Abstract B48: Xanthohumol from hops (Humulus lupulus L.) reactivates epigentically silenced genes by potential DNA methyltransferase inhibition

Abstract

Abstract Epigenetic silencing of tumor suppressor genes by DNA hypermethylation is now recognized as a hallmark of cancer. Thus, reversal of DNA hypermethylation by demethylating agents and DNA methyltranserase (Dnmt) inhibitors has been extensively investigated. Recently, several studies have shown that natural dietary compounds have the potential to reactivate epigenetically silenced genes by various mechanisms, which might have an impact on chemoprevention strategies. Xanthohumol (XN), a prenylated chalcone from hops, exerts a broad spectrum of chemopreventive actions and inhibits mammary carcinogenesis in vivo. In this study, we report for the first time that epigenetic deregulation of gene expression during carcinogenesis is a novel target of XN action. We demonstrated that XN reactivates a series of candidate genes silenced by DNA promoter hypermethylation in human cancer cell lines. By treating human breast and prostate cancer cell lines MCF-7 and LNCaP with subtoxic XN-concentrations for 48 and 72h, respectively, we observed significant dose-dependent re-expression of glutathione-S-transferase Π (GSTPi), Cyclin D2 and RassF1A mRNA, measured by quantitative RT-PCR. The demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) was used as positive control. To address if the XN-mediated mechanism of gene re-expression involved inhibition of Dnmt activity, we tested XN in an in vitro system using the bacterial methlytransferase M.SssI as an enzyme source. XN at concentrations ranging from 3.13–50 µM dose-dependently inhibited M.SssI activity with a halfmaximal inhibitory concentration of 35.2 µM (p<0.05). Computational modelling of XN docking to Dnmt1 indicates steric interaction with the active site. In addition, in MCF-7 and LNCaP cells treated with XN, expression of Dnmt1, 3a and 3b was inhibited by XN-treatment in a dose-dependent manner, both at the transcriptional (p<0.05) and protein level. Next, demethylating potential of XN was investigated. DNA of XN-treated MCF-7 and LNCaP cells was subjected to quantitative methylation analyis using the Sequenom Massarray platform. Whereas treatment for 72h with 1µM of 5-aza-dC significantly reduced DNA hypermethylation of promoter CpG islands of GSTPi, Cyclin D2 and RassF1A by 10–20% (p<0.05), XN-treatment with 5–10µM did not show general DNA demethylation. However, individual CpG sites in the analyzed regions were strongly demethylated upon XN-treatment. Ongoing studies investigate whether demethylation of these single CpG sites is crucial for active transcription, and whether this can be correlated to induced gene-expression. In conclusion, we demonstrated that XN reactivates the expression of hypermethlyated genes by inhibition of Dnmt activity, expression and demethylation of selected CpG sites, which represents a novel chemopreventive mechanism by XN. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B48.