Sylvia Aust

Sulfation of resveratrol in human liver: Evidence of a major role for the sulfotransferases SULT1A1 and SULT1E1

Sulfation of resveratrol, a polyphenolic compound present in grapes and wine with anticancer and cardioprotective activities, was studied in human liver cytosol. In the presence of 3′-phosphoadenosine-5′-phosphosulfate, three metabolites (M1–3) whose structures were identified by mass spectrometry and NMR as trans-resveratrol-3-O-sulfate, trans-resveratrol-4′-O-sulfate, and trans-resveratrol-3-O-4′-O-disulfate, respectively. The kinetics of M1 formation in human liver cytosol exhibited an pattern of substrate inhibition with a Ki of 21.3 ± 8.73 µM and a Vmax/Km of 1.63 ± 0.41 µL min−1mg−1 protein. Formation of M2 and M3 showed sigmoidal kinetics with about 56-fold higher Vmax/Km values for M3 than for M2 (2.23 ± 0.14 and 0.04 ± 0.01 µL min−1 mg−1). Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 is almost exclusively catalysed by SULT1A1 and only to a minor extent by SULT 1A2, 1A3 and 1E1, whereas M2 is selectively formed by SULT1A2. M3 is mainly catalysed by SULT1A2 and 1A3. In conclusion, the results elucidate the enzymatic pathways of resveratrol in human liver, which must be considered in humans following oral uptake of dietary resveratrol.

Subcellular localization of the ABCG2 transporter in normal and malignant human gallbladder epithelium

Epithelium of the gallbladder and biliary tract is exposed to high concentrations of potentially harmful exogenous and endogenous compounds excreted into primary bile. As the ATP-dependent efflux pump ABCG2 can prevent cellular accumulation of anticancer drugs, estrogen sulfate, xenobiotics, porphyrins, and sterols, its expression in the biliary tract might mediate protection by hindering their penetration. We therefore investigated the expression and subcellular distribution of ABCG2 in normal and malignant human gallbladder.After demonstrating ABCG2 expression in gallbladder epithelium by RT-PCR and Western blotting, we analyzed the subcellular localization of ABCG2 by indirect immunofluorescence in gallbladder adenocarcinoma specimens, and compared it to that in cholelithiasis, and normal gallbladder samples (n=54). In control, cholelithiasis, and well-differentiated tumor samples (grade 1, T1–3), ABCG2 is present at the luminal membrane of epithelial cells, which was proven by colocalization of apical-bound TRITC-labeled lectin (wheat germ agglutinin). In poorly differentiated gallbladder adenocarcinomas, intracellular ABCG2, in addition to luminal ABCG2 immunoreactivity, was found in 13/21 carcinoma samples (grade 2 and 3, T2-4, P<0.01). In 3/11 of grade 3 tumors, ABCG2 was present in the cytoplasmatic compartment only (P<0.01). In proliferating bile ducts of cholangiocarcinomas, ABCG2 showed an analogous staining pattern with presence in cytosolic compartments. However, the apical marker enzyme neutral endopeptidase remained on the membrane in all samples. To study whether phosphatidylinositol 3-kinase (PI3K) signaling might be necessary for ABCG2 membrane insertion, we treated freshly isolated human gallbladder epithelial cells with the PI3K inhibitor wortmannin. As assessed by indirect immunofluorescence, this maneuver redistributes ABCG2 to intracellular compartments. In conclusion, our data suggest a protective role for ABCG2 in well-differentiated gallbladder epithelial cells. Cytoplasmatic accumulation of ABCG2 in poorly differentiated carcinomas might coincide with malfunctioning of PI3K-signaling pathways during tumor progression.

The melatonin receptor subtype MT1 is expressed in human gallbladder epithelia

Abstract: Based on the fact that human bile and, particularly gallbladder bile, contains high physiological levels of the antioxidant melatonin, the aim of this study was to investigate whether the melatonin receptor MT1 is present in human gallbladder. Expression and localization of MT1 was assessed by RT‐PCR, Western blotting and immunofluorescence analysis in gallbladder samples from patients with cholelithiasis and with advanced gallbladder carcinoma. Additionally, we monitored mRNA expression of the two key enzymes of melatonin synthesis, i.e. arylalkylamine‐N‐acetyltransferase (AANAT) and hydroxyindole‐O‐methyltransferase (HIOMT). MT1 mRNA and protein were present in all cholelithiasis (n = 10) and gallbladder carcinoma (n = 5) samples. As indicated from RT‐PCR and Western blot studies, MT1 is located in gallbladder epithelia. Epithelial expression was further proven by immunofluorescence staining of MT1 in paraffin‐embedded cholelithiasis and gallbladder carcinoma sections. Analysis of AANAT and HIOMT mRNA expression showed that HIOMT mRNA is present in gallbladder. Surprisingly, AANAT was not detectable under conditions where it was found in a human colon specimen. The absence of AANAT suggests that in human gallbladder, HIOMT might be involved in the formation of 5‐hydroxytryptamine products other than melatonin. In summary, our results provide the first evidence for the presence of MT1 in human gallbladder epithelia. Therefore, in addition to its profound antioxidative effects in the biliary system, melatonin might also act through MT1‐mediated signal transduction pathways. Thereby, it might be involved in the regulation of gallbladder function.

Melatonin Modulates Acid/Base Transport in Human Pancreatic Carcinoma Cells

Melatonin was found to improve pancreatic organ function in diseased animals. To study whether pancreatic bicarbonate secretion is stimulated by melatonin, investigations were done in two human ductal pancreatic adenocarcinoma cell lines MIA PaCa-2 (MIA) and PANC-1 (PANC). Using the fluorescence pH-sensor BCECF-AM, we monitored melatonin effects on basal intracellular pH (pH_i), and on pH_i recovery after intracellular alkalinization produced by the removal of extracellular HCO₃ ^-/CO₂. Exposure to 1µM melatonin for 24hrs and presence of the indoleamine during the experiment increases the basal pH_i. Moreover, pHi recovery and HCO₃ ^- secretion are facilitated after the alkaline load. These findings are in line with the observed increase in mRNA expression of the Na⁺/HCO₃ ^--cotransporter SLC4A4b for the uptake and the Cl^-/HCO₃ ^--exchanger SLC26A6 for the secretion of HCO₃ ^-. The reduction in Na⁺/H⁺- exchanger SLC9A1 mRNA would favor pH_i recovery after alkalinization, but it does not explain the initial increase in pHi. This controversial effect and the requirement for continuous presence of melatonin throughout the experiment suggest that nontranscriptional signalling may contribute to the effects of melatonin on acid/base movements. In summary, we show a stimulatory effect of melatonin on bicarbonate secretion in the pancreatic cancer cell lines which may help to prevent duodenal damage.

Somatostatin receptor scintigraphy with 111In-DOTA-lanreotide and 111In-DOTA-Tyr3-octreotide in patients with stage IV melanoma: in-vitro and in-vivo results.

The overexpression of somatostatin receptors (SST-Rs) on various tumour cells provides the molecular basis for the successful use of radiolabelled SST analogues in clinical oncology. The objective of the study was to evaluate the tumour binding of 111In-1,4,7,10-tetraazacyclo-dodecane-N,N′,N′′,N′′′-tetraacetic acid-lanreotide (111In-DOTA-LAN) and 111In-DOTA-tyrosine3-octreotide (111In-DOTA-Tyr3-OCT) in patients with stage IV melanoma. In addition, we evaluated the potential antiproliferative effect of SST analogues, together with an assessment of the functionality of SST-Rs, on four melanoma cell lines. Twenty-three patients with advanced metastatic melanoma underwent scintigraphy. Thirty-eight of 61 lesions (62%) were positively imaged with 111In-DOTA-LAN, whereas 23 (37%) were negative. With 111In-DOTA-Tyr3-OCT, 10 of the 23 documented lesions (43%) were positive and 13 (56%) were negative. In vitro, cell lines showed no growth inhibition in the presence of SST analogues and no influence on cell cycle distribution was found with the addition of SST analogues to cultured cells. In addition, no functional surface SST-Rs could be demonstrated on these cell lines. Taken together, our results demonstrate the visualization of metastatic melanoma in a high percentage of patients, probably due to binding of SST analogues to SST-Rs on tumour vessels or infiltrating immune cells. Judging from our data, however, there is no evidence of functional SST-R expression on melanoma cells.