In vitro degradation and antitumor activity of oxime bond-linked daunorubicin-GnRH-III bioconjugates and DNA-binding properties of daunorubicin-amino acid metabolites |
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Authors: | Orbán Erika Mezo Gábor Schlage Pascal Csík Gabriella Kulić Zarko Ansorge Philipp Fellinger Erzsébet Möller Heiko Michael Manea Marilena |
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Institution: | (1) Research Group of Peptide Chemistry, Hungarian Academy of Sciences, E?tv?s Lor?nd University, P?zm?ny P. stny. 1/A, 1117 Budapest, Hungary;(2) Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University of Konstanz, Universit?tsstrasse 10, 78457 Constance, Germany;(3) Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry and Zukunftskolleg, University of Konstanz, Universit?tsstrasse 10, 78457 Constance, Germany;(4) Department of Biophysics and Radiobiology, Semmelweis Medical University, Budapest, Hungary;(5) NMR Spectroscopy, Department of Chemistry, University of Konstanz, 78457 Constance, Germany;(6) Department of Anatomy, Cell and Developmental Biology, E?tv?s Lor?nd University, 1117 Budapest, Hungary; |
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Abstract: | Bioconjugates with receptor-mediated tumor-targeting functions and carrying cytotoxic agents should enable the specific delivery
of chemotherapeutics to malignant tissues, thus increasing their local efficacy while limiting the peripheral toxicity. In
the present study, gonadotropin-releasing hormone III (GnRH-III; Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2) was employed as a targeting moiety to which daunorubicin was attached via oxime bond, either directly or by insertion of
a GFLG or YRRL tetrapeptide spacer. The in vitro antitumor activity of the bioconjugates was determined on MCF-7 human breast
and HT-29 human colon cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their degradation/stability
(1) in human serum, (2) in the presence of cathepsin B and (3) in rat liver lysosomal homogenate was analyzed by liquid chromatography
in combination with mass spectrometry. The results show that (1) all synthesized bioconjugates have in vitro antitumor effect,
(2) they are stable in human serum at least for 24 h, except for the compound containing an YRRL spacer and (3) they are hydrolyzed
by cathepsin B and in the lysosomal homogenate. To investigate the relationship between the in vitro antitumor activity and
the structure of the bioconjugates, the smallest metabolites produced in the lysosomal homogenate were synthesized and their
binding to DNA was assessed by fluorescence spectroscopy. Our data indicate that the incorporation of a peptide spacer in
the structure of oxime bond-linked daunorubicin–GnRH-III bioconjugates is not required for their antitumor activity. Moreover,
the antitumor activity is influenced by the structure of the metabolites (daunorubicin–amino acid derivatives) and their DNA-binding
properties. |
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