The mechanism for anthracycline-induced inhibition of collagen biosynthesis

Abstract

One of the recognized side effects accompanying anti-neoplastic anthracyclines administration is poor wound healing resulting from impairment 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 [EC 3.4.13.9] cleaves imidodipeptides containing C-terminal proline, providing large amount of proline for collagen synthesis. Therefore, we compared the effect of daunorubicin and doxorubicin on prolidase activity and collagen biosynthesis in confluent cultured human skin fibroblasts. We have found that daunorubicin and doxorubicin coordinately induced the inhibition of prolidase activity (IC50=0.3 and 10 μM, respectively) and collagen biosynthesis (IC50=1 and 15 μM, respectively) in cultured human skin fibroblasts. The inhibitory effect of daunorubicin or doxorubicin on prolidase activity and collagen biosynthesis was not due to anti-proliferative activity of these drugs as shown by cell viability tetrazoline test. The decrease in prolidase activity due to the treatment of confluent cells with the anthracyclines was not accompanied by any difference in the amount of enzyme protein recovered from these cells as shown by Western immunoblot analysis. It may be suggested that the inhibition is a post-translational event. Since prolidase is metalloprotease, requiring manganese for catalytic activity, and anthracyclines are known as chelators of divalent cations, we considered that the chelating ability of anthracyclines might be an underlying mechanism for the anthracyclines-induced inhibition of prolidase activity. In order to determine the ability of daunorubicin or doxorubicin to form complexes with manganese (II), potentiometric method was employed based on the measurement of protonation constant by pH-metric titrated assay. We have found that both anthracyclines form stable complexes with manganese (II). The composition of the daunorubicin–Mn(II) complex was calculated as 3:1 while that of doxorubicin–Mn(II) complex was 2:1. The constant stability value for the investigated complexes were calculated as βav=(1.74±0.01)×1023 for daunorubicin, and βav=(1.99±0.025)×1011 for doxorubicin. The higher ability of daunorubicin vs. doxorubicin to chelate manganese and inhibit prolidase activity may explain the potential mechanism for its greater potency to inhibit collagen biosynthesis.