Gonadotropin releasing hormone-III (
GnRH-III), a native
isoform of the human
GnRH isolated from sea lamprey, specifically binds to
GnRH receptors on
cancer cells enabling its application as targeting moieties for anticancer drugs. Recently, we reported on the identification of a novel
daunorubicin-
GnRH-III conjugate (
GnRH-III-[4Lys(Bu), 8Lys(Dau=Aoa)] with efficient in vitro and in vivo antitumor activity. To get a deeper insight into the mechanism of action of our lead compound, the cellular uptake was followed by confocal
laser scanning microscopy. Hereby, the drug
daunorubicin could be visualized in different subcellular compartments by following the localization of the drug in a time-dependent manner. Colocalization studies were carried out to prove the presence of the drug in lysosomes (early stage) and on its site of action (nuclei after 10 min). Additional flow cytometry studies demonstrated that the cellular uptake of the bioconjugate was inhibited in the presence of the competitive
ligand triptorelin indicating a receptor-mediated pathway. For comparative purpose, six novel
daunorubicin-
GnRH-III bioconjugates have been synthesized and biochemically characterized in which 6Asp was replaced by D-Asp, D-Glu and D-Trp. In addition to the analysis of the in vitro
cytostatic effect and cellular uptake, receptor binding studies with 125I-
triptorelin as radiotracer and degradation of the
GnRH-III conjugates in the presence of rat liver lysosomal homogenate have been performed. All derivatives showed high binding affinities to
GnRH receptors and displayed in vitro
cytostatic effects on HT-29 and MCF-7
cancer cells with IC50 values in a low micromolar range. Moreover, we found that the release of the active drug metabolite and the cellular uptake of the bioconjugates were strongly affected by the
amino acid exchange which in turn had an impact on the antitumor activity of the bioconjugates.