In Vitro Inhibitory Potential of Different Anthocyanin-Rich Berry Extracts in Murine CT26 Colon Cancer Cells.

Anti-oxidant, -inflammatory, and -carcinogenic activities of bioactive plant constituents, such as anthocyanins, have been widely discussed in literature. However, the potential interaction of anthocyanin-rich extracts with routinely used chemotherapeutics is still not fully elucidated. In the present study, anthocyanin-rich polyphenol extracts of blackberry (BB), bilberry (Bil), black currant (BC), elderberry (EB), and their respective main anthocyanins (cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-sambubioside) were investigated concerning their cytotoxic and DNA-damaging properties in murine CT26 cells either alone or in combination with the chemotherapeutic agent SN-38. BB exerted potent cytotoxic effects, while Bil, BC, and EB only had marginal effects on cell viability. Single anthocyanins comprised of the extracts could not induce comparable effects. Even though the BB extract further pronounced SN-38-induced cytotoxicity and inhibited cell adhesion at 100-200 µg/mL, no effect on DNA damage was observed. In conclusion, anti-carcinogenic properties of the extracts on CT26 cells could be ranked BB >> BC ≥ Bil ≈ EB. Mechanisms underlying the potent cytotoxic effects are still to be elucidated since the induction of DNA damage does not play a role.

Alternaria alternata Mycotoxins Activate the Aryl Hydrocarbon Receptor and Nrf2-ARE Pathway to Alter the Structure and Immune Response of Colon Epithelial Cells.

After ingestion of food commodities, the gastrointestinal tract (GIT) poses the first barrier against xenobiotics and pathogens. Therefore, it is regularly confronted with external stressors potentially affecting the inflammatory response and the epithelial barrier. Alternaria mycotoxins such as alternariol (AOH) and altertoxin II (ATX-II) are frequently occurring food and feed contaminants that are described for their immunomodulatory capacities. Hence, this study aimed at exploring the effect of AOH and ATX-II as single compounds or binary mixtures on the immune response and epithelial homeostasis in noncancerous colon epithelial cells HCEC-1CT. Both toxins suppressed mRNA levels of proinflammatory mediators interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and secretion of IL-8, as well as mRNA levels of the matrix metallopeptidase 2 (MMP-2). Binary combinations of AOH and ATX-II reduced the response of the single toxins. Additionally, AOH and ATX-II modified immunolocalization of transmembrane proteins such as integrin ß1, zona occludens 1 (ZO-1), claudin 4 (Cldn 4), and occludin (Ocln), which support colonic tissue homeostasis and intestinal barrier function. Moreover, the cellular distribution of ZO-1 was affected by ATX-II. Mechanistically, these effects could be traced back to the involvement of several transcription factors. AOH activated the nuclear translocation of the aryl hydrocarbon receptor (AhR) and the nuclear factor erythroid 2-related factor 2 (Nrf2), governing cell metabolic competence and structural integrity. This was accompanied by altered distribution of the NF-κB p65 protein, an important regulator of inflammatory response. ATX-II also induced AhR and Nrf2 translocation, albeit failing to substantiate the effect of AOH on the colonic epithelium. Hence, both toxins coherently repress the intestinal immune response on the cytokine transcriptional and protein levels. Furthermore, both mycotoxins affected the colonic epithelial integrity by altering the cell architecture.

Immunomodulatory Properties of Blackberry Anthocyanins in THP-1 Derived Macrophages.

An anthocyanin-rich diet is considered to protect against chronic inflammatory processes although the bioavailability of anthocyanins is regarded as rather low. Moreover, the immunomodulatory role of anthocyanins is not fully understood yet. In the present study, fractions of blackberry (Rubus fruticosus) juice were investigated in plasma-relevant concentrations with respect to their immunomodulatory properties in lipopolysaccharide (LPS)-challenged THP-1-derived macrophages. The complex blackberry extract acted ineffective as well as potential degradation products. Cyanidin-3O-glucoside (Cy3glc), the main constituent of blackberry anthocyanins, diminished TNF-α levels at a concentration of 0.02 µg/mL, indicating protective effects as measured with quantitative RT-PCR and multiplex cytokine assays. LPS-boosted activity of transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) of differentiated THP-1 reporter gene cells was marginally inhibited by Cy3glc. LPS-induced microRNA-155 was further increased, supporting the evidence of protection. Of note, fractions obtained from blackberry juice, in particular cyanidin-3O-(6″-dioxalylglucoside), were displaying potential pro-inflammatory properties as these elevated IL-6 and TNF-α levels. In conclusion, highly purified anthocyanin fractions of blackberry juice display both anti- and pro-inflammatory properties at plasma-relevant concentrations depending on their structure and substitution pattern.

Combinatory effects of cereulide and deoxynivalenol on in vitro cell viability and inflammation of human Caco-2 cells.

Deoxynivalenol (DON), one of the most abundant mycotoxins in cereal products, was recently detected with other mycotoxins and the emetic bacterial toxin cereulide (CER) in maize porridge. Within a cereal-based diet, co-exposure to these toxins is likely, hence raising the question of combinatory toxicological effects. While the toxicological evaluation of DON has quite progressed, consequences of chronic, low-dose CER exposure are still insufficiently explored. Information about the combinatory toxicological effects of these toxins is lacking. In the present study, we investigated how CER (0.1-100 ng/mL) and DON (0.01-10 µg/mL) alone and in a constant ratio of 1:100 (CER:DON) affect the cytotoxicity and immune response of differentiated human intestinal Caco-2 cells. While DON alone reduced cell viability only in the highest concentration (10 µg/mL), CER caused severe cytotoxicity upon prolonged incubation (starting from 10 ng/mL after 24 h and 48 h, 2.5 ng/mL and higher after 72 h). After 72 h, synergistic effects were observed at 2.5 ng/mL CER and 0.25 µg/mL DON. Different endpoints of inflammation were investigated in interleukin-1ß-stimulated Caco-2 cells. Notably, DON-induced interleukin-8 transcription and secretion were diminished by the presence of 10 and 25 ng/mL CER after short-term (5 h) incubation, indicating immunosuppressive properties. We hypothesise that habitual consumption of cereal-based foods co-contaminated with CER and DON may cause synergistic cytotoxic effects and an altered immune response in the human intestine. Therefore, further research concerning effects of co-occurring bacterial toxins and mycotoxins on the impairment of intestinal barrier integrity, intestinal inflammation and the promotion of malnutrition is needed.

Bioavailability, metabolism, and excretion of a complex Alternaria culture extract versus altertoxin II: a comparative study in rats.

Despite the frequent infection of agricultural crops by Alternaria spp., their toxic secondary metabolites and potential food contaminants lack comprehensive metabolic characterization. In this study, we investigated their bioavailability, metabolism, and excretion in vivo. A complex Alternaria culture extract (50 mg/kg body weight) containing 11 known toxins and the isolated lead toxin altertoxin II (0.7 mg/kg body weight) were administered per gavage to groups of 14 Sprague Dawley rats each. After 3 h and 24 h, plasma, urine and feces were collected to determine toxin recoveries. For reliable quantitation, an LC-MS/MS method for the simultaneous detection of 20 Alternaria toxins and metabolites was developed and optimized for either biological matrix. The obtained results demonstrated efficient excretion of alternariol (AOH) and its monomethyl ether (AME) via feces (> 89%) and urine (> 2.6%) after 24 h, while the majority of tenuazonic acid was recovered in urine (20 and 87% after 3 and 24 h, respectively). Moreover, modified forms of AOH and AME were identified in urine and fecal samples confirming both, mammalian phase-I (4-hydroxy-AOH) and phase-II (sulfates) biotransformation in vivo. Despite the comparably high doses, perylene quinones were recovered only at very low levels (altertoxin I, alterperylenol, < 0.06% in urine and plasma, < 5% in feces) or not at all (highly genotoxic, epoxide-holding altertoxin II, stemphyltoxin III). Interestingly, altertoxin I was detected in all matrices of rats receiving altertoxin II and suggests enzymatic de-epoxidation in vivo. In conclusion, the present study contributes valuable information to advance our understanding of the emerging Alternaria mycotoxins and their relevance on food safety.

Activation of the Nrf2-ARE pathway by the Alternaria alternata mycotoxins altertoxin I and II.

The mycotoxins altertoxin I and II (ATX I and II) are secondary metabolites produced by Alternaria alternata fungi and may occur as food and feed contaminants, especially after long storage periods. Although the toxic potential of altertoxins has been previously investigated, little is known about the pathways that play a role in their intracellular metabolism. In order to identify potential targets of ATX I and ATX II, the two toxins were tested for interaction with the nuclear factor erythroid-derived 2-like 2/antioxidant response element (Nrf2/ARE) pathway in mammalian cells. This pathway can be activated by various stressors resulting in the expression of enzymes important for metabolism and detoxification. In the present study, only ATX II triggered a concentration-dependent increase in Nrf2-ARE-dependent luciferase expression. Consistently, confocal microscopy revealed an ATX II-induced increase in Nrf2 signal in HT29 intestinal cells. In agreement with these data, ATX II induced the transcription of γ-glutamate cysteine ligase, the key enzyme in catalyzing GSH synthesis of the cells and which is regulated by Nrf2. Further investigations demonstrated that ATX II induced a concentration-dependent depletion of the cellular GSH levels after short incubation time (3 h) and an increase after longer incubation time (24 h). In conclusion, it was demonstrated that ATX II can interact at several levels of the Nrf2-ARE pathway in mammalian cells and that ATX I does not share the same mechanism of action.

Impact of phase I metabolism on uptake, oxidative stress and genotoxicity of the emerging mycotoxin alternariol and its monomethyl ether in esophageal cells.

Studies on the genotoxicity of Alternaria mycotoxins focus primarily on the native compounds. Alternariol (AOH) and its methyl ether (AME) have been reported to represent substrates for cytochrome P450 enzymes, generating hydroxylated metabolites. The impact of these phase I metabolites on genotoxicity remains unknown. In the present study, the synthesis and the toxicological effects of the metabolites 4-hydroxy alternariol (4-OH-AOH) and 4-hydroxy alternariol monomethyl ether (4-OH-AME) are presented and compared to the effects of the parent molecules. Although the two phase I metabolites contain a catecholic structure, which is expected to be involved in redox cycling, only 4-OH-AOH increased reactive oxygen species (ROS) in human esophageal cells (KYSE510), 4 times more pronounced than AOH. No ROS induction was observed for 4-OH-AME, although the parent compound showed some minor impact. Under cell-free conditions, both metabolites inhibited topoisomerase II activity comparable to their parent compounds. In KYSE510 cells, both metabolites were found to enhance the level of transient DNA-topoisomerase complexes in the ICE assay. Although the level of ROS was significantly increased by 4-OH-AOH, neither DNA strand breaks nor enhanced levels of formamidopyrimidine-DNA-glycosylase (FPG)-sensitive sites were observed. In contrast, AOH induced significant DNA damage in KYSE510 cells. Less pronounced or even absent effects of hydroxylated metabolites compared to the parent compounds might at least partly be explained by their poor cellular uptake. Glucuronidation as well as sulfation appear to have only a minor influence. Instead, methylation of 4-OH-AOH seems to be the preferred way of metabolism in KYSE510 cells, whereby the toxicological relevance of the methylation product remains to be clarified.

Cereulide and Deoxynivalenol Increase LC3 Protein Levels in HepG2 Liver Cells.

Food contaminants of bacterial or fungal origin frequently contaminate staple foods to various extents. Among others, the bacterial toxin cereulide (CER) and the mycotoxin deoxynivalenol (DON) co-occur in a mixed diet and are absorbed by the human body. Both toxins exert dis-tinctive mitotoxic potential. As damaged mitochondria are removed via autophagy, mitochondrial and lysosomal toxicity were assessed by applying low doses of single and combined toxins (CER 0.1-50 ng/mL; DON 0.01-5 µg/mL) to HepG2 liver cells. In addition to cytotoxicity assays, RT-qPCR was performed to investigate genes involved in lysosomal biogenesis and autophagy. CER and DON caused significant cytotoxicity on HepG2 cells after 5 and 24 h over a broad concentration range. CER, alone and in combination with DON, increased the transcription of the autophagy related genes coding for the microtubule associated protein 1A/1B light chain 3 (LC3) and sequestome 1 (SQSTM1) as well as LC3 protein expression which was determined using immunocytochemistry. DON increased LC3 protein expression without induction of gene transcription, hence it seems plausible that CER and DON act on different pathways. The results support the hypothesis that CER induces autophagy via the LC3 pathway and damaged mitochondria are therefore eliminated.

Markers for DNA damage are induced in the rat colon by the Alternaria toxin altertoxin-II, but not a complex extract of cultured Alternaria alternata.

Mycotoxins produced by Alternaria spp. act genotoxic in cell-based studies, but data on their toxicity in vivo is scarce and urgently required for risk assessment. Thus, male Sprague-Dawley rats received single doses of a complex Alternaria toxin extract (CE; 50 mg/kg bw), altertoxin II (ATX-II; 0.21 mg/kg bw) or vehicle by gavage, one of the most genotoxic metabolites in vitro and were sacrificed after 3 or 24 h, respectively. Using SDS-PAGE/Western Blot, a significant increase of histone 2a.X phosphorylation and depletion of the native protein was observed for rats that were exposed to ATX-II for 24 h. Applying RT-PCR array technology we identified genes of interest for qRT-PCR testing, which in turn confirmed an induction of Rnf8 transcription in the colon of rats treated with ATX-II for 3 h and CE for 24 h. A decrease of Cdkn1a transcription was observed in rats exposed to ATX-II for 24 h, possibly indicating tissue repair after chemical injury. In contrast to the observed response in the colon, no markers for genotoxicity were induced in the liver of treated animals. We hereby provide the first report of ATX-II as a genotoxicant in vivo. Deviating results for similar concentrations of ATX-II in a natural Alternaria toxin mixture argue for substantial mixture effects.

Antioxidant Effects of Elderberry Anthocyanins in Human Colon Carcinoma Cells: A Study on Structure-Activity Relationships.

SCOPE: Glycosylation is a way to increase structure-stability of anthocyanins, yet compromises their bioactivity. The study investigates the antioxidant activity of purified cyanidin (Cy)-based anthocyanins and respective degradation products in Caco-2 clone C2BBe1 aiming to identify structure-activity relationships. RESULTS AND METHODS: Cyanidin 3-O-glucoside (Cy-3-glc) and cyanidin 3-O-sambubioside (Cy-3-sam) proved to be most potent regarding antioxidant properties and protection against hydrogen peroxide (H2 O2 )-induced reactive oxygen species (ROS)-levels measured with the dichloro-fluorescein (DCF) assay. Cyanidin 3-O-sambubioside-5-O-glucoside (Cy-3-sam-5-glc) and cyanidin 3-O-rutinoside (Cy-3-rut) were less efficient and not protective, reflecting potential differences in uptake and/or degradation. Following ranking in antioxidant efficiency is suggested: (concentrations ≤10 × 10-6  M) Cy-3-glc ≥ Cy-3-sam > Cy-3-sam-5-glc ≈ Cy-3-rut ≈ Cy; (concentrations ≥50 × 10-6  M) Cy-3-glc ≈ Cy-3-sam ≥ Cy > Cy-3-sam-5-glc ≈ Cy-3-rut. Cy and protocatechuic acid (PCA) reduced ROS-levels as potent as the mono- and di-glycoside, whereas phloroglucinol aldehyde (PGA) displayed pro-oxidant properties. None of the degradation products protected from oxidative stress. Gene transcription analysis of catalase (CAT), superoxide-dismutase (SOD), glutathione-peroxidase (GPx), heme-oxygenase-1 (HO-1), and glutamate-cysteine-ligase (γGCL) suggest no activation of nuclear factor erythroid 2-related factor 2 (Nrf2). CONCLUSION: More complex residues and numbers of sugar moieties appear to be counterproductive for antioxidant activity. Other mechanisms than Nrf2-activation should be considered for protective effects.

Consumption of anthocyanin-rich beverages affects Nrf2 and Nrf2-dependent gene transcription in peripheral lymphocytes and DNA integrity of healthy volunteers.

Recently, we demonstrated that the consumption of a bolus of bilberry extract modulates the transcription of Nrf2-regulated genes in peripheral blood lymphocytes (PBL) of healthy volunteers, accompanied by decreased DNA-damage. In the present study, we addressed the question whether consumption of consumer-relevant amounts of anthocyanin-rich beverages can achieve similar effects. The impact of three different anthocyanin-rich beverages on Nrf2-dependent gene transcription as well as and the status of DNA-damage in whole blood was investigated. After a polyphenol-reduced diet, five healthy male subjects consumed a bolus (700 mL) of respective test beverages with blood sampling up to 8 h after intake. All beverages affected the transcription of Nrf2, HO-1 and NQO-1, but showed different potencies and persistence of effects. Consumption of red fruit juice significantly reduced total DNA strand breaks (with formamidopyrimidine-DNA-glycosylase-(fpg) treatment) after 8 h in blood samples of the volunteers, suggesting antioxidant and DNA protective effects, albeit transcript levels of Nrf2-dependent genes had reached the basal state. The amount of basic DNA strand breaks (damage without oxidative DNA strand breaks) remained unchanged during the monitoring period. In contrast, a beverage prepared from grape skin extract significantly increased basic and total DNA strand breaks 2 h after intake, underlining the necessity of further investigations regarding composition, safety and consumer´s acceptance of respective products to exclude undesired adverse effects. Consumption of a bolus of anthocyanin-rich beverages affected Nrf2 and Nrf2-dependent gene transcription in human PBL and DNA integrity, which is indicative for systemic effects.

Long-Term Consumption of Anthocyanin-Rich Fruit Juice: Impact on Gut Microbiota and Antioxidant Markers in Lymphocytes of Healthy Males.

Polyphenols are considered protective against diseases associated with oxidative stress. Short-term intake of an anthocyanin-rich fruit juice resulted in significantly reduced deoxyribonucleic acid (DNA) strand-breaks in peripheral blood lymphocytes (PBLs) and affected antioxidant markers in healthy volunteers. Consequently, effects of long-term consumption of fruit juice are of particular interest. In focus was the impact on nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2), the Nrf2-regulated genes NAD(P)H quinone oxidoreductase 1 (NQO-1) and heme oxygenase 1 (HO-1) as well as effects on the gut microbiota. In a nine-week placebo-controlled intervention trial with 57 healthy male volunteers, consumption of anthocyanin-rich juice significantly increased NQO-1 and HO-1 transcript levels in PBLs compared to a placebo beverage as measured by real-time polymerase chain reaction (PCR). Three Nrf2-promotor single nucleotide polymorphisms (SNPs), analyzed by pyrosequencing, indicated an association between individual Nrf2 transcript levels and genotype. Moreover, the Nrf2 genotype appeared to correlate with the presence of specific microbial organisms identified by 16S-PCR and classified as Spirochaetaceae. Furthermore, the microbial community was significantly affected by the duration of juice consumption and intake of juice itself. Taken together, long-term consumption of anthocyanin-rich fruit juice affected Nrf2-dependent transcription in PBLs, indicating systemic effects. Individual Nrf2 genotypes may influence the antioxidant response, thus requiring consideration in future intervention studies focusing on the Nrf2 pathway. Anthocyanin-rich fruit juice had an extensive impact on the gut microbiota.

First insights into Alternaria multi-toxin in vivo metabolism.

Alternaria mycotoxins frequently contaminate agricultural crops and may impact animal and human health. However, data on mammalian metabolism and potential biomarkers of exposure for human biomonitoring (HBM) are scarce. Here, we report the preliminary investigation with respect to metabolism and excretion of Alternaria toxins in Sprague Dawley rats. Four animals were housed in metabolic cages for 24 h after gavage administration of an Alternaria alternata culture extract containing ten known toxins. LC-MS/MS analysis of 17 Alternaria toxins in urine and fecal samples allowed to gain first insights regarding xenobiotic metabolism and excretion rates. Alternariol (6-10%), alternariol monomethyl ether (AME, 6-7%) and tenuazonic acid (up to 55%) were recovered in urine and fecal samples (9%, 87%, 0.3%, respectively), while perylene quinones administered at comparatively high levels, were either determined at very low levels (up to 0.5% altertoxin I in urine and 15% in feces; 0.2% alterperylenol in urine and 3% in feces) or not at all (altertoxin II, stemphyltoxin III). AME-3-sulfate, which was not present in the administered extract, was determined in urine, representing up to 23% of the AME intake. Critical evaluation of the applied sample preparation protocol and LC-MS/MS analysis revealed interesting preliminary results and information crucial for improving follow-up experiments.

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.

The secondary Fusarium metabolite aurofusarin induces oxidative stress, cytotoxicity and genotoxicity in human colon cells.

Aurofusarin (AURO), a dimeric naphthoquinone, is produced by Fusarium fungi. Although frequently found in food and feed, toxicological studies are limited. Hence, the in vitro toxicity of AURO was investigated in the colon adenocarcinoma cell line HT29 and the non-tumorigenic colon cells HCEC-1CT. Cytotoxic effects were found at concentrations ≥1 µM by evaluating mitochondrial activity (WST-1) and cellular proliferation (sulforhodamine B assay). 10 µM of AURO induced a decrease of cells in the S-phase, measured by flow cytometry. Confocal microscopy revealed AURO-mediated increase of intracellular p53 protein. In accordance, DNA-damage was seen in the comet assay (≥1 µM) together with enhanced levels of formamidopyrimidine-DNA-glycosylase (fpg)-sensitive sites, indicative for oxidative stress. An increase of intracellular reactive oxygen species was observed in the dichlorofluorescein (DCF) assay (≥5 µM). The GSSG/GSH ratio was elevated, but no impact on redox-sensitive Nrf2-dependent genes (Nrf2, γ-GCL, NQO1) was found at the gene expression level. However, induction of cytochrome P450 monooxygenase (CYP) 1A1 was measured at the gene expression and protein level. In conclusion, these in vitro data suggest that, when co-occurring, AURO might be considered as a potential contributor to the overall toxicity of respective Fusarium mycotoxin mixtures.

Response of intestinal HT-29 cells to the trichothecene mycotoxin deoxynivalenol and its sulfated conjugates.

The sulfated forms of the Fusarium toxin deoxynivalenol (DON), deoxynivalenol-3-sulfate (DON-3-Sulf) and deoxynivalenol-15-sulfate (DON-15-Sulf) were recently described, however little is known about their mechanism of action in mammalian cells. DON-3-Sulf and DON-15-Sulf were taken up by HT-29 colon carcinoma cells, although to a lesser extent compared to DON. All three compounds were found to enhance the intracellular ROS level in the dichlorofluorescein assay (≥ 1µM), even though substantial differences were observed in their cytotoxic potential. In silico modelling highlighted that DON-sulfates do not share the classical mechanism of action of DON, being unable to fit into the ribosomal pocket and trigger the classical ribotoxic stress response. However, DON-3-Sulf and DON-15-Sulf sustained a distinctive proliferative stimulus in HT-29 and activated autophagy. The mechanisms of action of DON-3-Sulf and DON-15-Sulf suggest a potential interplay between the onset of ribosomal inhibition and autophagy activation as an alternative and/or complementary mode of action for DON and its sulfated analogues.

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.

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.

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 affect protein kinase C activity and the onset of apoptosis in human colon carcinoma cells.

Polyphenol-rich apple extracts have been reported to suppress human colon cancer cell growth in vitro. The protein kinase C (PKC) is among the signaling elements known to play an important role in colon carcinogenesis. In the present study, we investigated whether apple polyphenols affect PKC activity and induce apoptosis in the human colon carcinoma cell line HT29. A polyphenol-rich apple juice extract (AE02) was shown to inhibit cytosolic PKC activity in a cell-free system. In contrast, incubation of HT29 cells for 1 or 3 h with AE02 up to 2 mg/mL did not affect the cytosolic PKC activity. After prolonged incubation (24 h), cytosolic PKC activity was modulated, albeit a u-shaped curve of effectiveness was observed, with an initial inhibitory effect followed by the recurrence and even induction of enzyme activity. Concomitantly, in the cytosol, a significant decrease of the protein levels of PKCalpha, PKCbetaII, and PKCgamma together with a significant increase of a proapoptotic PKCdelta fragment was observed. However, the effects on the protein levels of these PKC isoforms in the cytosol were not associated with translocation between the different cellular compartments but might instead result from the onset of apoptosis. Indeed, the treatment with AE02 was shown to induce apoptosis by the activation of caspase-3, DNA fragmentation, and cleavage of poly(ADP ribose) polymerase. So far, identified and available constituents of the apple extract did not contribute substantially to the observed effects on PKC and apoptosis induction. In summary, apple polyphenols were found to inhibit PKC activity in a cell-free system. However, our results indicate that within intact cells PKC does not represent the primary target of apple polyphenols but appears to be affected in the course of apoptosis induction.