IN VITRO DEVELOPMENT OF 2,4-DOPA, A TYROSINASE-ACTIVATED PRODRUG OF POTENTIAL USE IN THE TREATMENT OF MALIGNANT MELANOMA.

摘要

Tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, E.C. 1.14.18.1) is found in the melanosomes of normal and malignant pigment cells and catalyzes the hydroxylation of L-p-tyrosine in the meta-position to form L-3,4-DOPA and, ultimately, melanin pigment. In the present studies, tyrosinase was demonstrated to hydroxylate 2,4-DOPA (2,4-dihydroxyphenylalanine) in the meta-position to generate 6-hydroxy-DOPA (2,4,5-trihydroxyphenylalanine). 2,4-Dopamine was also tested as a substrate for tyrosinase and was hydroxylated in the meta-position to generate 6-hydroxydopamine (2,4,5-trihydroxyphenylethylamine) as product. Both reactions were accelerated by the inclusion of L-3,4-DOPA which is the natural cosubstrate for tyrosinase. The products, 6-hydroxy-DOPA and 6-hydroxydopamine, are well-known neurotoxins that are taken up into catecholamine neurons and express neurotoxicity primarily through the production of toxic oxy-radicals.;MJY-Alpha mammary tumor and L-1210 leukemia were studied to examine non-tyrosinase-mediated effects. No toxicity was observed against either MJY-alpha mammary tumor or L-1210 leukemia cultures.;C-1300 neuroblastoma cells do not contain tyrosinase but do contain tyrosine hydroxylase, the enzyme which catalyzes the hydroxylation of L-p-tyrosine to L-3,4-DOPA within catecholamine neurons. Neuroblastoma cultures were tested to examine the possibility that tyrosine hydroxylase might generate 6-hydroxy-DOPA from 2,4-DOPA, with resultant cytotoxicity. 2,4-DOPA was shown to be non-specifically cytotoxic to neuroblastoma cultures. The cytotoxicity of 2,4-DOPA could not be blocked by either alpha-methyl-p-tyrosine or 3-iodo-tyrosine, two potent inhibitors of tyrosine hydroxylase. Since the cytotoxicity against the neuroblastoma cells appeared to be non-specific, it is hoped that catecholamine neurons might not be at risk during treatment with 2,4-DOPA.;These studies indicate that 2,4-DOPA functions as a tyrosinase-activated prodrug which may potentially be of use in the treatment of malignant melanoma.;2,4-DOPA was tested in cell culture to assess its potential as a tyrosinase-targeted prodrug against melanoma. Treatment with 2,4-DOPA produced cytotoxicity against both B-16 and Cloudman melanoma cultures. In experiments with B-16 melanoma cultures, 2,4-DOPA inhibited the synthesis of DNA, RNA, and protein in a dose- and time-dependent manner.