Up-regulation of Melan-A/MART-1 antigen expression during human melanoma cell differentiation
aSection of Molecular Medicine, Institute of Neurobiology and Molecular Medicine, National Research Council, Italy, bDepartment of Experimental Medicine and Biochemical Science - University of Rome “Tor Vergata”and, Rome, Italy, cInstitute of Biochemistry and Clinical Biochemistry, Catholic University of Rome “Sacro Cuore”, Rome, Italy, dDepartment of Chemical Science, Laboratory of Biochemistry, University of Catania, Catania, Italy
BACKGROUND AND AIM: Melan-A/MART-1 is a melanocytic differentiation antigen, expressed in melanocytes and melanoma cells, which is recognized by cytotoxic T-lymphocytes (CTL). It is of interest for clinicians as potential immunotherapeutic target and it is relevant for pathologists as a diagnostic marker. We have previously investigated the effect of cyanidin-3-O-beta-glucopyranoside (C-3-G), a flavonoid of the anthocyanin class which is present in both fruits and vegetables of human diets, on proliferation and differentiation of TVM-A12 human melanoma cell line obtained from a metastatic lesion. A single dose of C-3-G, at low concentration corresponding to those achieved with food intake, has been able to reduce cell proliferation without affecting cell viability and to induce melanoma cell differentiation characterized by a strong increase in dendritic outgrowth. In this work, attempting to correlate the Melan-A/MART-1 expression with melanoma differentiation, we studied the intracellular distribution of this antigen during TVM-A12 cell differentiation induced by C-3-G. METHODS. The effect of C-3-G treatment on cell morphology was analyzed by optical and scanning electron microscopy. The expression of Melan-A/MART-1 antigen in differentiating TVM-A12 human melanoma cells was investigated by: a) cytofluorimetric analysis, b) confocal laser scanning microscopy (CLSM) and c) Western Blot analysis, using the Melan-A (A103) mouse monoclonal antibody. RESULTS. After C-3-G treatment, a dose and time dependent increase in the percentage of positive cells as well as in the mean fluorescence intensity was recorded by cytofluorimetric analysis. The observation of Melan-A/MART-1 intracellular distribution showed a dramatic increase of antigen expression in cells exhibiting morphological features of differentiated melanoma, as revealed by CLSM after indirect immunofluorescence labelling. CLSM observation of Melan-A/MART-1 expression in 100% of normal melanocytes confirmed the correlation between increasing in the expression of this antigen and a more differentiated and less malignant phenotype. The increase of antigen expression in differentiated melanoma cells was also revealed by Western Blot analysis. CONCLUSIONS. Our results demonstrate that C-3-G is able to revert human melanoma cells from the proliferating to the differentiated state and that the acquisition of differentiated phenotype is correlated with an up-regulation of Melan-A/MART-1 antigen expression. Because this antigen can be recognized by CTL, C-3-G represents an attractive candidate in the development of novel strategies for both treatment and immunotherapy of melanoma through consumption of C-3-G in an appropriate cancer prevention diet.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 797 (Manifestations of cancer).