Alternariol (AOH), a mycotoxin formed by Alternaria alternata, has been reported to possess genotoxic properties. However, the underlying mechanism of action is unclear. Here, we tested the hypothesis that interactions with DNA-topoisomerases play a role in the DNA-damaging properties of AOH. First we compared DNA-damaging properties of AOH with other Alternaria mycotoxins such as AOH monomethyl ether (AME), altenuene and isoaltenuene. AOH and AME significantly increased the rate of DNA strand breaks in human carcinoma cells (HT29, A431) at micromolar concentrations, whereas altenuene and isoaltenuene did not affect DNA integrity up to 100 microM. Next, we selected AOH as the most DNA-damaging Alternaria metabolite for further studies of interactions with DNA topoisomerases. In cell-free assays, AOH potently inhibited DNA relaxation and stimulated DNA cleavage activities of topoisomerase I, IIalpha and IIbeta. Stabilisation of covalent topoisomerase II-DNA intermediates by AOH was also detectable in cell culture, and here, the IIalpha isoform was preferentially targeted. AOH is thus characterised as a poison of topoisomerase I and II with a certain selectivity for the IIalpha isoform. Since topoisomerase poisoning and DNA strand breakage occurred within the same concentration range, poisoning of topoisomerase I and II might at least contribute to the genotoxic properties of AOH.
In the present study, we investigated the effect of anthocyanidins on human topoisomerases I and II and its relevance for DNA integrity within human cells. Anthocyanidins bearing vicinal hydroxy groups at the B-ring (delphinidin, DEL; cyanidin, CY) were found to potently inhibit the catalytic activity of human topoisomerases I and II, without discriminating between the IIalpha and the IIbeta isoforms. However, in contrast to topoisomerase poisons, DEL and CY did not stabilize the covalent DNA-topoisomerase intermediates (cleavable complex) of topoisomerase I or II. Using recombinant topoisomerase I, the presence of CY or DEL (> or = 1 microM) effectively prohibited the stabilization of the cleavable complex by the topoisomerase I poison camptothecin. We furthermore investigated whether the potential protective effect vs topoisomerase I poisons is reflected also on the cellular level, affecting the DNA damaging properties of camptothecin. Indeed, in HT29 cells, low micromolar concentrations of DEL (1-10 microM) significantly diminished the DNA strand breaking effect of camptothecin (100 microM). However, at concentrations > or = 50 microM, all anthocyanidins tested (delphinidin, cyanidin, malvidin, pelargonidin, and paeonidin), including those not interfering with topoisomerases, were found to induce DNA strand breaks in the comet assay. All of these analogues were able to compete with ethidium bromide for the intercalation into calf thymus DNA and to replace the minor groove binder Hoechst 33258. These data indicate substantial affinity to double-stranded DNA, which might contribute at least to the DNA strand breaking effect of anthocyanidins at higher concentrations (> or = 50 microM).
The anthocyanidin delphinidin (DEL) has recently been shown to inhibit human topoisomerase I and II, without stabilizing the covalent DNA/topoisomerase intermediate [Habermeyer, M., Fritz, J., Barthelmes, H. U., Christensen, M. O., Larsen, M. K., Boege, F., and Marko, D. (2005) Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity. Chem. Res. Toxicol. 18, 1395-404]. In the present study, we demonstrated that DEL affects the catalytic activity of topoisomerase IIalpha in a redox-independent manner. Furthermore, this potent inhibitory effect is not limited to a cell-free system, but is also of relevance within intact cells. DEL at micromolar concentrations was found to significantly decrease the level of topoisomerase IIalpha/DNA intermediates stabilized by the topoisomerase II poison doxorubicin in the human colon carcinoma cell line (HT29). In addition, DEL diminished the DNA-damaging properties of topoisomerase II poisons in HT29 cells without affecting the level of sites sensitive to formamidopyrimidine-DNA-glycosylase. However, the preventive effect on DNA damage exhibited an apparent maximum at a concentration of 10 microM DEL, followed by a recurrence of DNA damage at higher DEL concentrations. Furthermore, the incubation of HT29 cells with 10 microM DEL resulted in a decrease of etoposide (ETO)-induced DNA strand breaks. However, the level of ETO-stabilized covalent topoisomerase/DNA intermediates did not affect DEL, indicating an additional mechanism of action. An impact of DEL on genes involved in the repair of DNA double-strand breaks and the onset of apoptosis has to be considered. In conclusion, the natural food constituent DEL represents, depending on the concentration range, a protective factor against the DNA-damaging effects of topoisomerase II poisons in vitro. Further studies are needed to clarify whether in vivo a high DEL intake might compromise the therapeutic outcome of these anticancer agents.
Oligomeric procyanidins were found to inhibit the protein tyrosine kinase activity of the epidermal growth factor receptor (EGFR). The inhibitory potency was found to increase with the degree of oligomerisation (PA2 > PC1 >> PB1 = PB2 = PB3 = PB4 >> (-)-epicatechin). To address the question whether the interference with the activity of isolated EGFR preparations also plays a role within intact cells, effects on the phosphorylation status of the EGFR, as a measure of its activity, were determined in human colon carcinoma cells. Treatment of HT29 cells with the trimeric procyanidin PC1 resulted in a decrease of the EGFR autophosphorylation already at low micromolar concentrations. A respective procyanidin tetramer (PA2) failed to affect substantially the receptor phosphorylation status by up to 50 microM, indicating that the effectiveness of oligomeric procyanidins against EGFR activity within intact cells might be limited with increasing degree of polymerisation. Nevertheless, oligomeric procyanidins were identified as potential inhibitors of the EGFR, which might be of interest with respect to chemoprevention. However, PC1 and PA2 were also identified as potent inhibitors of the catalytic activity of human topoisomerase I and II, demanding further studies to elucidate whether the interference of procyanidins with topoisomerases might impair DNA integrity, thus limiting their usefulness in terms of chemoprevention.
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