Metastatic
melanoma is resistant to conventional
therapies.
N-propionyl-4-S-cysteaminylphenol (NPrCAP), an N-protected
sulfur-
amine analog of
tyrosine, is a good substrate for
tyrosinase and is selectively incorporated into
melanoma cells, causing cytotoxicity in vitro and in vivo. We have recently shown that intratumoral
injections of NPrCAP suppress not only the growth of primary B16F1
melanoma tumors but also of secondary, re-challenged
tumors. The participation of CD8(+) T cells has been suggested for the NPrCAP-mediated anti-B16
melanoma immunity. In this study, the molecular mechanism of the NPrCAP cytotoxicity and immunogenicity was examined. The
phenol NPrCAP was shown to be activated by mushroom
tyrosinase to the ortho-
quinone N-propionyl-4-S-cysteaminyl-1,2-benzoquinone (NPrCAQ), and the structure was confirmed by reducing it to the corresponding
catechol. NPrCAQ reacted rapidly with biologically relevant
sulfhydryl compounds such as
cysteine,
glutathione and
bovine serum albumin. The NPrCAQ-
thiol adduct formation was proven with a model
thiol N-acetylcysteine by spectroscopic methods. The production and release of NPrCAQ-
protein adducts was verified in B16F1
melanoma cells in vitro and in B16F1
melanoma-bearing mice in vivo through the detection of 5-S-cysteaminyl-3-S-cysteinylcatechol after
acid hydrolysis of the
protein fraction. These results suggest that the
phenol NPrCAP, acting as a prohapten, can be activated in
melanoma cells by
tyrosinase to the
quinone-
hapten NPrCAQ, which binds to melanosomal
proteins through their
cysteine residues to form possible neo-
antigens, thus triggering the immunological response. NPrCAP thus represents a potential new approach to
immunotherapy against metastatic
melanoma.