msh--4-nle-7-phe-alpha- and Skin-Neoplasms

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.

Topics: alpha-MSH; Animals; Endocytosis; Fluorescent Dyes; Gene Expression; Humans; Injections, Intravenous; Ligands; Male; Melanoma, Experimental; Mice; Mice, Nude; Microscopy, Fluorescence; Molecular Imaging; Neoplasm Proteins; Peptidomimetics; Receptor, Melanocortin, Type 1; Skin Neoplasms

Topics: alpha-MSH; Anticarcinogenic Agents; Humans; Injections, Subcutaneous; Male; Middle Aged; Nevus, Pigmented; Skin Neoplasms

Several properties of the MSH receptor in solid melanotic and amelanotic mouse M2R tumour isografts were studied in C57BL mice. Using cell membrane fractions prepared from such tumours and the superpotent [Nle4,D-Phe7]alpha MSH analogue, the affinity and receptor contents of the two tumour variants were found to be similar. When occupied by MSH, the receptor-MSH complex (R.MSH) was readily soluble in cholate. In the solubilized form, R.MSH was extremely stable and dissociated to an extent of only 30% within 12 days at 4 degrees C. While this high stability can be maintained in the pH range of 7.0-8.5, the solubilized R.MSH complex becomes increasingly unstable below pH 7.0 and totally dissociates at a pH < 6.0. In the membrane-bound form, the R.MSH complex shows a parallel pH stability profile which is shifted down by approximately two pH units. In addition to low pH, the R.MSH complex becomes unstable and totally dissociates in the presence of 10 mM EGTA, suggesting that the calcium-sensitive function of the receptor is still associated with the receptor in the detergent-soluble state. The R.MSH complexes in the soluble and membrane-bound forms are also totally resistant to proteolytic digestion by V8 protease, but were slowly digested by trypsin. Treatment of R.MSH with 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride or bis (sulphosuccinimidyl) suberate led to covalent crosslinking of MSH to the receptor molecule. The electrophoretic mobility on SDS-PAGE of the 43/46 kD doublet of the receptor-MSH conjugate (R*MSH) was identical to the photoaffinity labelled MSH receptor product described earlier in cultured M2R cells. However, the efficiency of production of the crosslinked product was approximately 30%, much higher than that achieved previously by photoaffinity labelling. Using rabbit polyclonal anti-alpha MSH antibodies, the R*MSH conjugate was identifiable on Western immunoblots. These results provide a basis for further development of procedures for purification of the MSH receptor molecule and studying its protein structure.

Topics: alpha-MSH; Animals; Antibodies; Blotting, Western; Cross-Linking Reagents; Detergents; Drug Stability; Iodine Radioisotopes; Melanocyte-Stimulating Hormones; Melanoma, Amelanotic; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Rabbits; Receptors, Pituitary Hormone; Skin Neoplasms; Solubility; Structure-Activity Relationship; Tumor Cells, Cultured