Expression of sulfotransferases and sulfatases in human breast cancer: Impact on resveratrol metabolism
Introduction
Breast cancer is a major cause of cancer death in women worldwide. Chemoprevention in combination with anticancer treatment is therefore important to reduce morbidity and mortality. Evidence from epidemiological and experimental studies indicates that natural constituents of the diet may well act as chemopreventive agents and inhibit mammary carcinogenesis [1]. One of such compounds is resveratrol (3,4′,5-trihydroxy-trans-stilbene), which is produced by several plants, berries, fruits and is mainly found in the skin of grapes and red wine [2]. The antiproliferative property of resveratrol has been demonstrated in vitro against hormone-dependent and hormone-independent breast cancer cells due to the induction of apoptosis via down-regulation of NF-kappa B, Bcl-2, inhibition of ribonucleotide reductase, and DNA polymerase [3]. In addition, resveratrol is an excellent antioxidant and radical scavenger, a fact that plays a crucial role in the anticancer effects of stilbene derivates [4]. Resveratrol was also shown to inhibit cyclooxygenases (COX-1, COX-2) [5], which partly explains why this compound also reduces the occurrence of colon cancer and breast cancer. In addition to these in vitro data, recent experiments have showed significantly less tumor growth in human breast cancer xenografts in vivo, supporting the use of this polyphenol as a potential chemotherapeutic agent. Furthermore, a recent human study analyzing the relationship between dietary intake of resveratrol and breast cancer risk using data from a case control-study on 369 cases and 602 controls also showed a significant inverse association for this polyphenol [6]. Interestingly, resveratrol is active against tumor cells but has not shown any organ-specific cytotoxicity in animal models [7].
Data from our laboratory revealed that resveratrol is metabolized in the human breast cancer cell lines MDA-MB-231 and ZR-75–1 to resveratrol-3-O-sulfate, dependent on sulfotransferase (SULT) 1A1 expression [8]. Using recombinant enzymes we were also able to show that sulfation of resveratrol is catalyzed by SULT1A1 and to a lesser extent by SULT1A2, 1A3 and 1E1 [9].
Only a few studies conducted thus far have been focused on SULT expression in breast cancer tissues. While Suzuki and coworkers demonstrated the expression of SULT1E1 by real-time PCR in breast cancer tissues derived from laser capture microdissection [10], a study conducted by Aust et al. revealed the expression of SULT1A1, SULT1A2, SULT1A3, and SULT1E1 in malignant and non-malignant breast tissue using conventional RT-PCR [11]. Expression of SULT1A1 and SULT1A3, but not SULT1E1, was confirmed in mammary tissue-derived cytosol from 24 healthy persons by Western blot analysis [12].
Whether resveratrol is also sulfated in breast tumor is not yet known. The aim of the present study was to investigate the metabolism of resveratrol in malignant human breast cancer specimens and its adjacent non-cancerous tissue. Adjacent breast tissues were selected as reference for human breast cancer samples because the activity and expression of drug-metabolizing enzyme systems may vary greatly between individuals due to genetic, environmental and physiological factors [13]. A further aim of the study was to identify the chemical structure of isolated biotransformation products by liquid chromatography/mass spectrometry (LC/MS) and NMR. Furthermore, the mRNA expression of the major isoenzymes responsible for the observed metabolism and the corresponding hydrolytic enzymes in human breast cancer in comparison to normal tissue were investigated by quantitative real-time PCR and correlated with protein expression on paraffin-embedded sections from the same patients as measured by indirect immunofluorescence.
Our results demonstrated that resveratrol sulfation was significantly higher in breast cancer specimens compared to non-malignant samples as a result of significantly higher expression of STS in control tissue specimens. This information is important as intracellular biotransformation may affect anticancer activity of resveratrol.
Section snippets
Materials
Resveratrol (3,4′,5-trihydroxy-trans-stilbene), sulfatase type V: from Limpets (Patella vulgata), adenosine-3′-phosphate-5′-phosphosulfate (PAPS, 78% purity) and dithiothreitol (DTT) were obtained from Sigma–Aldrich (Munich, Germany). Methanol and water were of HPLC grade and purchased from Merck, Darmstadt, Germany. The Pierce Micro BCA Protein Assay Reagent Kit (Rockford, IL, USA) was used to determine protein content. All other chemicals and solvents were commercially available, of
Metabolism of resveratrol in human breast cancer tissue samples
Cytosol from 13 individual human breast tumor and adjacent non-tumor specimens were incubated with 1 μM resveratrol for 60 min at 37 °C and subsequently analyzed by HPLC. In the presence of PAPS, one biotransformation product was identified (tr = 28.4) in addition to resveratrol (tr = 31.5 min). No metabolite formation was seen in the absence of PAPS (data not shown). Treatment of samples with sulfatase prior to HPLC analysis led to the disappearance of this metabolite. The concomitant increase of
Discussion
In the present study we investigated the metabolism of resveratrol in breast cancer samples and corresponding non-malignant breast tissue specimens. Incubation of resveratrol in cytosolic tissue fractions revealed the presence of resveratrol-3-O-sulfate, whose formation is strongly dose-dependent. While sulfation clearly prevailed in the lower substrate concentrations (0.5–10 μM), an increase of the initial applied dose (25–100 μM) led to a dramatic reduction of resveratrol sulfation by up to
Conflicts of interest
All authors (Michaela Miksits, Katrin Wlcek, Martin Svoboda, Theresia Thalhammer, Isabella Ellinger, Gabriele Stefanzl, Charles N. Falany, Thomas Szekeres and Walter Jaeger) have disclosed no financial relationship and conflict of interest for the submitted manuscript entitled: “Expression of sulfotransferases and sulfatases in human breast cancer: impact on resveratrol metabolism”.
Acknowledgements
This study was supported by grants of the Jubiläumsfonds der Österreichischen Nationalbank (12600 to W.J.), FWF (P21083-B11 to W.J.), OVCAD STREProject (FP-6-018698 to M.S.), FFG-Bridge (818094 to I.E. and T.T.), and PHS (GM38953 to C.N.F.).
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