Dorothee Rogoll

The Ussing type chamber model to study the intestinal transport and modulation of specific tight-junction genes using a colonic cell line.

Polyphenols in apples, such as various hydroxycinnamic acids and flavonoids, have positive health effects that strongly depend on their bioavailability. In order to show that the Ussing-type chamber is a useful model to study metabolism, transport, and tightness of cell monolayers in one experimental setup, monolayers of the T84 colon carcinoma cell line mounted in Ussing-type chambers were incubated in the presence of physiological concentrations of various hydroxycinnamic acids (including ferulic, isoferulic, cinnamic, and hydrocinnamic acids) and flavonoids for 4 h. Concentrations of each tested polyphenol in the apical chamber, basolateral chamber, and those associated with the cells were then determined using HPLC with DAD (HPLC-DAD). The transport studies showed that the amounts of the tested polyphenols that passed from the apical to the basolateral side of the T84 monolayers depended on their polarity. Metabolites, such as glucuronides and sulfates of ferulic acid, were also detected at measurable levels by HPLC-ESI-MS/MS in the model system, but only when they were supplied at supra-physiological concentrations (>100 microM). In addition, the transepithelial resistance (TER) of T84 monolayers was measured before and after the addition of polyphenols, with and without short-term exposure to apical sodium caprate (C10), a tight junction (TJ) modulator. Exposure to C10 induced a decrease in TER that was reversible by incubation with polyphenols. However, no increase in paracellular permeability of tested polyphenols was observed after apical C10 exposure, so C10 did not promote fluxes of hydroxycinnamic acids across the monolayers. Further, real-time PCR analysis of the T84 colon cell line showed that ferulic and isoferulic acids induced significant increases in expression of the TJ components zonula occludens-1 (ZO-1) and claudin-4 transcription, but reductions in occludin expression. In contrast, caffeic and p-coumaric acids had no significant effects on the transcription of either ZO-1 or occludin. Our results provide confirmation that T84 cells could be used as model system to simulate the intestinal mucosa, and that polyphenols are able to increase the TER of C10-treated and -untreated T84 monolayers.

LOH and copy neutral LOH (cnLOH) act as alternative mechanism in sporadic colorectal cancers with chromosomal and microsatellite instability

BACKGROUND AND AIMS Tumor suppressor genes are often located in frequently deleted chromosomal regions of colorectal cancers (CRCs). In contrast to microsatellite stable (MSS) tumors, only few loss of heterozygosity (LOH) studies were performed in microsatellite instable (MSI) tumors, because MSI carcinomas are generally considered to be chromosomally stable and classical LOH studies are not feasible due to MSI. The single nucleotide polymorphism (SNP) array technique enables LOH studies also in MSI CRC. The aim of our study was to analyse tissue from MSI and MSS CRC for the existence of (frequently) deleted chromosomal regions and tumor suppressor genes located therein. METHODS AND RESULTS We analyzed tissues from 32 sporadic CRCs and their corresponding normal mucosa (16 MSS and 16 MSI tumors) by means of 50K SNP array analysis. MSS tumors displayed chromosomal instability that resulted in multiple deleted (LOH) and amplified regions and led to the identification of MTUS1 (8p22) as a candidate tumor suppressor gene in this region. Although the MSI tumors were chromosomally stable, we found several copy neutral LOHs (cnLOH) in the MSI tumors; these appear to be instrumental in the inactivation of the tumor suppressor gene hMLH1 and a gene located in chromosomal region 6pter-p22. DISCUSSION Our results suggest that in addition to classical LOH, cnLOH is an important mutational event in relation to the carcinogenesis of MSS and MSI tumors, causing the inactivation of a tumor suppressor gene without copy number alteration of the respective region; this is crucial for the development of MSI tumors and for some chromosomal regions in MSS tumors.

Modulation of Nrf2-dependent gene transcription by bilberry anthocyanins in vivo

In a human pilot intervention study (healthy + ileostomy probands), the questions were addressed whether in vivo consumption of an anthocyanin-rich bilberry (Vaccinium myrtillius L.) pomace extract (BE) affects (i) the transcription of Nrf2-dependent genes in peripheral blood mononuclear cells (PBMC), indicative for systemic effects, and (ii) the level of oxidative DNA damage in these cells. In healthy test subjects transcripts of NAD(P)H quinone oxidoreductase 1 (NQO1) were significantly elevated throughout the observation period (1-8 h), whereas transcription of heme oxygenase 1 (HO-1) and Nrf2 was significantly decreased. NQO1 and HO-1 transcription remained unchanged in the ileostomy probands, whereas Nrf2-transcription was suppressed in both groups. Decrease in oxidative DNA damage was observed 2 h after BE consumption again only in healthy subjects. In vitro studies using a reporter gene approach (CHO) and qPCR (HT29) indicate that not the intact anthocyanins/anthocyanidins are the activating constituents but the intestinal degradation product phloroglucinol aldehyde (PGA). Taken together, consumption of anthocyanin-rich BE was found to modulate Nrf2-dependent gene expression in PBMCs indicative for systemic activity. Limitation of the effect to healthy test subjects suggests a role of colonic processes for bioactivity, supported by the results on Nrf2-activating properties of the intestinal anthocyanin degradation product PGA.

Gastrointestinal absorption and metabolism of apple polyphenols ex vivo by the pig intestinal mucosa in the Ussing chamber.

Polyphenols contained in food have various positive effects on human health. The absorption and metabolism of polyphenols in the intestinal tract needs to be studied to estimate these effects. The Ussing chamber technique was used to investigate the transport behavior of apple polyphenols through pig small intestinal mucosa, which served as a model for human gastrointestinal mucosa. The identities and concentrations of polyphenols and their metabolites in the half-chambers (luminal and basolateral) within an incubation period of 4 h were determined by HPLC-MS/MS and HPLC-DAD (DAD = diode-array detection). Flux values were also measured. It was found that 5-caffeoylquinic acid and caffeic acid were absorbed and translocated to the basolateral side (1.9 and 3.7%, respectively), but other compounds, including glycosides of phloretin and quercetin, were observed without translocation. A Ussing chamber utilizing pig small intestinal mucosa is a suitable model for assessing the effect of apple polyphenols on mucosal integrity and nutrition absorption across porcine mucosa.

Human intervention study to investigate the intestinal accessibility and bioavailability of anthocyanins from bilberries

We investigated the importance of the large intestine on the bioavailability of anthocyanins from bilberries in humans with/without a colon. Low bioavailability of anthocyanins in plasma and urine was observed in the frame of this study. Anthocyanins reached the circulation mainly as glucuronides. Analysis of ileal effluents (at end of small intestine) demonstrated that 30% of ingested anthocyanins were stable during 8h passage through the upper intestine. Only 20% degradants were formed and mostly intact anthocyanins were absorbed from the small intestine. Higher amounts of degradants than anthocyanins reached the circulation after bilberry extract consumption in both groups of subjects. Comparison of the bioavailability of anthocyanins in healthy subjects versus ileostomists revealed substantially higher amounts of anthocyanins and degradants in the plasma/urine of subjects with an intact gut. The results suggested that the colon is a significant site for absorption of bioactive components such as anthocyanins and their degradation products.

Encapsulation of anthocyanins from bilberries – Effects on bioavailability and intestinal accessibility in humans

Anthocyanins are flavonoids that have been suggested to provide beneficial health effects. The biological activity of anthocyanins is influenced by their pharmacokinetic properties, but anthocyanins are associated with limited bioavailability in humans. In the presented study, we investigated how the encapsulation of bilberry extract (BE), a source of anthocyanins, with either whey protein or citrus pectin influences the bioavailability and intestinal accessibility of anthocyanins in humans. We performed an intervention study that analyzed anthocyanins and their degradation products in the urine, plasma, and ileal effluent of healthy volunteers and ileostomists (subjects without an intact colon). We were able to show, that whey protein encapsulation modulated short-term bioavailability and that citrus pectin encapsulation increased intestinal accessibility during passage through the small intestine and modulated the formation of the degradation product phloroglucinol aldehyde (PGAL) in human plasma.

Abstract 5179: Inhibition of U937-cell adhesion to human endothelial cells by glycosylated lysozyme mutants

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The evascularization of cells is an important step in defense reactions and the spreading of metastases. During inflammation, the endothelium of the affected tissue becomes activated and interacts with cells bearing cognate structures such as sialyl Lewis X (sLex) substances. In this study, we present the sLex-antagonist hLysII/IV-FUCTVI by which a significant inhibition of adhesion is achieved at 1ng/ml (0.07 nM), making it the most potent known inhibitors of adhesion described to date. In our study we overexpressed the glycosylated human lysozyme mutant hLysII/IV in CHO cells stably expressing fucosyltransferases FUCIII, IV, V, VI, or VII. Digestional analysis with endo- and exoglycosidases showed that hLysII/IV-FUCTVI contains predominantly biantennary complex N-glycans with a variable number of lactosamine repeats; these were terminally sialylated and fucosylated, suggesting the presence of the sLex antigen. In contrast, hLysII/IV-FUCTIII and IV were not terminally sialylated and hLysII/IV-FUCV and VII were not terminally fucosylated. In a static adhesion assay, binding of U937 to activated HUVEC cells was exclusively blocked in the presence of medium supernatant containing hLysII/IV-FUCTVI and by affinity-purified hLysII/IV-FUCTVI. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5179. doi:1538-7445.AM2012-5179

Differential Cathelicidin Expression in Duodenal and Gastric Biopsies from Tanzanian and German Patients

Background Epithelial surfaces such as the gastrointestinal mucosa depend on expression of antimicrobial peptides like cathelicidin for immune defence against pathogens. The mechanisms behind mucosal cathelicidin regulation are incompletely understood. Methods Cathelicidin expression was analysed in duodenal, antral and corpus/fundic mucosal biopsies from African and German patients. Additionally, cathelicidin expression was correlated with Helicobacter pylori (HP) infection and the inflammatory status of the mucosa. Results High cathelicidin transcript abundance was detected in duodenal biopsies from African subjects. On the contrary, cathelicidin mRNA expression was either undetectable or very low in tissue specimens from German patients. Also, in the antrum and corpus/fundus regions of the stomach significantly higher cathelicidin transcript levels were measured in Tanzanian compared to German patients. In gastric biopsies from African patients cathelicidin expression was increased in HP positive compared to HP negative subjects. Additionally, the inflammatory status measured by IL-8 expression correlated well with the HP infection status. Conclusions A higher duodenal and gastric cathelicidin expression in African (compared with European) individuals may be due to upregulation by antigenic stimulation and may confer a higher resistance against enteric infections.

Abstract 2248: Oncogenic actions of HDGF (hepatoma-derived growth factor) in colorectal cancer: Implication for tumor biology/ patient prognosis and modulation by butyrate

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL HDGF was identified as an oncogene relevant for colon cancer carcinogenesis by cDNA-array analysis, RT-PCR and immunohistochemistry. The analysis were performed in colonic adenoma (Geki2), carcinoma (HT29) and metastasis (SW620) cell lines, and in 50 colorectal cancer tissues and corresponding normal mucosa. By incubation of the cell lines with the short chain fatty acid butyrate (4mM), it was possible to (down-) regulate the HDGF-expression level. In the patients we analysed, HDGF overexpression was not associated with a worse prognosis or any other clinical parameter. To study the mechanisms of action of HDGF we stably transfected a HDGF-siRNA containing plasmid into HT29 colon carcinoma cells, resulting in a almost complete inactivation of HDGF-expression. This blocking leads to altered growth characteristics and cell differentiation and the cells exhibit potent pro-apoptotic properties. An affymetrix-expression array was performed and first results will me shown at the meeting. In conclusion, HDGF might be a potential therapeutic target for human colorectal cancer and a modulation of expression is possible by the short chain fatty acid butyrate. These findings may have major implications in the treatment of colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2248. doi:10.1158/1538-7445.AM2011-2248