Influence of conjugation of doxorubicin to transferrin on the iron uptake by K562 cells via receptor‐mediated endocytosis
Authors: BÉRCZI Alajos , RUTHNER Monika , SZŰTS Viktória , FRITZER Monika , SCHWEINZER Esther , GOLDENBERG Hans 
Published: March 2005

The influence of conjugation of doxorubicin to holotransferrin on the receptor‐mediated endocytosis of and on the iron uptake from transferrin was studied using K562 cells. 125I‐labelled transferrin and doxorubicin‐transferrin conjugates were used in the binding, dissociation, and ligand‐exchange experiments at 0°C, and 59Fe, 125I‐labelled (double‐labelled) ligands were used in the endocytosis, iron uptake, and recycling experiments at 37°C. The binding affinity of conjugates was about half of that of transferrin. Binding of 125I‐labelled ligands was blocked by both unlabelled ligands to the same degree, however, it was not blocked at all by an 8000‐fold excess of doxorubicin. After saturation bindings, slightly more 125I‐labelled conjugates dissociated from the surface of cells than transferrin. Exchange of 125I‐labelled ligands for unlabelled ligands resulted in different EC50 values (defined as the concentration of unlabelled ligand at which half as much radioligand is exchanged for unlabelled ligand as would be exchanged at infinitely high concentration of unlabelled ligand under similar assay conditions). While transferrin exchanged transferrin with an EC50 value close to the binding affinity, conjugates exchanged conjugates with much lower efficiency. The heterolog exchange experiments yielded EC50 values inbetween the two extrema. For studying iron uptake, K562 cells were loaded with the double‐labelled ligands either at 37°C (endosome‐loading only) or at 0°C (surface‐loading only). Results obtained for the endocytosis of, the iron uptake from, and the recycling of double‐labelled ligands indicate that (a) the rate of iron uptake is smaller from conjugates than from transferrin, (b) there are at least two parallel recycling processes for both ligand · receptor complexes, and (c) each time constant characterizing the different steps of iron uptake via receptor‐mediated endocytosis is smaller for conjugates than for transferrin (or, the half times characterizing the different steps are higher for conjugates than for transferrin). Endocytosis and iron uptake were unaffected by free doxorubicin (12.5 μM) or colchicin (1 mM). Benzyl alcohol (30 mM) slowed down the rate of both endocytosis and iron uptake, while dithiothetiol (5 mM) decreased the rate of iron uptake and increased the rate of endocytosis. N‐Ethylmaleimide (1 mM) completely stopped both endocytosis and iron uptake. The results suggest that the binding of conjugates to the surface of cells is governed by the binding of the transferrin part of conjugates to the transferrin receptor. However, conjugation of doxorubicin to transferrin seems to influence all properties of transferrin. A minor alteration in the structure may modify the binding affinity and stability of conjugates as well as decrease the accessibility of competing ligand to displace the bound ligand. Either of them could be responsible for the longer endocytosis and recycling time for conjugates. Our results also point to the importance of both the sulfhydryl groups and the fluidity of plasma membrane in the iron uptake via receptor‐mediated endocytosis.