Metabolism of resveratrol in breast cancer cell lines: impact of sulfotransferase 1A1 expression on cell growth inhibition.

Resveratrol is a polyphenolic compound present in grapes and wine with anticancer activities that undergoes pronounced metabolism in humans. In order to determine whether metabolism of resveratrol also occurs in tumor cells and whether biotransformation has any impact on cytotoxicity, metabolism experiments were conducted with hormone-dependent ZR-75-1 and hormone-independent MB-MDA-231 human breast cancer cells. Along with resveratrol, it was possible to identify one metabolite, namely, resveratrol-3-O-sulfate in both cell lines. Its concentration in the cytoplasm and culture medium was 5.4- to 9-fold higher in ZR-75-1 cells than in MDA-MB-231 cells, concomitant with a 3.1-fold higher IC(50) value in the ZR-75-1 cell line (74 microM compared to 38 microM). By using RT-PCR, expression of sulfotransferase (SULT)1A1 mRNA, but not of other SULTs investigated, showed a close correlation with resveratrol 3-O-sulfate formation which was particularly high in ZR-75-1 and very low in MDA-MD-231 cells. In conclusion, we demonstrate that SULT1A1-based biotransformation reduces the anticancer activity of resveratrol in breast cancer cells, which must be considered in humans following oral uptake of dietary resveratrol as a chemopreventive agent.

Altered expression of the hormone- and xenobiotic-metabolizing sulfotransferase enzymes 1A2 and 1C1 in malignant breast tissue.

Cytosolic sulfotransferases (SULTs) catalyze the biotransformation of steroid hormones as well as drugs and environmental toxins. Mostly, sulfonation leads to an inactivation of parent compounds, although formation of more toxic and cancerogenic metabolites also occurs. To assess possible alterations in the SULT enzyme expression pattern between malignant and non-malignant tissue, we studied the presence of 9 SULT enzymes of family 1 and 2 by semi-quantitative RT-PCR. Forty-two specimens from ductal and lobular breast carcinomas, lymph node metastasis, mastopathy and normal breast tissue were derived from 29 patients. Substantial expression of SULT 1A1, 1A2, 1A3, 1B1, 1C1, 1E1, 2A1, 2B1a and 2B1b mRNAs was observed in malignant and non-malignant tissue, although the pattern of the individual SULTs varied between the patients, and SULT1C1 mRNA was present in a greater number of malignant than non-malignant tissues (p<0.05). A major finding was that unspliced SULT1A2 mRNA, containing the complete intron between exons 7 and 8, was found in 4 of 16 non-malignant specimens, but was undetectable in the 26 malignant samples investigated. Taken together, the presence of various SULT enzymes in normal, premalignant and malignant breast tissue suggests an important role of SULT-mediated biotransformation in the breast. While the increased expression of SULT1C1 in malignant tissue seems to reflect tumor dedifferentiation, our finding of unspliced SULT1A2 mRNA in non-malignant tissue offers additional aspects regarding the search for breast cancer risk factors.

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 In-1,4,7,10-tetraazacyclo-dodecane-N,N',N'',N'''-tetraacetic acid-lanreotide (In-DOTA-LAN) and In-DOTA-tyrosine-octreotide (In-DOTA-Tyr-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 In-DOTA-LAN, whereas 23 (37%) were negative. With In-DOTA-Tyr-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.

The peroxisome proliferator-activated receptor gamma ligand 15-deoxy-Delta12,14-prostaglandin J2 induces vascular endothelial growth factor in the hormone-independent prostate cancer cell line PC 3 and the urinary bladder carcinoma cell line 5637.

Cyclopentenone-prostaglandin derivatives, including the peroxisome-proliferator activated receptor gamma (PPARgamma) ligand 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), inhibit tumor cell growth in vitro and in vivo. As 15d-PGJ2 was found to stimulate the expression of vascular endothelial growth factor (VEGF) in endothelial cells, we investigated whether 15d-PGJ2 induces this angiogenic factor in the human androgen-independent PC 3 prostate and the 5637 urinary bladder carcinoma cell line. In PC 3 cells, 15d-PGJ2 caused a dose-dependent increase in VEGF mRNA expression, as determined by RT-PCR. Stimulation started after 6 h, and after 72 h, VEGF mRNA expression reached a maximum of 3.3+/-0.3 U, 4.4+/-0.3 U and 6.1+/-0.1 U with 1, 5 and 10 microM 15d-PGJ2, respectively. Between 12-72 h, VEGF protein production was stimulated by up to 2-fold with 5 and 10 microM 15d-PGJ2 as assessed by ELISA in PC 3 cell-conditioned medium. In 5637 cells, 15d-PGJ2 did not alter VEGF mRNA expression for up to 72 h. Thereafter, VEGF mRNA expression was transiently increased from 2.3+/-0.8 U in control cells to 4.6+/-0.5 U in 1 microM and 5.9+/-0.6 U in 5 microM 15d-PGJ2-treated cells. VEGF protein production was only moderately stimulated (1.7-fold). 10 microM 15d-PGJ2 had no effect on VEGF mRNA expression in 5637 cells, but effectively reduced viability in both cell lines. 15d-PGJ2 also increased PPARgamma mRNA expression in both cell lines. While in PC 3 cells, stimulation of PPARgamma mRNA expression occurred after 72 h, in 5637 cells, a transient stimulation took place after 6 h (4-fold). We demonstrated that 15d-PGJ2 induces VEGF in PC 3 and 5637 cancer cells. This might be important if PG-analogues are considered as antitumor agents.

Pancreatic stellate/myofibroblast cells express G-protein-coupled melatonin receptor 1.

In chronic pancreatitis and pancreatic cancer, progressive fibrosis with the accumulation of extracellular matrix occurs. The main extracellular matrix-producing cell types are retinoid-storing pancreatic stellate cells (PSCs) of mesenchymal origin. Similar to liver stellate cells, quiescent PSCs undergo activation and acquire a myofibroblast-like phenotype in response to pro-fibrogenic mediators (reactive oxygen species, cytokines and toxic metabolites). Activated PSCs differ in their differentiation stage and are characterized by the expression of glial fibrillary-acidic protein, alpha-smooth muscle actin, and nestin. As G-protein-coupled receptors were described to regulate PSC differentiation, we investigated tissue samples from patients with pancreatitis and ductal pancreatic adenocarcinoma for the expression of G-protein-coupled melatonin receptors MT1 and MT2 by double immunofluorescence staining. We show that MT1, but not MT2, is occasionally expressed in PSCs in normal tissue, while in the diseased tissue MT1 is found at high rates in activated PSCs at all stages, and, additionally, in ductal epithelial cells. It is speculated that MT1 activation by its ligand melatonin regulates proliferation and differentiation of PSCs. Prevention of myofibroblast formation by MT1 activation could explain favourable effects of the pineal hormone melatonin on the outcome of pancreatic fibrosis in animal models.

Estrogen sulfotransferase (SULT1E1) expression in benign and malignant human bone tumors.

BACKGROUND AND OBJECTIVES: 17beta-estradiol regulates growth and differentiation in normal and malignant bone. E2 is inactivated to 17beta-estradiol-sulfate through estrogen sulfotransferase (SULT1E1). RESULTS: In an explorative study, SULT1E1 mRNA expression was assessed in a broad range of samples from benign, primary and secondary malignant bone tumors. We detected SULT1E1 mRNA in 31/50 tumor samples (10/19 malignant, 6/13 benign tumors; 15/18 metastases). Significantly more SULT1E1-positive samples were found in metastases than in primary bone tumors (P = 0.019). Yet, there was no difference between malignant and benign primary tumors (P = 0.718). SULT1E1 mRNA levels were not related to patients' age, gender, tumor location, stage, grading, and chemotherapy pretreatment. Relative SULT1E1 mRNA levels did not correlate with that of estrogen-receptor alpha (ERalpha) as assessed by quantitative TaqMan PCR (10 malignant, 8 benign tissue samples). In the latter, ERalpha mRNA, but not SULT1E1 mRNA levels were significantly lower than in the malignant samples (P = 0.006 and P = 0.71, respectively). Also, pronounced expression of SULT1E1 mRNA but not of ERalpha mRNA was observed in osteosarcoma (MG-63, HOS) and Ewing's sarcoma (TC-71) cells, while human osteoblasts and BMSC contained ERalpha but not SULT1E1 mRNA. CONCLUSION: Frequent expression of SULT1E1 mRNA in various human bone tumors suggests that sulfonation might be important to control E2 levels and activity.

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 HCO(3) (-)/CO(2). Exposure to 1 microM melatonin for 24 hrs and presence of the indoleamine during the experiment increases the basal pH(i). Moreover, pHi recovery and HCO(3) (-) secretion are facilitated after the alkaline load. These findings are in line with the observed increase in mRNA expression of the Na(+)/HCO(3) (-)-cotransporter SLC4A4b for the uptake and the Cl(-)/HCO(3) (-)-exchanger SLC26A6 for the secretion of HCO(3) (-). 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.

Biotransformation of melatonin in human breast cancer cell lines: role of sulfotransferase 1A1.

The biologically active melatonin metabolite, 6-hydroxymelatonin (6-OHMel), is conjugated to form 6-hydroxymelatonin sulfate (6-OHMelS). To elucidate the role of the sulfotransferase (SULT) enzyme 1A1, considerably expressed in normal and malignant human breast cells, we measured the formation of 6-OHMelS by ELISA in hormone-dependent MCF-7 and hormone-independent MDA-MB231 (MDA) breast cancer cell lines after stable transfection with SULT1A1. In parent MDA cells, low SULT1A1 mRNA expression was associated with moderate 6-OHMelS formation as determined after application (24 hr) of 0.1 microM 6-OHMel. As expected, overexpression of SULT1A1 in MDA cells resulted in a 2.9- and 110-fold increase in 6-OHMelS in the cytosol and cellular supernatant respectively. Furthermore, 6.3- and 115-fold increases were observed after 0.5 microM, and 12.6- and 101-fold increases after 1 microM 6-OHMel respectively. In MCF-7 cells, because of high basal SULT1A1 expression, only two- to threefold increases in 6-OHMelS were observed after transfection with the enzyme. In total, 866 and 539 pmol/mg protein 6-OHMelS were formed from 1 microM 6-OHMel in SULT1A1 overexpressing MDA and MCF-7 cells, respectively, whereas application of 1 microM melatonin produced only <1% of 6-OHMelS. Possible interactions with the SULT1A1 substrate tamoxifen (tam), an anti-estrogen applied in the therapy of breast cancer, were also studied. A concentration of 1 microM tam increased 6-OHMelS formation by approximately threefold in the presence of 1 microM melatonin or 1 microM 6-OHMel respectively. However, no alterations were detected after application of 1 microM 4-hydroxy-tamoxifen. In summary, we demonstrate the importance of SULT1A1 for the biotransformation of 6-OHMel in human breast cancer cells. Our data further suggest that tam can modulate melatonin biotransformation.

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.

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.