Genotoxic effect of doxorubicin-transferrin conjugate on human leukemia cells

Abstract

Doxorubicin (DOX) is an effective anthracycline antibiotic against a wide spectrum of tumors and hematological malignancies. It mainly interacts with DNA, but can also generate reactive oxygen species (ROS), which damage cell components. Unfortunately, numerous side effects, such as severe cardiotoxicity and bone-marrow suppression, limit its use. To reduce this obstacle and improve its pharmacokinetics, we conjugated DOX to transferrin (TRF), a human plasma protein. In our study, we compared the effect of DOX and the doxorubicin-transferrin conjugate (DOX-TRF) on human leukemic lymphoblasts (CCRF-CEM), and on normal peripheral blood mononuclear cells (PBMC). In parallel, experiments were carried out on two human chronic myeloid leukemia (CML) cell lines derived from K562 cells, of which one was sensitive and the other resistant to doxorubicin (K562/DOX). By use of the alkaline comet assay, the effect of the agents on the induction of DNA damage in normal human cells and human leukemia cells was determined. Oxidative and alkylating DNA damage were assayed by a slightly modified comet assay that included the use of the DNA-repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg). To investigate whether DNA breaks are the result of apoptosis, we examined the induction of DNA fragmentation visualized as oligosomal ladders after simple agarose electrophoresis under neutral conditions. Modifications of the genome induced by the different drugs were analyzed following assessment of the cell-cycle phase. The DOX-TRF conjugate caused more DNA damage than the free drug, the degree of DNA fragmentation being dependent on the duration of treatment and the cell type analyzed. With neutral agarose electrophoresis we showed that the test compounds caused the formation of a characteristic DNA-ladder pattern. Furthermore, the DOX-TRF conjugate generated a higher percentage of apoptotic cells in the subG1 fraction and blocked more cells in the G2/M phase of the cell cycle than did free DOX. In summary, both agents induced DNA damage in cancer cells, but the DOX-TRF conjugate generated more genotoxic effects and apoptosis than the unconjugated drug.

Keywords: Cell cycle; DNA damage; Doxorubicin–transferrin conjugate; Free radicals.