Abstract 2531: Vescalagin, a polyphenolic ellagitanin specifically inhibiting the alpha isoform of human DNA topoisomerase II
Authors: Auzanneau Céline , Montaudon Danièle , Puyo Stéphane , Jacquet Rémi , Elkaoukabi-Chaibi Assia , Quideau Stéphane , De Giorgi Francesca , Ichas François , Pourquier Philippe 
Published: June 2012

Abstract
Polyphenolic ellagitanins are natural compounds that are often associated with the therapeutic activity of plant extracts used in traditional medicine. They display cancer-preventing activity in animal models at concentrations found in diets by a mechanism which remains unclear. Potential targets have been proposed, including topoisomerases II (Top2) which are involved in the regulation of replication, transcription and chromosome segregation. Top2α and Top2β, the two human isoforms of Top2, are the target of anticancer agents such as etoposide (VP-16). Antitumoral activity of VP-16 is primarily due the inhibition of top2α, whereas inhibition of top2β is responsible for the development of secondary malignancies, pointing towards the need for more selective top2α inhibitors. In this study, vescalagin, methylvescalagin, acutissimin B (acuB), and epiacutissimin B (epiB) were tested for their selectivity against Top2α and Top2β. Using kinetoplast DNA as a substrate, we showed that Top2α-mediated decatenation was inhibited by ≈ 50% by all 4 compounds at 1 µM, whereas only minor inhibition of top2β could be observed (&lt;20%). At 10 and 100 µM, inhibition of Top2α remained selective for vescalagin, but not for the other compounds, acuB and epiB being slightly more selective towards Top2β. Selective Top2α inhibition by vescalagin was further investigated in cells. Preliminary results show that downregulation of Top2α by siRNA renders human erythroleukemic CEM cells ≈ 4-fold resistant to 1µM vescalagin, whereas downregulation of Top2β increased CEM resistance only by 2-fold. This is in accordance with the fact that VP-16-resistant CEM VM1 cells are also resistant to vescalagin (&gt; 100 fold). We confirmed that vescalagin was a catalytic inhibitor of Top2α, since no double-strand break formation or phosphorylation of H2AX could be observed in treated CEM cells (50 µM, 2h). This was confirmed by the absence of Top2α-DNA cleavage complexes formation as measured by the Immuno-Complex of Enzyme (ICE) assay. Since polyphenolic ellagitanin are known to induce ROS, we investigated the potential contribution of ROS on vescalagin-induced cytotoxicity. Indeed, vescalagin treatment induced single-strand breaks (SSB) in both CEM and CEM VM1 cell lines. We showed that treatment with N-acetylcystein (NAC) prevented the formation of vescalagin-induced SSB in both cell lines but had no significant effect on vescalagin-induced cytotoxicity in CEM or CEM-VM1 cells. This precludes the role of ROS in vescalagin induced cytotoxicity by specific inhibition of Top2α. Together, our data suggest that vescalagin is a new polyphenolic ellagitanin compound which specifically inhibits Top2α in vitro and in cells at pharmacological concentrations.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2531. doi:10.1158/1538-7445.AM2011-2531