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METABOLOMIC ANALYSIS OF RESVERATROL-INDUCED EFFECTS ON THE HUMAN BREAST CANCER CELL LINES MCF-7 AND MDA-MD-231 W. Jaeger*1, A. Gruber2, B. Giessrigl3, G. Krupitza3, D. Sonntag2 1 Department of Clinical Pharmacy and Diagnostics, University of Vienna, Austria, 2 BIOCRATES Life Sciences AG, Innsbruck, Austria; 3Institute of Clinical Pathology, Medical University of Vienna, Austria INTRODUCTION Breast cancer is a major cause of cancer death in women worldwide. Evidence from epidemiological and experimental studies indicates that natural constituents of the diet may well act as chemopreventive agents and inhibit mammary carcinogenesis. One of such compounds is resveratrol (RES), 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. OH HO OH The antiproliferative property of RES has been demonstrated in vitro against breast cancer cells due to the induction of apoptosis via downregulation of NF-kappa B, Bcl-2, inhibition of ribonucleotide reductase, and DNA polymerase. In addition, recent experiments have shown significantly less tumor growth in human breast cancer xenografts in vivo, supporting the use of this polyphenol as a potential chemotherapeutic agent. Although gene and protein expression have been extensively profiled in breast cancer cells after RES treatment, there are no data about metabolic alterations that characterize the effects of this compound. Most breast cancer biomarker discovery studies are based on the qualitative or quantitative changes of large biopolymers (e.g. DNA, RNA, glycans and proteins). In contrast, metabolic biomarker discovery approaches that focus an small molecules (below 1 kDa) have received significantly less attention, despite the fact that the identification and quantification of specific metabolites in tumor cells provides highresolution biochemical snapshots depicting the functional endpoints of drug disposure (Fig.1). There are two approaches to metabolomics, i.e., targeted metabolomics and metabolic profiling. While both approaches are complementary, targeted metabolomics, i.e., the identification and quantification of defined sets of structurally known and biochemically annotated metabolites, takes advantage of our detailed understanding of most biochemical pathways (Fig.2). RESULTS & DISCUSSION Sample cohorts Fig. 1: Omics technologies used for the development of molecular diagnostics Metabolic profiling (e.g. full scan LC-MS) Targeted metabolomics (ID / quantitation by MS/MS) Differential pattern information Metabolite concentration shifts Identification of relevant metabolites Functional annotation Resveratrol significantly reduced cell viability in the cancer cell lines MCF-7 ZR and MDA-MB231, yielding IC50 of 68.3 ± 2.6 and 67.6 ± 4.1 µM, respectively. Cell growth inhibition was accompanied by substantial metabolic changes, which were dose-dependent and similar in both cell lines. Results obtained for MCF-7 cells were chosen to demonstrate the metabolic effects of resveratrol on mammary carcinoma cells. The most interesting findings were related to the following metabolite classes: Amino acids: Resveratrol induced degradation or release of amino acids (Fig. 3). Enzymatical conversion of tryptophan to the bioactive metabolite serotonin was stimulated, whereas methionine underwent increased non-enzymatical oxidation to methionine-sulfoxide. Taurine, on the other hand, was substantially released from the cells, which is often caused by cell swelling and the occurrence of reactive oxygen species (Lambert, 2007). Fig. 2: Metabolic profiling versus targeted metabolomics EFFECT OF RESVERATROL ON THE METABOLISM OF Polyamines: Upon treatment with resvertarol, a profound modulation of polyamine biosynthesis could be seen in mammary cancer cell lines (Fig. 4). While the synthesis of putrescine from ornithine by ornithine decarboxylase, seemed to be inhibited, an up to 8-fold increased synthesis of spermidine from putrescine was observed, indicating activation of spermidine synthase. Interestingly, synthesis of spermine from spermidine was not stimulated, but rather inhibited. Putrescine and spermidine are essential for a variety of cellular processes related to signal transduction, growth and differentiation. Therefore, resveratrol-induced changes in polyamine metabolism could be directly linked to cell vitality (Takao et al, 2006). Conversion of putrescine to metabolically active spermidine and spermine occurs early during cell proliferation, and the activity of the two rate-limiting enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SADMC) is increased in proliferating cells (Milovic et al, 2000). Resveratrol and the analog (Z)-3,5,4`trimethoxystilbene have been shown to reduce ODC and SADMC activities by depletion of the polyamines putrescine and spermidine, hence exerting the cytotoxic effects by depleting the intracellular pool of polyamines (Wolter et al, 2003; Schneider et al, 2003). Arachidonic acid: A pronounced increase in extracellular arachidonic acid (AA) and its metabolite 12SHETE could be observed with high doses of resveratrol (Fig. 5). AA is released from cell membrane phospholipids upon activation of phospholipase A2 and subsequently converted to 12S-HETE by the action of 12-lipoxygenase. Increased levels of 12S-HETE may therefore indicate oxidative stress in tumor cells under resveratrol treatment. (Nazarewicz et al, 2007). Furthermore, resveratrol also reduced prostaglandin E2 (PGE2) levels (from >4 to 0 pmol/mio cells), thus confirming that this polyphenol is an inhibitor of cyclooxygenase 2 (Murias et al. 2005). AMINO ACIDS Stimulation of tryptophan degradation Tryptophanhydroxylase Tryptophan Serotonin Cell culture medium MCF-7 cells Release of taurine from cells Cell swelling ROS Leakage Taurine Cell culture medium MCF-7 cells Cell culture medium MCF-7 cells Formation of methionine-sulfoxide ROS OBJECTIVE To investigate possible metabolic alterations in MCF-7 and MDA-MD-231 breast cancer cells after different doses of resveratrol treatment focusing on the following classes of analytes: Methionine Methionine sulfoxide Fig. 3: Stimulation of serotonin synthesis, taurine release and methionine-sulfoxide formation by resveratrol POLYAMINES A Arginine amino acids Cell culture medium MCF-7 cells biogenic amines and polyamines eicosanoids and oxidized polyunsaturated fatty acids (PUFAs) CONCLUSION Arginase Ornithine A Methionine Ornithine decarboxylase B Putrescine Spermidine synthase Cell culture medium S-Adenosylmethionine B Decarboxylated S-Adenosylmethionine MCF-7 cells Spermidine Spermine synthase C Cell Culture MCF-7 and MDA-MD-2131 cells were cultured in phenol red-free D-MEM/F-12 (GIBCO 21041-025) containing 10% heat inactivated FCS and 1% P/S. For the experiment cells were seeded in 15 cm plates. After 24 h, medium was changed to 15 mL phenol red-free D-MEM/F-12 (GIBCO 21041-025) containing 10% DCC-stripped FCS and 1% P/S. Cells were treated with resveratrol (0 µM, 25 µM, 50 µM, 100 µM) for 72 hours (DMSO final concentration: 0.1 %). C Spermine METHODS Cell culture medium MCF-7 cells Fig. 4: Modulation of polyamine synthesis by resveratrol ARACHIDONIC ACID REFERENCES CELL MEMBRANE PHOSPHOLIPIDS Phospholipase A2 Lambert, I, Am J Physiol Cell Physiol 2007;293:C390-C400 Milovic V et al, Cancer Letters 2000;154:195-200 Murias et al, Bioorganic & Medicinal Chemistry 2004;12:5571-5578 Nazarewicz R et al, Arch Biochem Biophys 2007;468,114-120 Takao K et al, Int. Journal of Biochemistry & Cell Biology 2006;38:621-628 Wolter F et al, Carcinogenesis 2003;24(3):469-474 Schneider Y et al, Int. J Cancer 2003;107(2): 189-196 Mobilization of arachidonic acid Targeted Metabolomics By using a high-throughput liquid chromatography-based mass spectrometry platform, we applied targeted metabolomics for the identification and quantitation of almost 70 analytes of 4 different classes in cell pellets and in cell culture medium. MRM detection was performed using a 4000 Q TRAP® tandem mass spectrometry instrument (AB Sciex), and data were finally exported for biochemical analysis. 12-Lipoxygenase Cell culture medium SADMC The metabolic effects of resveratrol on breast cancer cell lines could be revealed using targeted metabolomics. Biogenic amines and polyamines, namely serotonin, putrescine, spermidine, and methionine-sulf-oxide, as well as arachidonic acid and its metabolite 12S-HETE were identified as potential markers for the pharmacological response of resveratrol, which must be considered in humans following oral uptake of dietary resveratrol or of other substances intented to be chemopreventive agents. Cell culture medium Fig. 5: Release of arachidonic acid from membrane lipids by lipase A2 and increased production of 12S-HETE ACKNOWLEDGEMENT We would like to thank the Method Development and Contract Research groups of BIOCRATES Life Sciences AG for their valuable work and great effort within this project.