Melanin counter act puromycin-induced inhibition of collagen and DNA biosynthesis in human skin fibroblasts

Abstract

Puromycin is an experimental anti-tumor antibiotic acting through inhibition of protein synthesis. Because of its untoward side effects (as inner ear and renal lesions) the antibiotic was not approved for clinical trials. The mechanism underlying the organ specificity of the side effect is not understood. In view of the fact that a number of drugs form with melanin complexes that affect their pharmacological activity, we determined whether puromycin interacts with melanin and how this process affects biosynthesis of collagen in cultured human skin fibroblasts. Our results indicate that puromycin forms complexes with melanin. The amount of puromycin bound to melanin increases with increase of initial drug concentration. The Scatchard plot analysis of the drug binding to melanin has shown that at least two classes of independent binding sites are implicated in the puromycin-melanin complex formation: one class of strong binding sites with the association constant K 1 = 1.84 × 10 6 M −1, and the second class of weak binding sites with the association constant K 2 = 5.26 × 10 3 M −1. The number of total binding sites were n 1 = 0.1260 and n 2 = 0.2861 μmol puromycin per 1 mg melanin. We found that puromycin induced inhibition of collagen and DNA biosynthesis (IC 50 ∼ 2 μM). Melanin at 100 μg/ml produced about 20% inhibition of DNA synthesis, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 μg/ml) to puromycin - treated cells (2 μM) abolished the inhibitory action of puromycin on collagen and DNA biosynthesis. We have suggested that IGF-I receptor expression, involved in collagen metabolism, may be one of the targets for puromycin - induced inhibition of collagen biosynthesis. It was found that melanin abolished puromycin induced decrease in the expression of IGF-I receptor as well MAP kinases expression: ERK1 and ERK2 as shown by Western immunoblot analysis. These data suggest that tissue specific pharmacological activity of puromycin may depend on the melanin abundance in tissues.