Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors

Abstract

We developed two solid-phase reagent systems for microscopic visualization and characterization of melanocyte-stimulating hormone ( The search for a molecular marker for melanoma cancer cells is pivotal for the development of modalities for the early detection and management of melanoma. The identification of a specific universal antigen associated with melanoma has remained elusive. Usually, good antibody candidates specific for a particular melanoma specimen fail to recognize other tumor variants from different patients and sometimes even from the same individual (1, 2). The occurrence of melanotic as well as amelanotic tumors further illustrates the great degree of melanoma variance and the problem of unambiguous detection. A group of acidic cytosolic proteins, commonly referred to as S-100 proteins, have been claimed to be a useful marker for melanoma (3, 4). An immunoassay based on this protein has been used to screen formalin fixed melanoma tumors in vitro, stressing its importance as a good diagnostic tool for melanoma, especially in case of metastatic lymph node melanoma. Unfortunately, a large number of normal tissues also contain S-100 proteins in substantial amounts (5), making it a rather complex issue in distinguishing the truly positive from the truly negative specimens. Furthermore, the cytosolic localization of these proteins suggests that antibody-directed targeting of modalities of therapeutic relevance might not be feasible. For this purpose, the identification of specific cell membrane markers is better suited because of their rather facile accessibility by cell-specific targeting agents.We recently described the development of melanotropin conjugates that could detect the presence of melanotropin receptors on a number of mouse and human melanoma cell lines, melanotic as well as amelanotic, that we investigated (6, 7). These conjugates were composed of multiple copies of both a potent melanotropin analog and a fluorophore that were conjugated to a biologically inert macromolecule in a pendant fashion. The multivalency of the ligand molecule in these conjugates afforded specific binding between the conjugate and the melanotropin receptors, presumably by establishing simultaneous interactions with a number of melanocytestimulating hormone (MSH) receptors on the cell membrane. In addition, these macromolecular composites also allowed visualization of certain phenomena, like capping and internalization, that are associated with certain ligand-receptor binding.Here we describe another class of multivalent melanotropic ligands that can detect the presence or absence of specific MSH binding sites on melanoma cells in a very simple and straightforward manner. These methods provide simple alternatives to the use of fluorescence techniques previously described by and recently by us (6, 7). The specificity of these newer solid-phase reagents for MSH-binding sites is addressed and established. MATERIALS AND METHODSN, N-Dimethylaminopyridine was obtained from Aldrich. DTT, mercaptoethanol, 4-( p-...

Anti-inflammatory and anti-invasive effects of α-melanocyte-stimulating hormone in human melanoma cells

“…However, stable transfection of these cells with the wildtype MC-1R showed that these cells were then able to respond to a-MSH with a significant reduction in invasion. The HBL melanoma cell line has previously been demonstrated to express 1000 -3000 MSH receptor-binding sites per cell , and A375 and C8161 melanoma cell lines have also been demonstrated to have MSH receptors (Baumann et al, 1997) and MSH binding sites (Sharma et al, 1996). In comparison, normal human cutaneous melanocytes are reported to express between 300 and 1000 binding sites per cell (Donatien et al, 1992;De Luca et al, 1993).…”

Bench-top to clinical therapies: A review of melanocortin ligands from 1954 to 2016

“…also developed both latex bead and polyamide bead conjugates to NDP-MSH analogs and achieved similar results as Schwyzer et al . [263-265]. The latex beads were considered microspheres (∼1 μm in diameter) and the polyamide beads were classified as macrospheres (40 to 100 μm in diameter).…”

Novel targeting strategy based on multimeric ligands for drug delivery and molecular imaging: homooligomers of α-MSH

“…However, dynorphin and substance P agents did not bind to any cells. 4,5 Binding data indicated a significant hysteresis in binding kinetics with rapid on rates and immeasurably slow off rates. Such kinetics were ascribed to the phenomenon of cooperative affinity.…”