Abstract
F14512 is a novel anti-tumor molecule based on an epipodophyllotoxin core coupled to a cancer-cell vectoring spermine moiety. This polyamine linkage is assumed to ensure the preferential uptake of F14512 by cancer cells, strong interaction with DNA and potent inhibition of topoisomerase II (Topo II). The antitumor activity of F14512 in human tumor models is significantly higher than that of other epipodophyllotoxins in spite of a lower induction of DNA breakage. Hence, the demonstrated superiority of F14512 over other Topo II poisons might not result solely from its preferential uptake by cancer cells, but could also be due to unique effects on Topo II interactions with DNA. To further dissect the mechanism of action of F14512, we used Drosophila melanogaster mutants whose genetic background leads to an easily scored phenotype that is sensitive to changes in Topo II activity and/or localization. F14512 has antiproliferative properties in Drosophila cells and stabilizes ternary Topo II/DNA cleavable complexes at unique sites located in moderately repeated sequences, suggesting that the drug specifically targets a select and limited subset of genomic sequences. Feeding F14512 to developing mutant Drosophila larvae led to the recovery of flies expressing a striking phenotype, "Eye wide shut," where one eye is replaced by a first thoracic segment. Other recovered F14512-induced gain- and loss-of-function phenotypes similarly correspond to precise genetic dysfunctions. These complex in vivo results obtained in a whole developing organism can be reconciled with known genetic anomalies and constitute a remarkable instance of specific alterations of gene expression by ingestion of a drug. "Drosophila-based anticancer pharmacology" hence reveals unique properties for F14512, demonstrating the usefulness of an assay system that provides a low-cost, rapid and effective complement to mammalian models and permits the elucidation of fundamental mechanisms of action of candidate drugs of therapeutic interest in humans.
Highlights
Topoisomerase II (Topo II) is an essential enzyme that handles topological reactions in all living cells and is required for different aspects of DNA metabolism, from the resolution of constraints resulting from packaging of DNA into chromatin, to its transcription and replication and, most importantly, the condensation [1] and segregation of sister chromatids in the course of cell division
The results reported here demonstrate that, in addition to halting Drosophila cell growth in culture, F14512 induces unique features including rapid adherence, flattening and multi-nucleation but, at the concentrations used here, without the signs of apoptosis that are the hallmarks of etoposide and TOP-53, its closest relative
We note that the rapid appearance of multinucleated cells induced by F14512 treatment is reminiscent of the effects of dexrazoxane (ICRF-187), a potent Topo II catalytic inhibitor [54], suggesting the possibility of similar mechanisms of action
Summary
Topoisomerase II (Topo II) is an essential enzyme that handles topological reactions in all living cells and is required for different aspects of DNA metabolism, from the resolution of constraints resulting from packaging of DNA into chromatin, to its transcription and replication and, most importantly, the condensation [1] and segregation of sister chromatids in the course of cell division (reviewed in [2,3,4]) As such, it constitutes a target of choice for anticancer drugs ([5,6,7] and references therein). Accumulation of these lesions, which cannot be handled by the cellular DNA repair machinery, usually induces apoptotic pathways that result in the onset of cell death within approximately 48–72 hours in the case of dividing cells [14,15,16,17,18]
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