Anthocyanins and structurally related flavonoids: cellular mechanisms and their relevance for DNA integrity in-vitro and in-vivo
Zusammenfassung der Projektergebnisse
Scope of the project was to gain a more detailed insight into the mode of action of anthocyanins and anthocyanidins with special emphasis on signaling cascades such as the MAPK- and the PI3K-cascade, pathways pivotal in the regulation of proliferation, differentiation and apoptosis. DEL potently inhibited the tyrosine kinase activity of a variety of receptors of these pathways such as the ErbB-receptors, the vascular epidermal growth factor (VEGF) receptors and the insulin like growth factor receptor 1. Furthermore, the activity of the ErbB- and VEGF-receptors was also inhibited in the intact cell system. In addition, anthocyanin-rich extracts potently inhibited the receptor tyrosine kinases (RTKs) activity as well however with low specificity. Comparison of the free aglycon DEL with the complex anthocyanin-rich mixtures identified identical inhibition profiles for RTK inhibition. Therefore, anthocyanidins and anthocyanins were characterized as broad band inhibitors of a panel of RTKs. In intact cells, ErbB3 and VEGFR3 seemed especially sensitive to inhibition by anthocyanidins and anthocyanins. Moreover a potential transfer of the inhibitory signal of DEL on ErbB3 to downstream elements of the signaling cascade PI3K/PKB was traceable in human vulva carcinoma cells. After long time treatment with DEL activation of the caspase cascade, known to induce apoptosis, was observed representing an important mechanism of action of DEL. Due to reports on the formation of hydrogen peroxide under in-vitro conditions, accumulation of hydrogen peroxide by the anthocyanidin delphinidin (DEL) and different anthocyanin-rich extracts was tested, indicating a substantial accumulation of hydrogen peroxide which could be suppressed by the addition of catalase. Flavonoids have been ascribed DNA-damaging properties. Inhibition of topoisomerase activity is considered as a potential mode of action inducing DNA-strand breaks. Human topoisomerases are enzymes regulating DNA topology by forming stabilized intermediary complexes with the DNA. In the present project the anthocyanidins DEL and cyaniding (CY) were identified as inhibitors of topoisomerase activity contributing to their DNA-strand breaking properties. However the intermediary complex between DNA and topoisomerase is not stabilized by DEL and CY, indicating that these flavonoids do not act as “classical” topoisomerase poisons. Moreover, using recombinant human topoisomerase I, the presence of CY or DEL (≥ 1 µM) effectively prohibited the stabilization of the cleavable complex by the topoisomerase I poison camptothecin. Anthocyanidins have been reported to represent potent antioxidants. However, anthocyanidins as well as their glycosides failed to reduce oxidative DNA damage in human colon tumor cells. Findings on the formation of hydrogen peroxide in cell culture medium in the presence of anthocyanins/ anthocyanidins raised the question whether experimental artifacts might play a role for the lack of antioxidative efficacy in-vitro. We observed that suppression of hydrogen peroxide accumulation in the cell culture medium by catalase avoided putative oxidative DNA damage at high DEL concentrations. Furthermore DEL failed to suppress the DNA-damaging effects of the redox cycler menadion when catalase is absent. When the accumulation of hydrogen peroxide was suppressed DEL clearly displayed antioxidative properties effectively suppressing the effects of menadion. In a cell-free test system for topoisomerase IIα DEL led to a concentration dependent suppression of etoposid (ETO)- or doxorubicin (DOX)-induced DNA-damage, indicating a protective effect of DEL against topoisomerase II poisons. Similar results were obtained for the intact cell. A major milestone of the project was to investigate whether a treatment with anthocyanins affects the potency of topoisomerase I poison irinotecan to stabilize the cleavable complex formation in the colon of Wistar rats as main target organ of secondary polyphenols. In a pilot animal study we could show that the intraperitoneally treatment of Wistar rats with irinotecan, a topoisomerase I poison, led to a significant increase of covalent topoisomerase I/DNA intermediates in stomach, small intestine, colon and liver. In contrast, the oral treatment of Wistar rats with CY, cyaniding-3- glycoside or an anthocyanin-rich blackberry extract respectively did not increase the amount of topoisomerase I/DNA intermediates in the colon of Wistar rats, supporting the hypothesis that anthocyanins act as pure catalytic topoisomerase inhibitor and not as classical topoisomerase poison. Moreover in the colon of Wistar rats pretreated with CY or blackberry extract followed by irinotecan treatment the amount of topoisomerase I bound to DNA was significantly decreased. These results support the hypothesis that anthocyanins/anthocyanidins potentially decrease the effect of topoisomerase poisons during cancer chemotherapy, a finding with probable impact for future risk/benefit assessment.
Projektbezogene Publikationen (Auswahl)
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(2008) Comparison of delphinidin, quercetin and (-)-epigallocatechin-3-gallate as inhibitors of the EGFR and the ErbB2 receptor phosphorylation. Mol Nutr Food Res. 52, 815-822
Fridrich, D.; Teller, N.; Esselen, M.; Pahlke, G.; Marko, D.
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(2007) Limited stability in cell culture medium and hydrogen peroxide formation affect the growth inhibitory properties of delphinidin and its degradation product gallic acid. Mol Nutr Food Res. 51, 1163-1172
Kern, M.; Fridrich, D.; Reichert, J.; Skrbek, S.; Nussher, A.; Hofem, S.; Vatter, S.; Pahlke, G.; Rüfer, C.; Marko, D.
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(2008) Impact of delphinidin on the maintenance of DNA integrity in human colon carcinoma cells. J Agric Food Chem. 56, 8891-8896
Fritz, J.; Roth, M.; Holbach, P.; Esselen, M.; Marko, D.
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(2009) Delphinidin inhibits a broad spectrum of receptor tyrosine kinases of the ErbB and VEGFR family. Mol Nutr Food Res. 53, 1075-1083
Teller, N.; Thiele, W.; Boettler, U.; Sleeman, J.; Marko, D.
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(2009) Delphinidin modulates the DNA-damaging properties of topoisomerase II poisons. Chem Res Toxicol. 22, 554-564
Esselen, M.; Fritz, J.; Hutter, M.; Marko, D.
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(2009) Do anthocyanins and anthocyanidins, cancer chemopreventive pigments in the diet, merit development as potential drugs? Cancer Chemother Pharmacol. 64, 201-211
Thomasset, S.; Teller, N.; Cai, H.; Marko, D.M.; Berry, D.P.; Steward, W.P.; Gescher, A.J.
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(2009) Suppression of the kinase activity of receptor tyrosine kinases by anthocyanin-rich mixtures extracted from bilberries and grapes. J Agric Food Chem. 57, 3094-3101
Teller, N.; Thiele, W.; Marczylo, T.H.; Gescher, A.J.; Boettler, U.; Sleeman, J.; Marko, D.
