The Aza-Analogous Benzo[c]phenanthridine P8-D6 Acts as a Dual Topoisomerase I and II Poison, thus Exhibiting Potent Genotoxic Properties.

Georg Aichinger,Bernd Clement,Tamara N Steinhauer,Doris Marko,Falk-Bach Lichtenberger,Inken Flörkemeier,Giorgia Del Favero

doi:10.3390/molecules25071524

Georg Aichinger, Bernd Clement + Show 5 more

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https://doi.org/10.3390/molecules25071524

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Abstract

The benzo[c]phenanthridine P8-D6 was recently found to suppress the catalytic activity of both human topoisomerase (Topo) I and II. Concomitantly, potent cytotoxic activity was observed in different human tumor cell lines, raising questions about the underlying mechanisms in vitro. In the present study, we addressed the question of whether P8-D6 acts as a so-called Topo poison, stabilizing the covalent Topo–DNA intermediate, thus inducing fatal DNA strand breaks in proliferating cells. In HT-29 colon carcinoma cells, fluorescence imaging revealed P8-D6 to be taken up by the cells and to accumulate in the perinuclear region. Confocal microscopy demonstrated that the compound is partially located inside the nuclei, thus reaching the potential target. In the “in vivo complex of enzyme” (ICE) bioassay, treatment of HT-29 cells with P8-D6 for 1 h significantly enhanced the proportion of Topo I and II covalently linked to the DNA in concentrations ≥1 µM, indicating effective dual Topo poisoning. Potentially resulting DNA damage was analyzed by single-cell gel electrophoresis (“comet assay”). Already at 1 h of incubation, significant genotoxic effects were observed in the comet assay in concentrations as low as 1 nM. Taken together, the present study demonstrates the high Topo-poisoning and genotoxic potential of P8-D6 in human tumor cells.

Highlights

Benzo[c]phenanthridines have a wide-ranging pharmacological spectrum of action
We recently described P8-D6 to exert strong cytotoxic, pro-apoptotic, of action is of great importance
Results manner for cells incubated with P8-D6 at concentrations of ≥ 50 μM

Summary

Introduction

Benzo[c]phenanthridines have a wide-ranging pharmacological spectrum of action. Especially cytotoxic, and antimicrobial and anti-inflammatory effects have been previously described [1,2].Naturally occurring substances of this drug class, such as fagaronine, have been described to inhibit topoisomerase I and II by stabilizing the covalent DNA–enzyme intermediate [3]. Benzo[c]phenanthridines have a wide-ranging pharmacological spectrum of action. Cytotoxic, and antimicrobial and anti-inflammatory effects have been previously described [1,2]. Occurring substances of this drug class, such as fagaronine, have been described to inhibit topoisomerase I and II by stabilizing the covalent DNA–enzyme intermediate [3]. Due to poor yields and toxicological problems, synthesis of azo-analogous benzo[c]phenanthridines was previously optimized into a four step process [4,5]. The synthetic products were improved to enhance physicochemical properties and cytotoxicity. In the context of possible pharmaceutical applications, the compound

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