Apple procyanidins affect several members of the ErbB receptor tyrosine kinase family in vitro.

Complex polyphenol-rich extracts from apples are known to inhibit the activity of the epidermal growth factor receptor (EGFR) in vitro. The aim of the present study was to identify the bioactive constituents of the apple juice extract which contribute substantially to this potentially chemopreventive effect and to address the question whether the effect is specific to the EGFR or whether other members of the ErbB-receptor family might also be affected. Apple-derived dihydrochalcones and their respective glycosides were found to decrease EGFR activity under cell-free conditions with IC50-values ranging from 0.4 ± 0.1 to 267.0 ± 50.0 µM but showed no activity on human cancer cells. The concentration of quercetin or its glycosides in the extract was too low to contribute substantially to the EGFR-inhibitory properties. In contrast, fractions derived from the apple juice extract comprising ≥86% oligomeric procyanidins (OPCs) suppressed the activity of the EGFR in cell culture with an IC50 ∼ 100 µg mL(-1). In addition, the activity of further members of the ErbB-receptor family was potently inhibited, with ErbB3 receptor activity being most potently decreased (IC50 ∼ 10 µg mL(-1)). From the apple polyphenols identified so far OPCs were found to add the highest contribution to the inhibitory effects towards members of the ErbB-receptor family. Considering the crucial role of the ErbB-receptors in carcinogenesis, these results support the hypothesis that apple-derived OPCs as well as OPC-rich apple preparations might be of interest with respect to chemoprevention.

Comparison of delphinidin, quercetin and (-)-epigallocatechin-3-gallate as inhibitors of the EGFR and the ErbB2 receptor phosphorylation.

In the present study, delphinidin was found to suppress the phosphorylation of the epidermal growth factor receptor (EGFR) within human tumour cells (human colon carcinoma cell line (HT29), human vulva carcinoma cell line (A431)), albeit less effective than the flavonol quercetin. The higher potency of quercetin was also observed downstream on the level of the mitogen-activated protein kinase (MAPK) cascade. In addition, delphinidin, quercetin and (-)-epigallocatechin-3-gallate (EGCG) were found to suppress the phosphorylation of the ErbB2 receptor, with delphinidin exhibiting the strongest inhibitory properties. Their potency to suppress the ErbB2 receptor phosphorylation can be summarised as delphinidin > EGCG > quercetin. The effectiveness of delphinidin against the EGFR and the ErbB2 receptor was comparable, indicating a broader spectrum of activity against receptor tyrosine kinases. At low micromolar concentrations delphinidin showed some preference towards the ErbB2 receptor. In summary, quercetin and delphinidin appear to differ in their activity profile towards the ErbB receptor family members. Whereas quercetin was most effective against the EGFR, delphinidin exhibited some preference towards the ErbB2 receptor.

Oak ellagitannins suppress the phosphorylation of the epidermal growth factor receptor in human colon carcinoma cells.

The ellagitannins castalagin and vescalagin, and the C-glycosides grandinin and roburin E as well as ellagic acid were found to potently inhibit the growth of human colon carcinoma cells (HT29) in vitro. In a cell-free system these compounds were identified as potent inhibitors of the protein tyrosine kinase activity of the epidermal growth factor receptor (EGFR) with IC 50 values in the low nanomolar range. To address the question of whether the interference with the activity of the isolated EGFR also plays a role within intact cells, effects on the phosphorylation status of the EGFR, as a measure for its activity, were determined in HT29 cells. As exemplified for castalagin and grandinin, both the nonglycosylated and the glycosylated ellagitannins effectively suppressed EGFR phosphorylation, but only at concentrations > or =10 microM, thus, in a concentration range where growth inhibition was observed. These results indicate that the suppression of EGFR-mediated signaling might contribute to the growth inhibitory effects of these compounds present in oak-matured wines and spirits such as whiskey. In contrast, despite substantial growth inhibitory properties, ellagic acid did not significantly affect EGFR phosphorylation in HT29 cells up to 100 microM.

Limited stability in cell culture medium and hydrogen peroxide formation affect the growth inhibitory properties of delphinidin and its degradation product gallic acid.

In the present study we investigated the stability of anthocyanidins under cell culture conditions and addressed the question whether degradation products might contribute to the cellular effects assigned to the parent compounds. Substantial degradation was found already after 30 min, measured by HPLC/DAD. However, the decrease of detectable anthocyanidins exceeded by far the formation of the respective phenolic acids. From the formed phenolic acids only gallic acid (GA) exhibited growth inhibitory properties. However, also GA was found to be degraded rapidly. Furthermore, the incubation with delphinidin (DEL) or GA resulted in a substantial formation of hydrogen peroxide. The suppression of hydrogen peroxide accumulation by catalase modified significantly the growth inhibitory effects of DEL and GA, indicating that hydrogen peroxide formation might generate experimental artefacts. In summary, the results show that the phenolic acids formed by the degradation of cyanidin (CY), pelargonidin (PG), peonidin (PN) and malvidin (MV) do not contribute to the growth inhibitory effect of the parent compound. The degradation of DEL generates a phenolic acid with substantial growth inhibitory properties (GA). However, taken into account the small proportion of generated GA and its lacking stability, the contribution of GA to the growth inhibitory properties of DEL might be limited.

Apple polyphenols diminish the phosphorylation of the epidermal growth factor receptor in HT29 colon carcinoma cells.

Previously, we showed that an apple juice extract (AE) potently inhibits the protein tyrosine kinase (PTK) activity of the epidermal growth factor receptor (EGFR). In the present study, an apple pomace extract (APE) was found to exceed the EGFR inhibitory properties of AE in a cell-free system. The impact of the extracts on the phosphorylation status of the EGFR in intact cells (HT29) was sensitive to catalase, added to suppress the accumulation of hydrogen peroxide. In the absence of catalase, the formation of hydrogen peroxide was observed, achieving 1.1 +/- 0.1 microM (AE) and 1.5 +/- 0.1 microM (APE) after 45 min of incubation. In the presence of catalase, suppressing the hydrogen peroxide level to the solvent control, APE effectively suppressed EGFR phosphorylation, even exceeding the effects of AE. Both extracts inhibited the growth of HT29 cells, albeit the enhanced EGFR inhibitory properties of APE compared to AE were not reflected by a higher growth inhibitory potential. The results clearly show that the effect of apple extracts on the EGFR and cell growth are not simply artefacts of hydrogen peroxide formation. However, the formation of hydrogen peroxide has to be considered to modulate and/or mask cellular responses to apple extracts.

The epidermal growth factor receptor and human topoisomerases represent potential cellular targets of oligomeric procyanidins.

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.

Natural flavonoids are potent inhibitors of glycogen phosphorylase.

There is little known about effects to be expected from high intake of flavonoids with respect to regulation of glucose/glycogen homeostasis. Glucose/glycogen homeostasis is mainly regulated by glycogen synthase (GS) and glycogen phosphorylase (GP). We investigated effects of naturally occurring flavonoids with varying substitution pattern on the activity of isolated muscle GP. Almost all flavonoids tested inhibited phosphorylated, active GPa, as well as unphosphorylated, adenosine monophosphate-activated GPb. However, inhibition of GPa was two-to-fourfold stronger than that of GPb. The flavonol quercetin and the anthocyanidins cyanidin and delphinidin turned out to be the most potent inhibitors of GPa, with concentration values where enzymatic activity is 50% of the respective control in the low micromolar range (<5 microM). Furthermore, by comparing GPa inhibitory activity of typical representatives from all known flavonoid classes, structural elements of flavonoids required for effective GP inhibition could be identified.