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mediates its chemopreventive actions via modulation of mitogen- understood (28, 29). Intracellular polyamine levels are maintained activated protein kinase (MAPK) pathways. To examine p38 within very narrow limits because decreases of polyamine conMAPK–mediated actions, we used the specific inhibitor centrations interfere with cell growth whereas an excess seems to SB203580. This anti-inflammatory drug inhibits the catalytic be toxic (30). The three key enzymes of polyamine metabolism are activity of p38 MAPK by competitive binding in the ATP pocket ornithine decarboxylase and S-adenosylmethionine decarboxylase, (24). As shown in Fig. 4, incubation with resveratrol (50-200 Amol/L) the rate-limiting enzymes of polyamine biosynthesis, and SSAT, augmented phosphorylated p38 in a time- and dose-dependent which controls polyamine catabolism (31). Wolter et al. showed manner, both in Caco-2 and HCT-116 cells (f300% at 200 Amol/L that resveratrol-induced growth arrest of Caco-2 cells is accompaafter 16 hours; P 0.01), whereas p38 MAPK concentration nied by inhibition of polyamine biosynthesis as well as activation of remained unaffected. To characterize the role of p38 activation in polyamine catabolism (13). The peroxisome proliferator–activated receptor g (PPARg) is a nuclear receptor that acts as a tranresveratrol-mediated induction of SSAT, we pretreated Caco-2 and HCT-116 with p38 inhibitor SB203580 (10-20 Amol/L) for 1 hour scription factor controlling the expression of multiple genes and then added resveratrol (100 Amol/L) for another 24 hours. Both involved in cell growth, differentiation, and apoptosis of several in Caco-2-(Fig. 5A) and HCT-116-cells (Fig. 5B), coincubation with malignant cell lines, and therefore seems to play a crucial role in SB203580 significantly diminished resveratrol-induced SSAT acti- carcinogenesis (32, 33). To abolish PPARg-mediated functions, we vation [P 0.05 versus resveratrol (100 Amol/L) in Caco-2; P 0.01 transfected a dominant-negative mutant in Caco-2 cells. In this versus resveratrol (100 Amol/L) in HCT-116]. PPARg mutant, two charged amino acid residues (Leu468 and Glu471) in helix 12 of the ligand binding domain are mutated to Effects of resveratrol on PGC-1A and SIRT1 expression.

Western blot analysis was done to determine possible effects of alanine, whereupon the mutant retains ligand and DNA binding resveratrol on the expression of PPARg coactivator 1a and sirtuin but exhibits markedly reduced transactivation due to impaired homologue SIRT1, which exhibits PPARg-suppressive effects in coactivator recruitment (22). According to the current findings, an white adipocyte tissue. Resveratrol (50-200 Amol/L) led to a essential role for PPARg in enhancing SSAT enzyme activity is significant dose-dependent increase in both PGC-1a (f60% at assumed (21). In fact, we could show that, in contrast to Caco-2Amol/L; P 0.05; Fig. 6A) and SIRT1 (f140% at 200 Amol/L; wild type and Caco-2-empty vector cells, resveratrol failed to P 0.01; Fig. 6B) expression after 24 hours of incubation. increase SSAT activity in Caco-2-dnPPARg cells. The mitogenactivated protein kinase (MAPK) pathways have been recognized as a major signaling pathway by which cells transduce extracellular Discussion signals into an intracellular response. In several studies, resveratrol The present study clearly shows that resveratrol mediates was shown to mediate multiple functions by modulating MAPK growth inhibitory effects in colorectal cancer cell lines, at least pathways (26, 34, 35). We could show as well that incubation with partly, via polyamine degradation, whereas activation of transcrip- resveratrol causes phosphorylation, and thus activation, of p38 tion factor PPARg seems to play a pivotal role. The plant MAPK in colon cancer cells. Furthermore, combination of polyphenol resveratrol (3,4,5-trihydroxystilbene) exhibits multiple resveratrol with an inhibitor of p38 MAPK leads to an inhibition chemopreventive effects comprising cell growth inhibition (6, 25), of resveratrol-induced SSAT activation both in Caco-2 and HCT-116 induction of apoptosis (26), and prevention of angiogenesis (27), cells. Consequently, an activation of MAPK cascade by resveratrol whereby the underlying molecular mechanisms are only partly can be assumed. As previously described, our results point out an

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De novo ceramide biosynthesis is associated with resveratrol-induced inhibition of ornithine decarboxylase activity Sandra Ulrich a, Andrea Huwiler b, Stefan Loitsch a, Helmut Schmidt c, Jurgen M. Stein a,* ¨ a First Department of Internal Medicine-ZAFES, Johann Wolfgang Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany b Institute of General Pharmacology and Toxicology-ZAFES, Johann Wolfgang Goethe University, Frankfurt am Main, Germany c Institute of Clinical Pharmacology-ZAFES, Johann Wolfgang Goethe University, Frankfurt am Main, Germany

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* Corresponding author. Tel.: +49 69 6301 5917; fax: +49 69 6301 83112.

E-mail address: j.stein@em.uni-frankfurt.de (J.M. Stein).

Abbreviations: BrdU, bromodeoxyuridine; DMEM, Dulbecco’s modified Eagle’s medium; DFMO, alpha-difluoromethylornithine;

dnPPARg, dominant negative PPARg mutant; ELISA, enzyme-linked immunosorbent assay; MAPK, mitogen-activated protein kinase;

NSAID, non-steroidal anti-inflammatory drugs; ODC, ornithine decarboxylase; PAO, polyamine oxidase; PPARg, peroxisome-proliferator activated receptor g; RXR, retinoid X receptor; SAMDC, S-adenosylmethioninedecarboxylase; SMase, Sphingomyelinase; SPT, serine palmitoyltransferase; SSAT, spermine/spermidine acetyltransferase; TNF-a, tumor necrosis factor-a 0006-2952/$ – see front matter # 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.bcp.2007.04.001 282 biochemical pharmacology 74 (2007) 281–289

1. Introduction mycoplasma at monthly intervals. For experiments, the cells

were seeded onto plastic cell culture wells in serum containing Resveratrol, chemically known as 3,5,40 -trihydroxytransstil- medium and allowed to attach for 24 h. For the ODC activity bene, is a naturally occuring polyphenolic antioxidant assay the cells were synchronized in medium containing 1% compound, also classified as a phytoalexin, which are herbal FCS 24 h before treatment. Resveratrol, N-hexanoylsphingoantibiotics produced in response to environmental stress sine, N-acetylsphingosine, L-cycloserine, myriocin and manufactors including injuries, UV irradiation or fungal invasion [1]. mycin were obtained from Sigma–Aldrich (St. Louis, MO);

Dulbecco’s modified Eagle’s medium and OptimemTM I from Resveratrol was first detected in the root extract of the weed Polygonum cuspidatum [2], which has been known in Asian Gibco (Invitrogen, Carlsbad, CA); fetal calf serum, sodium folk medicine under the name Ko-jo-kon and was traditionally pyruvate solution, glutamine, penicillin and streptomycin used to treat liver, skin and circulatory diseases [3,4]. Anti- stock solutions from PAA Laboratories GmbH (Ontario, Canada); LipofectamineTM 2000 from Invitrogen (Carlsbad, CA).

carcinogenic properties of resveratrol were first reported by Jang et al. [5] demonstrating chemopreventive effectiveness

2.2. SDS-polyacrylamide gel electrophoresis and against all the three major steps of carcinogenesis, i.e.

immunoblot analysis initiation, promotion and progression. We and others provide several lines of evidence that resveratrol mediates these antiCaco-2 cells were seeded in 80 cm3 flasks; 24 h after plating, cells carcinogenic effects partly through the modulation of polyamine metabolism [6,7]. The major polyamines spermidine and were incubated with substances for different time intervals.

spermine, and their diamine precursor, putrescine are organic Whole cell extract was obtained according to the manufaccations with multiple functions in cell growth and cell death turer’s instructions (Active Motif, Rixensart, Belgium). Protein [8,9]. The intracellular polyamine pool size is controlled strictly was quantified with the Bio-Rad protein colorimetric assay.

by the combined action of de novo synthesis, catabolism, uptake After addition of sample buffer to the total cellular extract and and export of polyamines. This regulatory mechanism boiling samples at 95 8C for 5 min, protein was separated on a include reactions catalyzed by the biosynthetic enzymes 10% SDS-polyacrylamide gel. Protein was transferred onto ornithine decarboxylase (ODC), S-adenosylmethionine decar- nitrocellulose membrane (Schleicher&Schuell, Dassel, Gerboxylase (SAMDC) and spermidine/spermine synthases and by many) and the membrane was blocked for 1 h at room the catabolic spermidine/spermine acetyltransferase (SSAT) temperature with 3% skim milk in tris-buffered saline containand FAD-dependent polyamine oxidase (APAO) [10]. The ing 0.05% Tween 20 (TBST). Next, blots were washed and finding that agents that inhibit polyamine biosythesis can incubated overnight at 4 8C in TBST containing 3% skimmed prevent, or at least limit cell growth [6,11–13], together with the milk powder with a 1:500 dilution of primary antibodies for ODC fact, that polyamine concentrations are elevated in multiple and c-myc (all from Santa Cruz Biotechnology, Santa Cruz, cancer tissues [14,15], has made the polyamine metabolism a USA). The secondary, horseradish peroxidase-conjugated antipromising target for cancer chemoprevention and therapy. body (Santa Cruz Biotechnology) was diluted at 1:2000 and Ceramides are key compounds in the metabolism of incubated with the membrane for another 45 min in skim milk.

sphingolipids and are emerging as important second mes- After chemoluminescence reaction (ECL, Amersham Pharmasengers for various cellular processes including cell cycle cia Biotech, Buckinghamshire, UK), band were detected after arrest, differentiation and apoptosis (for review see Ref. [16]). exposure to Hyperfilm-MP (Amersham International plc, BuckCeramides can be produced via a de novo biosynthetic pathway inghamshire, UK). Blots were reprobed with b-actin antibody which is initiated by condensation of serine and palmitoyl- (Santa Cruz Biotechnologies, Santa Cruz, USA). For quantitative CoA catalyzed by serine palmitoyltransferase (SPT) as well as analysis, bands were detected and evaluated densitometrically by sphingomyelinase-mediated hydrolysis of sphingomyelin. by ProViDoc system (Desaga, Wiesloch, Germany), normalized Our aim was to study the potential involvement of ceramide for the density of b-actin.

biosynthesis in resveratrol mediated inhibition of ODC activity

2.3. Cell counts in colorectal cancer cells.

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nostics, Tokyo, Japan). This assay is a colorimetric immunoassay for quantification of cell proliferation based on the measurement of bromodeoxyuridine (BrdU) incorporation during DNA synthesis, and is a non-radioactive alternative to the [3H]-thymidine incorporation assay. Cells were grown in 96 well culture dishes (104 cells/well), incubated with C2- or C6-ceramide for different time intervalls, and then labelled with BrdU for a further 4 h. Incorporated BrdU was measured colorimetrically.

2.5. Lipid extraction and ceramide quantitation

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Fig. 2 – (A) Cell counts and (B) cell proliferation of Caco-2 cells 48 h after incubation without (control) or with C2-ceramide [10– 40 mmol/L] or C6-ceramide [10–40 mmol/L]. The ceramides lead to a conspicuous dose- and time-dependent reduction of cell counts as well as an inhibition of cell proliferation. Means W S.E., n = 3. (C) Cell counts and (D) cell proliferation of HT-29 cells 48 h after incubation without (control) or with C2-ceramide [10–30 mmol/L] or C6-ceramide [1–10 mmol/L]. The ceramides again lead to a conspicuous dose- and time-dependent reduction of cell counts as well as an inhibition of cell proliferation.

Means W S.E., n = 3; *p 0.05; **p 0.01; ***p 0.001 vs. control.

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Two major pathways may contribute to intracellular ceramide To determine whether the decrease in c-myc and ODC are the accumulation: namely the sphingomyelinase (SMase)-depen- cause of decreased growth rate or a result, we performed an dent catabolism of sphingomyelin, as well as de novo synthesis add-back experiment with exogenous spermine [50 mmol/L].

catalyzed through serine palmitoyltransferase. Hence, we For this we treated Caco-2-cells with spermine [50 mmol/L], tested whether selective pharmacological inhibitors of these resveratrol [50–100 mmol/L] and the combination of both and two key enzymes were able to prevent resveratrol-induced measured the cell counts after 48 h of incubation (Fig. 6). As inhibition of ODC-activity in Caco-2- and HT-29-cells. While spermine was able to counteract resveratrol-actions significo-incubation with the SMase inhibitor manumycin [1 mmol/L] cantly, we conclude that the observed reduction of cell counts causes no changes in resveratrol action, blockade of de novo after resveratrol-treatment is due to a reduction of intracelceramide synthesis with the SPT-inhibitors L-cycloserine lular polymine levels.

[1 mmol/L] and myriocin [5 mmol/L] counteracted inhibitory

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