituitary Hormone; Time Factors

Biocytin derivatives of a superpotent analogue of alpha-melanotropin, [Nle4,D-Phe7]-alpha-MSH, were prepared. [N alpha-Bct-Ser1, Nle4,D-Phe7]-alpha-MSH and [12-Bct-N alpha-dodecanoyl-Ser1,Nle4,D-Phe 7]- alpha-MSH were synthesized by solid-phase techniques, and the coupling of biotin and 12-aminododecanoic acid was achieved through their succinimido esters. These melanotropins possessed almost identical actions to [Nle4,D-Phe 7]- alpha-MSH as determined by several melanocyte bioassays. Both biocytin derivatives were highly potent agonists and exhibited prolonged biological activity as determined in the frog and lizard skin bioassays. Both biotinylated peptides were at least equipotent to alpha-MSH in stimulating Cloudman S91 mouse melanoma tyrosinase activity. The analogues were resistant to inactivation by alpha-chymotrypsin.

Topics: alpha-MSH; Animals; Biological Assay; Biotin; Lizards; Melanocyte-Stimulating Hormones; Melanoma; Monophenol Monooxygenase; Rana pipiens; Skin

Topics: Adenylyl Cyclases; Amino Acids; Animals; Chemical Phenomena; Chemistry, Physical; Lizards; Melanocyte-Stimulating Hormones; Melanoma; Rana pipiens; Skin; Skin Pigmentation; Time Factors

alpha-Melanocyte-stimulating hormone (alpha-MSH) reversibly darkens frog skins by stimulating melanosome movement (dispersion) within melanophores. Heat-alkali treatment of alpha-MSH results in prolonged biological activity of the hormone. Quantitative gas chromatographic analysis of the hydrolyzed heat-alkali-treated peptide revealed partial racemization particularly at the 4(methionine) and 7(phenylalanine) positions. [Nle4]-alpha-MSH, a synthetic analogue of alpha-MSH, reversibly darkens frog skins and also exhibits prolonged activity after heat-alkali treatment. Synthesis of [Nle4, D-Phe7]-alpha-MSH provided an analogue with prolonged biological activity identical to that observed with heat-alkali-treated alpha-MSH or [Nle4]-alpha-MSH. [Nle4, D-Phe7]-alpha-MSH was resistant to enzymatic degradation by serum enzymes. In addition, this peptide exhibited dramatically increased biological activity as determined by frog skin bioassay, activation of mouse melanoma adenylate cyclase, and stimulation of mouse melanoma cell tyrosinase activity. This Nle4, D-Phe7 synthetic analogue of alpha-MSH is a very porent melanotropin, 26 times as potent as alpha-MSH in the adenylate cyclase assay. The resistance of the peptide to enzymatic degradation and its extraordinarily potent and prolonged biological activity should make this analogue of alpha-MSH an important molecular probe for studying the melanotropin receptors of both normal and abnormal (melanoma) melanocytes.

Topics: alpha-MSH; Animals; Biological Assay; Cells, Cultured; Chemical Phenomena; Chemistry; Dose-Response Relationship, Drug; Melanocyte-Stimulating Hormones; Melanoma; Monophenol Monooxygenase; Structure-Activity Relationship