The mechanism of daunorubicin-induced inhibition of prolidase activity in human skin fibroblasts and its implication to impaired collagen biosynthesis.

Abstract

One of the recognized side effects accompanying antineoplastic anthracyclines administration is poor wound healing, resulting from impairement of collagen biosynthesis. However, the precise mechanism of anthracyclines-induced inhibition of collagen synthesis has not been established. We have suggested that prolidase, an enzyme involved in collagen metabolism may be one of the targets for anthracyclines-induced inhibition of synthesis of this protein. Prolidase [E.C. 3.4.13.9] cleaves imidodipeptides containing C-terminal proline, providing large amount of proline for collagen synthesis. We have found that daunorubicin (DNR) induced coordinately inhibition of prolidase activity (IC50 = 0.3 microM) and collagen biosynthesis (IC50 = 1 microM) in cultured human skin fibroblasts. The decrease in prolidase activity due to the treatment of confluent cells with DNR was not accompanied by any differences in the amount of the enzyme protein recovered from these cells as shown by western immunoblot analysis. Since prolidase is metaloprotease, requiring manganese for catalytic activity and anthracyclines are known as a chelators of divalent cations we considered that the chelating ability of anthracyclines may be an underlying mechanism for daunorubicin-induced inhibition of prolidase activity. In order to determine the ability of DNR to form complex with manganese (II), potentiometric method was employed based on the measurement of protonation constant by pH-metric titrated assay. We have found that DNR forms stable complex with manganese (II) and the composition of the complex of DNR with Mn (II) was calculated as 3:1. The constant stability value for the investigated complex was calculated as [beta(av) = (1.74 +/- 0.01) 10(23). The strong ability of DNR to chelate manganese may explain the potential mechanism for inhibition of prolidase activity, subsequently collagen biosynthesis and poor wound healing in patients administered DNR.