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«MODULATION OF POLYAMINE METABOLISM AS A CHEMOPREVENTIVE STRATEGY OF PHYTOCHEMICALS IN A CELL CULTURE MODEL OF COLORECTAL CANCERS Dissertation zur ...»

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3.6. The role of PPARg in resveratrol-induced inhibition of effects of resveratrol [100 mmol/L] on ODC-activity (Fig. 5). To ODC activity further verify the involvement of ceramide synthesis in resveratrol-mediated effects we treated Caco-2 cells with resveratrol [100 mmol/L] alone and in combination with L- As previously shown [20] the activation of transcription factor cycloserine and measured the protein levels of c-myc and ODC PPARg plays a crucial role in resveratrol-induced activation of after 24 h of incubation. As already shown in earlier studies [6] catabolic SSAT. So, we wanted to determine whether this resveratrol leads to a significant decrease of both c-myc receptor is also involved in ODC inhibition. In accordance to [***p 0.001] (Fig. 4A) and ODC [***p 0.001] (Fig. 4C) protein our recently published data, we now investigated the effects of levels, which could be significantly reduced [*p 0.05], when resveratrol [50–100 mmol/L] on ODC activity in Caco-2-wildceramide de novo synthesis was suppressed. type cells compared to Caco-2-cells transfected with either the biochemical pharmacology 74 (2007) 281–289 Fig. 3 – Influence of C2- and C6-ceramide on ODC activity in Caco-2- and HT-29-cells. Caco-2-cells were treated for 24 h with increasing concentrations of (A) C2-ceramide [10–40 mmol/L] and (B) C6-ceramide [10–40 mmol/L], HT-29-cells with (C) C2ceramide [10–30 mmol/L] and (D) C6-ceramide [1–10 mmol/L]. ODC activity was determined by 14CO2-release from labelled ornithine. Results (means W S.E.; n = 4) are expressed in enzyme units (picomole of released CO2) per milligram cellular protein per hour. *p 0.05; **p 0.01; ***p 0.001 vs. control.

empty vector or a dominant-negative PPARg mutant after 24 h. It is now well established that ceramides are important But in contrast to SSAT activation PPARg seems not to be second messengers for cell regulation which participate in essential for resveratrol-induced ODC inhibition as no signal transduction by activating specific serine/threonine differences could be observed, when PPARg mediated func- kinases, or by stimulating protein phosphatases. An increase tions are suppressed (Fig. 7). in intracellular ceramide concentrations could be induced by multiple exogenous agents comprising TNF-a, Fas ligand, 1a25-dihydroxyvitamin, chemotherapeutic agents, heat

4. Discussion stress and interleukin-1 [27,28]. Over the past few years there has been an escalating interest in exploring the role of Colorectal cancer is a major public health concern in all ceramide and its metabolites in tissue physiology and developed countries. Despite decades of advances in the pathophysiology. Typically, strategies that elevate cellular treatment and prevention of colorectal cancer, it remains the ceramide are being used for therapies aimed to arrest growth second most common cause of cancer death [21]. Hence, or promote apoptosis [29,30]. Interestingly, we could show interest in the concept and practice of chemoprevention that also the antiproliferative effects of resveratrol closely as an approach to arrest or reverse carcinogenesis at its correlate with a dramatic increase of endogenous ceramides.

earliest stages has increased greatly in the past few years Similar effects could be observed in a metastatic breast cancer cell model, when ceramide levels increased $5- and 10-fold [22]. Therefore, dietary polyphenols are of great interest due to their antioxidative and anticarcinogenic activities. after treatment with resveratrol 32 and 64 mmol/L, respecResveratrol, present in red wines, peanuts and grapes, tively, in comparison with untreated cells [31]. Treatment exhibits multiple chemopreventive effects comprising with C2- or C6-ceramide in turn, caused distinct growth inhibition of cell growth [23,24] and angiogenesis [25] inhibition in our colorectal cancer cell model. Sala et al.

as well as induction of apoptosis [26], whereby the under- hypothesize that the phenolic moiety is critical for the lying molecular mechanisms are only partly deciphered ceramide-associated growth-inhibitory effects of resveratrol [6,20]. [32]. While the activation of mitogen-activated protein kinase 286 biochemical pharmacology 74 (2007) 281–289 Fig. 4 – Western blot of c-myc (A) and ornithine decarboxylase protein (C) in Caco-2-cells after incubation with resveratrol [100 mmol/L] and cycloserine [1 mmol/L] alone and in combination for 24 h. Western blot of c-myc (B) and ornithine decarboxylase protein (D) in Caco-2 cells after incubation with increasing concentrations of C6-ceramide [10–40 mmol/L] for 6 h. A representative immunoblot of three independent experiments is shown. The graph presents the densitometric analysis. Means W S.E.; n = 3; Values not sharing a letter differ significantly; *p 0.05, **p 0.01.

p38 plays a crucial role in resveratrol-induced SSAT-activa- Nearly 70% of human colon cancers are associated with the tion [20], an involvement in ceramide-mediated actions is activation of proto-oncogene c-myc [36], a transcription factor discussed controversially [33–35] and requires further inves- that directly regulates the expression of ornithine decarboxtigations. ylase (ODC) by binding to a specific CACGTG sequence in the biochemical pharmacology 74 (2007) 281–289

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Sandra Ulrich, Bettina Jung, Stefan M. Loitsch, Jürgen M. Stein 1st Department of Medicine-ZAFES, Johann Wolfgang Goethe University, Frankfurt am Main





Key words:

Ursolic acid, apoptosis, colorectal cancer, Spermidine/spermine acetyltransferase, PPARγ * The abbreviations used are: BrdU, bromodeoxyuridine; CDK, cyclin dependent kinase;

DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxid; dnPPARγ, dominant negative PPARγ mutant; DTT, Dithiothreitol; EDTA, ethylenediaminetetraacetic acid; ELISA, Enzyme-linked immunosorbent assay; FADD, Fas Associated protein with Death Domain; PPARγ, peroxisome-proliferator activated receptor γ; RXR, retinoid X receptor; SSAT, spermine/spermidine acetyltransferase; TNF-α, tumor necrosis factor- α;

TRAIL, Tumor necrosis factor-related apoptosis-inducing ligand; UA, ursolic acid Address correspondence to : Jürgen Stein, MD, PhD, FEBG, 1st Department of Internal Medicine – ZAFES, Johann Wolfgang Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany, Telephone number: +49-69-6301-5917, Fax number: +49-69-6301E-mail: j.stein@em.uni-frankfurt.de

ABSTRACT

Previous studies could demonstrate that ursolic acid (UA), a pentacyclic triterpene found in berries and plants has antiproliferative as well as proapoptotic activities on cancer cells. The objective of this study was to elucidate the underlying molecular mechanisms of these chemopreventive effects.

We could show, that treatment with UA leads to a significant time- and dose-dependent cell growth inhibition of colorectal cancer cells, coincident with the upregulation of the cell cycle regulators cyclin E, p21WAF1/Cip1 and p27Kip1. In addition, UA significantly induces apoptosis, which is mediated by an increase of BAX/Bcl-2-protein-ratio as well as an upregulation of TRAIL protein which meets in an induction of caspase-3 activity. Furthermore, we could show that UA leads to a PPARγ-dependent induction of spermidine/spermine acetyltransferase (SSAT), a key enzyme of polyamine metabolism, which is associated with catabolism of intracellular polyamines and subsequent apoptotic responses.

In conclusion, the observed reduction of cell growth of colon cancer cell lines after treatment with ursolic acid presumably results from a large increase in the number of apoptotic cells.

The induction of the catabolic enzyme SSAT via PPARγ-dependent mechanisms therby seems to present the major molecular target in the induction of programmed cell death.

INTRODUCTION

Ursolic acid is a pentacyclic triterpenoid compound which naturally occurs in a large number of berries, medicinal herbs and other plants. It was identified as the major biological active incredient in a large number of plants which are used in traditional east asian folk medicine as drugs exhibiting hepatoprotective, antiinflammatory and anti-tumor effects (for review see 1).

Additionally, chemopreventive effects could be demonstrated in several cancer models comprising inhibition of proliferation as well as induction of apoptosis 2-4.

Cell division relies on the activation of cyclins, which bind to cyclin-dependent kinases (CDKs) to induce cell-cycle progression towards S phase and later to initiate mitosis. Since uncontrolled cyclin-dependent kinase activity is often the cause of human cancer, their function is tightly regulated by cell-cycle inhibitors such as the p21WAF1/Cip1 and p27Kip1 proteins 5. They have been described to regulate the G1-S transition by interacting with specific cyclin/cyclin dependent kinase complexes, and have also been shown to mediate growth arrest when overexpressed in the cell.

Apoptosis or programmed cell death is defined as an active physiologic process of cellular self-destruction, with specific morphologic and biochemical changes in the nucleus and cytoplasm 6. The signaling event leading to apoptosis can be divided into two distinct pathways, namely the intrinsic and extrinsic pathway. Engagement of the intrinsic pathway results in altered mitochondrial membrane permeability and the release of pro-apoptotic factors including cytochrome c, caspase-9 and second mitochondria-derived activator of caspases (Smac)/DIABLO into the cytosol. Cytochrome c binds to apoptosis-inducing factorApaf1) and procaspase-9 to form the “apoptosome”, which leads to activation of caspase-9 and subsequently caspase-3, resulting in apoptosis 7. A second caspase-independent pathway is characterized by the leakage of apoptosis-inducing factor AIF from mitochondria, resulting in direct chromatin condensation and DNA fragmentation 8. The extrinsic pathway is characterized by ligand fixation to death receptors present on the cell surface. These death receptors are members of the TNF receptor gene superfamily, which share similar, cysteine rich extracellular domains 9. Ligation of death receptors results in recruitment of adapter molecules such as FADD, which in turn recruits procaspase-8 to form the death inducingsignaling complex (DISC) 10. DISC releases caspase-8, which activates caspase-3 11.

Little is known about the underlying molecular mechanisms of ursolic acid related effects on cell proliferation and apoptosis in colorectal cancers. Thus the major aim of this study was to analyze modulatory effects on cell cycle regulating proteins as well as pro- and anti-apoptotic factors and to further characterize signal transduction pathways leading to this chemopreventive actions.

MATERIALS AND METHODS

Cell culture. Caco-2 cells of passages 20-30 were kept in Dulbecco´s modified eagle medium (DMEM), supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin, 1% sodium pyruvate and 1% nonessential amino acids. HCT-116 and HT-29 cells of passages 17were cultured in McCoy’s 5A supplemented with 10% FCS and 1% penicillin/streptomycin. Both cell lines were maintained at 37°C in an atmosphere of 95% air and 5% CO2. The cells were passaged weekly using Dulbecco´s PBS containing 0.25 % trypsin and 1 % EDTA. The medium was changed three times per week. Cells were screened for possible contamination with mycoplasma at monthly intervals. For experiments, the cells were seeded onto plastic cell culture wells in serum-containing medium and allowed to attach for 24h. For the SSAT activity assay the cells were synchronized in medium containing 1% FCS 24h before treatment. Ursolic acid, Fetal calf serum (FCS) and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO), Dulbecco’s modified Eagle’s medium (DMEM) and Geneticin (G418) from Gibco/Invitrogen (Carlsbad, CA); sodium pyruvate solution, glutamine, penicillin and streptomycin stock solutions from PAA Laboratories GmbH (Ontario, Canada); Lipofectamine™ 2000 from Invitrogen (Carlsbad, CA).

Cell Counts Cells were suspended and cultured on 96-well dishes at a density of 104/well (0.28 cm2).

Treatment started 24h after plating. At given time points following treatment cell numbers was assessed by crystal violet staining. Medium was removed from the plates and cells were fixed with 5% formaldehyde for 5 min. After washing with PBS cells were stained with 0.5% crystal violet for 10 min, washed again with PBS and unstained with 33% acetoic acid.

Absorption, which correlates with the cell number, was measured at 620 nm.

Cell Proliferation The effects of ursolic acid on DNA synthesis of cells were assessed using an cell proliferation ELISA kit (Roche Diagnostics, Mannheim, Germany). 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 ursolic acid for different time intervalls, and then labelled with BrdU for a further 4 hours. Incorporated BrdU was measured colorimetrically.

SDS-polyacrylamide gel electrophoresis and immunoblot analysis

Caco-2 cells were seeded in 80 cm2 flasks; 24h after plating, cells were incubated with substances for different time intervals. Western blot analysis using total protein extracts from cultured cells was performed as previously described 12. Protein was quantified with the BioRad (Bio-RAD Laboratories, Munich, Germany) protein colorimetric assay. Reprobing of blots for expression of β-actin was done routinely. Antibodies for p21WAF1/CIP1, p27KIP1, Cyclin E and BAX were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). AntiBcl-2 was purchased from Cell Signaling (Danvers, MO) and anti-TRAIL from Cayman Chemicals (Ann Arbor, MI). For quantitative analysis, bands were detected and evaluated densitometrically by ProViDoc system (Desaga, Wiesloch, Germany), normalized for the density of β-actin.

Cell death detection assay To determine and quantify the induction of apoptosis by ursolic acid in colon cancer cells, cytoplasmic histone-associated DNA fragments were measured using a commercially available Cell Death Detection ELISA kit (Roche Diagnostics, Mannheim, Germany). 24h after treatment with the mentioned substances, one µg of cell lysate was used for the ELISA procedure, following the manufacturer’s instructions. DNA fragmentation was quantified photometrically at 405 nm.



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