| Abstract
| - One of the major problems in cancer chemotherapy are the severe side effects that limit thedose of the anticancer drugs because of their unselectivity for tumor versus normal cells. Inthe present work, we show that coupling of anthracyclines to peptides is a promising approachto obtain selectivity. The peptide−drug conjugate was designed to bind to specific receptorsexpressed on the tumor cells with subsequent internalization of the ligand−receptor complex.Neuropeptide Y (NPY), a 36-amino acid peptide of the pancreatic polypeptide family, was chosenas model peptide because NPY receptors are overexpressed in a number of neuroblastomatumors and the thereof derived cell lines. Daunorubicin and doxorubicin, two widely usedantineoplastic agents in tumor therapy, were covalently linked to NPY via two spacers thatdiffer in stability: an acid-sensitive hydrazone bond at the 13-keto position of daunorubicinand a stable amide bond at the 3‘-amino position of daunorubicin and doxorubicin. Receptorbinding of these three conjugates ([C15]-NPY-Dauno-HYD, [C15]-NPY-Dauno-MBS, and [C15]-NPY-Doxo-MBS) was determined at the human neuroblastoma cell line SK-N-MC, whichselectively expresses the NPY Y1 receptor subtype, and cytotoxic activity was evaluated usinga XTT-based colorimetric cellular cytotoxicity assay. The different conjugates were able to bindto the receptor with affinities ranging from 25 to 51 nM, but only the compound containingthe acid-sensitive bond ([C15]-NPY-Dauno-HYD) showed cytotoxic activity comparable to thefree daunorubicin. This cytotoxicity is Y1 receptor-mediated as shown in blocking studies withBIBP 3226, because tumor cells that do not express NPY receptors were sensitive to freedaunorubicin, but not to the peptide−drug conjugate. The intracellular distribution wasinvestigated by confocal laser scanning microscopy. We found evidence that the active conjugate[C15]-NPY-Dauno-HYD releases daunorubicin, which is localized close to the nucleus, whereasthe inactive conjugate [C15]-NPY-Dauno-MBS is distributed distantly from the nucleus anddoes not seem to release the drug within the cell.
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