Synthesis, characterization and optimization of in vitro properties of NIR-fluorescent cyclic α-MSH peptides for melanoma imaging.

Melanoma are malignant tumors derived from melanocytes being responsible for the majority of skin cancer deaths with an increasing rate of incidence. The Melanocortin-1 receptor (MC1R) has been recognized as a molecular target for melanoma detection. Here, we report on the development and optimization of molecular probes which are based on novel conjugates of near-infrared (NIR) fluorescent indocyanine dyes and an MC1R-targeting peptide intended for optical fluorescence imaging enabling an early, specific, accurate and sensitive diagnosis of malignant melanomas. The introduction of anionic groups into the aromatic ring of the indolenine substructure of the conjugated dyes has shown to result in a strong fluorescence in aqueous solution and a concomitant increase of binding affinities of the peptide conjugates to the target receptor. The length and flexibility of the PEG chain introduced as a linker, as well as the nature of its attachment to the dye also affect the binding affinities, albeit to a lower extent. The conjugates have been successfully applied in the MC1R-specific staining of B16F10 melanoma cells, both in cell cultures and in microtome sections of solid tumors.

Psychosocial and Cultural Determinants of Interest and Uptake of Skin Cancer Genetic Testing in Diverse Primary Care.

BACKGROUND: Translational research in genomics has limited reach and requires efforts to broaden access and utility in diverse populations. Skin cancer is common and rates are rising, including among Hispanics. Germline variants in the melanocortin-1 receptor (MC1R) gene are common in the population and confer moderate risk for melanoma and basal cell cancers across skin types. Feedback about MC1R risk status may promote skin cancer risk awareness and risk reduction. AIMS: We examined the level of interest in pursuing MC1R testing, and patterns of interest across skin cancer perceived threat and control attitudes, cultural beliefs (family influence on health, health system distrust, cancer fatalism, skin cancer misconceptions), and health literacy. METHODS: We used a study website to inform primary care patients in Albuquerque, NM about the benefits and drawbacks of MC1R testing. Website logon, request of a saliva test kit, and return of the test kit (yes vs. no) were primary assessments of study interest and uptake. RESULTS: Of 499 participants provided with a test offer, 33% requested and returned the test. Lower family influence on participants' health was an important factor both overall and within ethnicity subgroups, and may indicate that primary care patients interested in skin cancer genetic testing see themselves as proactive health seekers, independent from family encouragement. Lower self-efficacy for skin cancer prevention was also an important characteristic of those who tested. CONCLUSION: As evidence for common genetic markers for skin cancer accumulates, these findings suggest characteristics of those most likely to pursue genetic testing for skin cancer risk.

A flow-through microfluidic system for the detection of circulating melanoma cells.

We report the fabrication of polyaniline nanofiber (PANI)-modified screen-printed electrode (PANI/SPE) incorporated in a poly-dimethylsiloxane (PDMS) microfluidic channel for the detection of circulating tumor cells. We employed this device to detect melanoma skin cancer cells through specific immunogenic binding of cell surface biomarker melanocortin 1 receptor (MC1R) to anti-MC1R antibody. The antibody-functionalized PANI/SPE was used in batch-continuous flow-through fashion. An aqueous cell suspension of ferri/ferrocyanide at a flow rate of 1.5 mL/min was passed over the immunosensor, which allowed for continuous electrochemical measurements. The sensor performed exceptionally well affording an ultralow limit of quantification of 1 melanoma cell/mL, both in buffer and when mixed with peripheral blood mononuclear cells, and the response was log-linear over the range of 10-9000 melanoma cells/10 mL.

Amplification of the Melanocortin-1 Receptor in Nephrotic Syndrome Identifies a Target for Podocyte Cytoskeleton Stabilization.

The melanocortin-1 receptor (MC1R) in podocytes has been suggested as the mediator of the ACTH renoprotective effect in patients with nephrotic syndrome with the mechanism of action beeing stabilization of the podocyte actin cytoskeleton. To understand how melanocortin receptors are regulated in nephrotic syndrome and how they are involved in restoration of filtration barrier function, melanocortin receptor expression was evaluated in patients and a rat model of nephrotic syndrome in combination with cell culture analysis. Phosphoproteomics was applied and identified MC1R pathways confirmed using biochemical analysis. We found that glomerular MC1R expression was increased in nephrotic syndrome, both in humans and in a rat model. A MC1R agonist protected podocytes from protamine sulfate induced stress fiber loss with the top ranked phoshoproteomic MC1R activated pathway beeing actin cytoskeleton signaling. Actin stabilization through the MC1R consisted of ERK1/2 dependent phosphorylation and inactivation of EGFR signaling with stabilization of synaptopodin and stressfibers in podocytes. These results further explain how patients with nephrotic syndrome show responsiveness to MC1R receptor activation by decreasing EGFR signaling and as a consequence restore filtration barrier function by stabilizing the podocyte actin cytoskeleton.

The alpaca melanocortin 1 receptor: gene mutations, transcripts, and relative levels of expression in ventral skin biopsies.

The objectives of the present study were to characterize the MC1R gene, its transcripts and the single nucleotide polymorphisms (SNPs) associated with coat color in alpaca. Full length cDNA amplification revealed the presence of two transcripts, named as F1 and F2, differing only in the length of their 5'-terminal untranslated region (UTR) sequences and presenting a color specific expression. Whereas the F1 transcript was common to white and colored (black and brown) alpaca phenotypes, the shorter F2 transcript was specific to white alpaca. Further sequencing of the MC1R gene in white and colored alpaca identified a total of twelve SNPs; among those nine (four silent mutations (c.126C>A, c.354T>C, c.618G>A, and c.933G>A); five missense mutations (c.82A>G, c.92C>T, c.259A>G, c.376A>G, and c.901C>T)) were observed in coding region and three in the 3'UTR. A 4 bp deletion (c.224 227del) was also identified in the coding region. Molecular segregation analysis uncovered that the combinatory mutations in the MC1R locus could cause eumelanin and pheomelanin synthesis in alpaca. Overall, our data refine what is known about the MC1R gene and provides additional information on its role in alpaca pigmentation.

Características clínicas de los pacientes con melanoma cutáneo en función de las variaciones en el gen del receptor 1 de la melanocortina (MC1R) / Clinical Characteristics of Patients With Cutaneous Melanoma According to Variants in the Melanocortin 1 Receptor Gene

INTRODUCCIÓN: Los pacientes con melanoma cutáneo portadores de polimorfismos en MC1R presentan unas características clínicas distintivas. El objetivo de este estudio ha sido caracterizar desde el punto de vista clínico las diferencias en el grado de afectación funcional del receptor debidas al número y tipo (R y r) de los polimorfismos del gen MC1R de los pacientes. MATERIAL Y MÉTODOS: Se seleccionaron 1.044 pacientes con melanoma diagnosticados en nuestro centro de forma consecutiva desde enero del año 2000 a partir de la base de datos de melanoma. Se clasificaron en 3 grupos según una puntuación asociada a los polimorfismos no sinónimos del gen MC1R y se compararon las frecuencias observadas de cada una de las variables epidemiológicas, fenotípicas, histológicas y los antecedentes personales y familiares de cáncer. RESULTADOS: Se observó que el desarrollo de melanoma antes de los 50 años (OR = 1,47), la localización en la cabeza y el cuello (OR = 3,04), poseer antecedentes de carcinoma basocelular y de carcinoma epidermoide cutáneo (OR = 1,70) y la presencia de nevus atípicos (OR = 1,74) y nevus asociados al melanoma (OR = 1,87) es predominantemente característico de pacientes con puntuación igual o mayor que 3.ConclusionesEl sistema de puntuación aplicado a los polimorfismos en MC1R nos ha permitido detectar relaciones en las que algunas características clínicas del paciente y de la presentación del melanoma presentan un efecto proporcional al grado de afectación funcional de la vía de la melanogénesis

INTRODUCTION: Patients with cutaneous melanoma who are carriers of polymorphisms in the melanocortin 1 receptor gene (MC1R) have distinctive clinical characteristics. The objective of this study was to determine the clinical characteristics associated with differing degrees of functional impairment of the melanocortin 1 receptor, as determined by the number and type (R and r) of MC1R polymorphisms. MATERIAL AND METHODS: In total, 1044 consecutive patients with melanoma diagnosed in our hospital after January 2000 were selected from the melanoma database. These patients were divided into 3 groups according to a score based on non synonymous MC1R polymorphisms. The frequencies of epidemiologic, phenotypic, and histologic variables and personal and family history of cancer were compared. RESULTS: Patients with a score of 3 or more were more likely to develop melanoma before the age of 50 years (odds ratio [OR] = 1.47), have a tumor on the head or neck (OR = 3.04), have a history of basal cell carcinoma or cutaneous squamous cell carcinoma (OR = 1.70), have atypical nevi (OR = 1.74), and have nevi associated with the melanoma (OR = 1.87). CONCLUSIONS: The use of a scoring system for MC1R polymorphisms allowed us to identify associations between the degree of functional impairment of the melanogenesis pathway and the clinical characteristics of the patients and melanoma presentation

Value of melanocytic-associated immunohistochemical markers in the diagnosis of malignant melanoma: a review and update.

Since the identification of S100 protein as an immunohistochemical marker that could be useful in the diagnosis of melanoma in the early 1980s, a large number of other melanocytic-associated markers that could potentially be used to assist in the differential diagnosis of these tumors have also been investigated. A great variation exists, however, among these markers, not only in their expression in some subtypes of melanoma, particularly desmoplastic melanoma, but also in their specificity because some of them can also be expressed in nonmelanocytic neoplasms, including various types of soft tissue tumors and carcinomas. This article reviews the information that is currently available on the practical value of some of the markers that have more often been recommended for assisting in the diagnosis of melanomas, including those that have only recently become available.

Synthesis and characterization of a melanoma-targeted fluorescence imaging probe by conjugation of a melanocortin 1 receptor (MC1R) specific ligand.

The incidence of malignant melanoma is rising more rapidly than that of any other cancer in the United States. The melanocortin 1 receptor (MC1R) is overexpressed in most human melanoma metastases, thus making it a promising target for imaging and therapy of melanomas. We have previously reported the development of a peptidomimetic ligand with high specificity and affinity for MC1R. Here, we have conjugated near-infrared fluorescent dyes to the C-terminus of this ligand via lysine-mercaptopropionic acid linkers to generate MC1R specific optical probes (MC1RL-800, 0.4 nM K(i); and MC1RL-Cy5, 0.3 nM K(i)). Internalization of the imaging probe was studied in vitro by fluorescence microscopy using engineered A375/MC1R cells and B16F10 cells with endogenous MC1R expression. The in vivo tumor targeting of MC1RL-800 was evaluated by intravenous injection of probe into nude mice bearing bilateral subcutaneous A375 xenograft tumors with low MC1R expression and engineered A375/MC1R tumors with high receptor expression. Melanotic B16F10 xenografts were also studied. Fluorescence imaging showed that the agent has higher uptake values in tumors with high expression compared to low (p < 0.05), demonstrating the effect of expression levels on image contrast-to-noise. In addition, tumor uptake was significantly blocked by coinjection of excess NDP-α-MSH peptide (p < 0.05). In conclusion, the MC1R-specific imaging probe developed in this study displays excellent potential for the intraoperative detection of regional node involvement and for margin detection during melanoma metastasis resection.

Radiofluorinated rhenium cyclized α-MSH analogues for PET imaging of melanocortin receptor 1.

In order to accomplish in vivo molecular imaging of melanoma biomarker melanocortin 1 receptor (MC1R), several α-melanocyte-stimulating hormone (α-MSH) analogues have been labeled with N-succinimidyl-4-¹8F-fluorobenzoate (¹8)F-SFB) and studied as positron emission tomography (PET) probes in our recent studies. To further pursue a radiofluorinated α-MSH peptide with high clinical translation potential, we utilized 4-nitrophenyl 2-¹8F-fluoropropionate (¹8F-NFP) to radiofluorinate the transition metal rhenium cyclized α-MSH metallopeptides for PET imaging of MC1R positive malignant melanoma. Metallopeptides Ac-d,Lys-ReCCMSH(Arg¹¹) (two isomers, namely RMSH-1 and RMSH-2) were synthesized using conventional solid phase peptide synthesis chemistry and rhenium cyclization reaction. The two isomers were then conjugated with ¹9F-NFP or ¹8F-NFP. The resulting cold or radiofluorinated metallopeptides, (¹8/¹9)F-FP-RMSH-1 and (¹8/¹9)F-FP-RMSH-2, were further evaluated for their in vitro receptor binding affinities, in vivo biodistribution, and small-animal PET imaging properties. The binding affinities of ¹9F-FP-RMSH-1 and ¹9F-FP-RMSH-2 were determined to be within low nanomolar range. In vivo studies revealed that both F-labeled metallopeptides possessed good tumor uptake in the B16F10 murine model with high MC1R expression, while possessing much lower uptake in A375M human melanoma xenografts. Moreover, ¹8F-FP-RMSH-1 displayed more favorable in vivo performance in terms of higher tumor uptake and much lower accumulation in the kidney and liver, when compared to that of ¹8F-FP-RMSH-2 at 2 h postinjection (p.i.). ¹8F-FP-RMSH-1 also displayed lower liver and lung uptake when compared with that of the same peptide labeled with ¹8F-SFB (named as ¹8F-FB-RMSH-1). Small animal PET imaging of ¹8F-FP-RMSH-1 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected, much lower tumor uptake and poorer tumor/normal organ contrast were observed for A375M model compared to those of the B16F10 model. ¹8F-FP-RMSH-1 also exhibited higher tumor uptake and better tumor retention when compared with ¹8F-FB-RMSH-1. ¹8F-FP-RMSH-1 demonstrates significant advantages over ¹8F-FB-RMSH-1 and ¹8F-FP-RMSH-2. It is a promising PET probe for imaging MC1R positive melanoma and MC1R expression in vivo.

[Acanthosis nigricans, papillomatosis mucosae and "tripe palms" in a patient with metastasized gastric carcinoma]. / Acanthosis nigricans, Papillomatosis mucosae und "tripe palms" bei einem Patienten mit metastasiertem Magenkarzinom.

HISTORY AND CLINICAL FINDINGS: A 48-year-old obese man presented with thickening, coarseness and hyperpigmentation of the skin, especially of the intertriginous areas, papillomatous to verrucous lesions of the lips and buccal oral mucosa, and hyperkeratosis of the palms ("tripe palms") and soles. He was obese, reported sleep apnea and had a history of hyperuricemia, mixed hyperlipidemia and previous myocardial infarction. He was on a maintenance dose of a proton pump inhibitor for chronic gastro-esophageal reflux. EXAMINATIONS: Immunohistochemical studies of the skin lesion revealed increased epidermal immunoreactivity for the melanocortin-1-receptor. Increased levels of tumor markers CA 19-9 (141100 U/ml), CA 72-4 (755 U/ml) and CEA (189 ng/ml) were found in the serum. Gastroscopic findings were suspicious of adenocarcinoma of the stomach: it was classified histologically as a signet-ring cell, non-mucinous adenocarcinoma. At the time of diagnosis the tumor had already metastasized to perigastric and peripancreatic lymph nodes with peritoneal carcinosis. TREATMENT AND COURSE: Since a curative resection was impossible a gastrojejunostomy was carried out. After this the patient received several courses of chemotherapy according to different schemes. Serum tumor marker levels and cutaneous signs regressed several times. CONCLUSIONS: Marked acanthosis nigricans -- especially when associated with further cutaneous markers of malignancy, e.g. mucocutaneous papillomatosis or so-called tripe palms -- calls for thorough search for malignant tumor, also if metabolic or endocrinological abnormalities co-exist. A pathogenetic role of a-melanocyte-stimulating hormone in the development of the skin changes is suggested.

Dimerization of the human melanocortin 1 receptor: functional consequences and dominant-negative effects.

The melanocortin 1 receptor (MC1R), a G(S)-protein-coupled receptor (GPCR), is a key regulator of proliferation and differentiation of epidermal melanocytes, and a determinant of human skin phototype and cancer risk. Homodimerization has been demonstrated for several GPCRs, but little information is available for MC1R. SDS-PAGE analysis of melanoma cells and heterologous cells expressing epitope-tagged MC1R revealed dimeric and oligomeric species in detergent-solubilized extracts, confirmed by co-immunoprecipitation of differentially tagged MC1R forms. Dimerization occurs early during MC1R biosynthesis, and is seen for mutants displaying intracellular retention. These mutants exerted dominant-negative effects on wild-type (WT) MC1R. Conversely, partial functional trans-complementation of selected loss-of-function mutants was observed. WT-MC1R lacks cooperativity in agonist binding, yet coexpression of WT and a C-terminal deletion mutant yielded a form of different pharmacological properties. The natural diminished function alleles R151C, R160W, and D294H, associated with red hair, displayed dimerization and heterodimerization with WT. Coexpression of WT and R151C or R160W reduced the density of binding sites on the plasma membrane of transfected cells, whereas D294H mediated a dominant-negative effect on functional coupling to adenylyl cyclase. Therefore, subtle changes of functional properties may be associated with different MC1R haplotypes, contributing to the complexity of skin phenotype.

Human hair follicles display a functional equivalent of the hypothalamic-pituitary-adrenal axis and synthesize cortisol.

The skin and its major appendages are prominent target organs and potent sources of key players along the classical hypothalamic-pituitary axis, such as corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and alpha melanocyte stimulating hormone (alpha-MSH), and even express key steroidogenic enzymes. Therefore, it may have established local stress response systems that resemble the hypothalamic-pituitary-adrenal (HPA) axis. However, functional evidence that this is indeed the case in normal human skin in situ has still been missing. We show that microdissected, organ-cultured human scalp hair follicles respond to CRH stimulation by up-regulating proopiomelanocortin (POMC) transcription and immunoreactivity (IR) for ACTH and alpha-MSH, which must have been processed from POMC. CRH, alpha-MSH, and ACTH also modulate expression of their cognate receptors (CRH-R1, MC1-R, MC2-R). In addition, the strongest stimulus for adrenal cortisol production, ACTH, also up-regulates cortisol-IR in the hair follicles. Isolated human hair follicles secrete substantial levels of cortisol into the culture medium, and this activity is further up-regulated by CRH. CRH also modulates important functional hair growth parameters in vitro (hair shaft elongation, catagen induction, hair keratinocyte proliferation, melanin production). Finally, human hair follicles display HPA axis-like regulatory feedback systems, since the glucocorticoid receptor agonist hydrocortisone down-regulates follicular CRH expression. Thus, even in the absence of endocrine, neural, or vascular systemic connections, normal human scalp hair follicles directly respond to CRH stimulation in a strikingly similar manner to what is seen in the classical HPA axis, including synthesis and secretion of cortisol and activation of prototypic neuroendocrine feedback loops.

Seeing the gene therapy: application of gene gun technique to transfect and decolour pigmented rat skin with human agouti signalling protein cDNA.

We developed a gene gun method for the transfer of human agouti signalling protein (ASP) cDNA to alter rat skin colour in vivo. Human ASP cDNA was cloned into a modified cytomegalovirus plasmid and delivered to the skin of Long-Evans rats by gene gun bombardment. Skin pigmentation, body weight and blood sugar of ASP cDNA-transfected rats were recorded against the control group, which were injected with plasmids encoding for green fluorescent protein. The treated skin showed lighter skin colour after 3 days of ASP gene transfection. This depigmentation effect was most prominent on day 14 and the skin gradually returned to its original pigmentation by day 28. Successful transfection of ASP gene in skin and hair follicles, as well as downregulation of melanocortin-1 receptor (MC1R) and tyrosinase expression upon treatment, was confirmed using immunohistochemistry and Western blot analysis. Body weight and blood sugar in the treated rats did not show statistically significant differences as compared to control groups. These observations demonstrate that gene transfer using the gene gun method can induce high cutaneous ASP production and facilitate a switch from dark to fair colour without systemic pleiotropic effects. Such a colour switch may be that ASP is acting in a paracrine fashion. In addition, this study verifies that ASP exerts its functions by acting as an independent ligand that downregulates the melanocyte MC1R and tyrosinase protein in an in vivo system. Our result offers new, interesting insights about the effect of ASP on pigmentation, providing a novel approach to study the molecular mechanisms underlying skin melanogenesis.

Autocrine inhibitory influences of alpha-melanocyte-stimulating hormone in malignant pleural mesothelioma.

Malignant pleural mesothelioma is a highly aggressive tumor arising from the mesothelial cells that line the pleural cavities. This tumor is resistant to most conventional anticancer treatments and appears to be very sensitive to growth-promoting influences of cytokines and growth factors. Identification of natural inhibitory pathways that control growth should aid discovery of novel therapeutic approaches. We hypothesized that alpha-melanocyte-stimulating hormone (alpha-MSH), which is produced by many cell types and antagonizes cytokines and growth factors, could be an endogenous inhibitory molecule in mesothelioma. Twelve mesothelioma cell lines were established from pleural effusions of patients with malignant mesothelioma. Mesothelioma cells were found to express mRNA for proopiomelanocortin and its processing enzymes; release alpha-MSH peptide into supernatants; and express melanocortin 1 receptor (MC1R), the high-affinity receptor for alpha-MSH. Immunoneutralization of MC1R in the cell lines enhanced expression of interleukin-8 (IL-8), IL-6, and transforming growth factor-beta. These molecules promote mesothelioma proliferation and are considered therapeutic targets in this tumor. Coincubation of mesothelioma cells with synthetic alpha-MSH significantly reduced cell proliferation. The present research shows an autocrine-inhibitory circuit based on alpha-MSH and its receptor MC1R. Activation of MC1R by selective peptides or peptidomimetics might provide a novel strategy to reduce mesothelioma cell proliferation by taking advantage of this endogenous inhibitory circuit.

Regulation of TNF-alpha secretion by a specific melanocortin-1 receptor peptide agonist.

The lack of specific pharmacological tools has impeded the evaluation of the role of each melanocortin receptor (MCR) subtype in the myriad physiological effects of melanocortins. 154N-5 is an octapeptide (MFRdWFKPV-NH(2)) that was first identified as an MC1R antagonist in Xenopus melanophores [J. Biol. Chem. 269 (1994) 29846]. In this manuscript, we show that 154N-5 is a specific agonist for human and murine MC1R. The peptide has negligible activity at MC3R and MC4R and is 25-fold less potent and a weak agonist at MC5R. 154N-5 was tested in both a cellular and an animal model of tumor necrosis factor-alpha (TNF-alpha) secretion. The inhibitory efficacy of 154N-5 on TNF-alpha secretion in both models was similar to the nonselective agonist NDP-alpha-melanocyte stimulating hormone (NDP-alphaMSH), thus, we conclude that inhibition of TNF-alpha secretion by melanocortin peptides is mediated by MC1R. 154N-5 is a valuable new tool for the evaluation of specific contribution of MC1R agonism to physiological and pathological processes.

Characterization of melanocortin receptors.

This unit describes a Scintillation Proximity Assay (SPA) for the measurement of ligand binding to melanocortin receptors (MCRs) using membranes prepared from cell lines stably expressing recombinant MCRs. It provides a facile method for determining the affinity of compounds at MC1R, MC3R, MC4R, or MC5R.