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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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The same effects could be observed in Caco-2 cells, which is in accordance with our earlier studies [108]. Next, we tried to investigate the role of PPARγ in resveratrol-induced activation of SSAT. As no complete PPARγ antagonist have been described hitherto, we transfected a dominant-negative mutant receptor in Caco-2 cells to abolish PPARγ-mediated functions. In this mutant highly conserved hydrophobic and charged residues (Leu468 and Glu471) in helix 12 of the ligand-binding domain were mutated to alanine. So, this mutant retains ligand and DNA binding, but exhibits markedly reduced transactivation due to impaired coactivator recruitment [109]. Resveratrol leads to a significant increase of SSAT activity (p0.05 vs. control) in Caco-2-wild type cells after 24h of incubation, which is in agreement with our previous data [75]. In Caco-2 empty vector cells, resveratrol also significantly increases SSAT-activity (p0.05 vs. control) whereas no effects could be observed when PPARγ-mediated functions are suppressed in Caco-2-dnPPARγ mutant cells. To investigate the effects of resveratrol on PPARγ ligand-dependent activity, we did a chimeric Gal4-PPARγ transactivation assay. Because the chimeric receptor contained only hinge region and ligand binding domain of the PPARγ, any effect of

RESULTS

resveratrol affecting kinase-sensitive AF1 domain was ruled out. After incubation with resveratrol, we could generate similar effects of PPARγ agonist pioglitazone on PPARγ activity (p0.01). To show evidence of resveratrol ability to increase PPARγ activity, we measured, after resveratrol treatment, the expression of cytokeratin 20, which is described to be a specific target gene of PPARγ activity in colorectal cancer cells [110]. Incubation with resveratrol led to an ~40% increase of cytokeratin 20 expression at after 72h (p0.001 vs.

control). We further did Western blot analysis to determine possible effects of resveratrol on translational level. However, no significant changes in PPARγ protein expression could be detected (unpublished data). In a next step Western blot analysis was done to determine possible effects of resveratrol on the expression of PGC1α (PPARγ coactivator 1α) and sirtuin homologue SIRT1, which exhibits PPARγ-suppressive effects in white adipocyte tissue. Resveratrol led to a significant dose-dependent increase in both PGC1α (~60%, p0.05) and SIRT1 (~140%, p0.01) expression after 24h of incubation.

There are several lines of evidence that resveratrol mediates its chemopreventive actions via modulation of mitogen-activated protein kinase (MAPK) pathways. To examine p38 MAPK-mediated actions, we used the specific inhibitor SB203580. This anti-inflammatory drug inhibits the catalytic activity of p38 MAPK by competitive binding in the ATP pocket [111]. Incubation with resveratrol augmented phosphorylated p38 in a time- and dose-dependent manner, both in Caco-2 and HCT-116 cells (~300% at 200 µmol/L after 16h;

p0.01), whereas p38 MAPK concentration remained unaffected. To characterize the role of p38 activation in resveratrol-mediated induction of SSAT, we pretreated Caco-2 and HCT-116 with p38 inhibitor SB203580 [10-20 µmol/L] for 1 hour and then added resveratrol for another 24 hours. Both in Caco-2 and HCT-116 cells, coincubation with SB203580 significantly diminished resveratol-induced SSAT activation (p0.05 vs. resveratrol in Caco-2; p0.01 vs. resveratrol in HCT-116). In summary, our data confirm our earlier studies showing that resveratrol-mediated gowth inhibition of colorectal cancer cells seem to involve SSAT-induced polyamine catabolism. Here, we further demonstrate that transcription factor PPARγ acts as a p38-dependent target in resveratrol-induced molecular mechanisms.

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Apart from inducing polyamine catabolism, resveratrol was also shown to inhibit key enzymes involved in the biosynthetic pathway of polyamine metabolism.

And again we were interested in the underlying molecular mechanisms leading to these effects. In detail, the aim of this work was to study the potential involvement of ceramide biosynthesis in resveratrol mediated inhibition of biosynthetic ODC activity in colorectal cancer cells. First of all we examined the effect of resveratrol [50-200 µmol/L] on the intracellular ceramide concentrations of Caco-2 cells using mass-spectometry. After 24h of incubation we could observe a significant dose-dependent up-regulation of C16-ceramide levels ~6.5-fold at 200 µmol/L (p0.001). Since natural ceramides are not permeant to cell membranes, our study has been carried out by using short chain cell-permeable analogs to determine the role of ceramides in these signal transduction pathways. Caco-2 and HT-29-cells were incubated with increasing concentrations of N-acetylsphingosine (C2-ceramide) and Nhexanoylsphingosine (C6-ceramide) [1-40 µmol/L] for 24-72h. After each time interval both cell proliferation ELISA (BrdU) and crystal violet staining were performed. Both in Caco-2- and HT-29-cells a significant time- and dosedependent decrease in cell proliferation and cell counts could be measured.

Resveratrol on the one hand induces intracellular ceramide synthesis and on the other hand reduces the protein levels of the protooncogenes ODC and cmyc, a transcription factor that directly regulates the expression of ODC [75]. To reveal a possible coherency, we first measured the effects of C2- and C6ceramides in Caco-2- and HT-29 cells on ODC activity after 24h of treatment which both caused a significant inhibition in a dose-dependent manner (p0.001). We further did Western blot analysis to measure effects on the protein levels of ODC and c-myc after treatment with the C6-ceramide. And actually a dose-dependent decrease both in c-myc (p0.001) and ODC (p0.05) protein levels comparable to the resveratrol-induced effects [75] could be observed after 6h of incubation. Two major pathways may contribute to intracellular ceramide accumulation: namely the sphingomyelinase (SMase)RESULTS dependent catabolism of sphingomyelin, as well as the de novo synthesis catalyzed through serine palmitoyltransferase (SPT). Hence, we tested whether selective pharmacological inhibitors of these two key enzymes were able to prevent resveratrol-induced inhibition of ODC activity. While co-incubation with the SMase inhibitor manumycin [1 µmol/L] causes no changes in resveratrol action, blockade of de novo ceramide synthesis with the SPT-inhibitors Lcycloserine [1 mmol/L] and myriocin [5 µmol/L] counteracted inhibitory effects of resveratrol on ODC-activity. To further verify the involvement of ceramide synthesis in resveratrol-mediated effects we treated Caco-2 cells with resveratrol alone and in combination mith L-cycloserine and measured the protein levels of c-myc and ODC after 24h of incubation. As already shown in earlier studies resveratrol leads to a significant decrease of both c-myc (p0.001) and (p0.001) protein levels, which could be significantly reduced (p0.05), when ceramide de novo synthesis was suppressed. To determine whether the decrease in c-myc and ODC are the cause of decreased growth rate or a result, we performed an add-back experiment with exogenous polyamines. For this we treated Caco-2 cells with spermine [50 µmol/L], resveratrol and the combination of both and measured cell counts after 48h of incubation. As spermine was able to counteract resveratrol-actions significantly, we conclude that the observed reduction of cell counts after resveratroltreatment is due to a reduction of intracellular polyamine levels.





As shown in MANUSCRIPT I the activation of transcription factor PPARγ plays a crucial role in resveratrol-induced activation of catabolic SSAT. So we wanted to determine whether this receptor is also involved in ODC inhibition. In accordance to MANUSCRIPT I, we now investigated the effects of resveratrol on ODC activity in Caco-2-wildtype cells compared to Caco-2-cells transfected with either the empty vector or a dominant-negative PPARγ mutant after 24h. But in contrast to SSAT activation PPARγ seems not to be essential for resveratrolinduced ODC inhibition as no differences could be observed, when PPARγ mediated functions are suppressed.

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2.3 Ursolic acid induces apoptosis through PPARγ mediated SSAT-activation in colon cancer cells (MANUSCRIPT III) In addition to polyphenols, triterpenoid compounds, e.g. ursolic acid, were also described to show potent chemopreventive and anticarcinogenic properties in malignant cell lines. 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 these chemopreventive actions.

Caco-2-, HCT-116- and HT29-cells were incubated with increasing concentrations of ursolic acid [5-30 µmol/L] for 24 h, 48 h and 72 h. After each time interval both cell proliferation ELISA (BrdU) and crystal violet staining were performed. While not effective in Caco-2 cells after 24h of incubation ursolic acid leads to significant time- and dose-dependent decrease in cell counts (p0.001) as well as to a significant inhibition of cell proliferation (p0.001) in all cell lines after 48h at the latest. To decipher the molecular mechanisms leading to cell growth inhibition we started to measure the expression status of several cell cycle regulating proteins, whereby the most prominent effects could be observed with the cell cycle inhibitors p21WAF1/Cip1 and p27Kip1. While ursolic acid leads to a significant dose-dependent increase of p21WAF1/Cip1 protein levels already after 24h of incubation (~ 30%, p0.05), an increase of p27Kip1 levels could not be observed until 48h of treatment (~ 40%, p0.05). Another interesting change could be detected in the expression levels of cyclin E, which is essential for progression through the G1-phase of the cell cycle and for initiation of DNA replication by interacting with and activating its catalytic partner, the CDK 2 and therefore can be considered as a promotor of cell replication and proliferation. In contrast to our expectations, we could observe a significant dose-dependend increase in the protein levels of cyclin E in Caco-2 cells after 24h of incubation with ursolic acid (~ 40%, p0,05).

To evaluate a possible influence of apoptosis induction on the cell growth inhibition of Caco-2, HCT-116 and HT-29 cells, we investigated DNA fragmentation as a marker of programmed cell death. Thereby, ursolic acid RESULTS causes a significant dose-dependent increase of DNA-fragments after 24h of incubation [p0,001 vs. control in all cell lines]. Additionally, we measured caspase-3-activity in Caco-2 cells at the same point of time as a further marker of apoptotic actions. Again, ursolic acid seems to exhibit pro-apoptotic properties, as we could also observe a significant dose-dependent increase of caspase-3-activity in Caco-2-cells [p0.001 vs. control]. To specify the underlying molecular mechanisms leading to apoptosis after incubation with ursolic acid, we examined several apoptosis regulating proteins in Caco-2 cells by Western blot analysis. To analyse the effects on the intrinsic pathway we have chosen members of the Bcl-2 family of proteins, which are known to regulate membrane permeability and cytochrome c release from mitochondria.

While ursolic acid leads to an upregulation of proapoptotic BAX protein levels (up to ~20%), the expression of the antiapoptotic Bcl-2 was diminished after 24h of incubation (about ~40%). These single effects result in a significant increase of the BAX/Bcl-2 protein ratio up to 60% (p0.001), which is generally known to trigger apoptosis. We further measured protein levels of TRAIL (TNFrelated apoptosis-inducing ligand) an immunological inducer of extrinsic mechanisms leading to programmed cell death. This ligand, binding to specific death receptors on the cell surface, was also significantly upregulated after 24h of incubation with ursolic acid in a dose-dependent manner (p0.05). As resveratrol was shown to mediate its chemopreventive effects at least partly through the modulation of polyamine metabolism (MANUSCRIPT I + II), we were interested if ursolic acid modulates similar intracellular mechanisms. Hence, we examined the effects of ursolic acid on ODC and SSAT activity in Caco-2wildtype cells compared to Caco-2-cells transfected with either an empty vector or a dominant negative PPARγ mutant to investigate effects mediated by PPARγ. In contrast to resveratrol, ursolic acid only leads to a significant increase of SSAT activity (p0,001 vs. control) in Caco-2-wildtype cells after 24 h of incubation but does not simultaneously inhibit ODC activity (unpublished data). In Caco-2-empty vector cells ursolic acid also significantly increases SSAT-activity (p0,001 vs. control), whereas no effects could be observed when PPARγ mediated functions are suppressed in Caco-2-dnPPARγ mutant cells.

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3 DISCUSSION

3.1 Resveratrol-induced modulation of polyamine metabolism The phytoalexin resveratrol (3,4’,5-trihydroxystilbene) exhibits multiple chemopreventive effects comprising cell growth inhibition [108;112], induction of apoptosis [113], and prevention of angiogenesis [114], whereby the underlying molecular mechanisms are only partly understood (MANUSCRIPT IV + MANUSCRIPT V). One of our theories is concerned with the role of polyamines or polyamine metabolism respectively. Intracellular polyamine levels are maintained within very narrow limits because decreases of polyamine concentrations interfere with cell growth, whereas an excess seems to be toxic [115]. The three key enzymes of polyamine metabolism are ODD and SAMDC, the rate-limiting enzymes of polyamine biosynthesis, and SSAT, which controls polyamine catabolism [116]. Wolter et al. showed that resveratrol-induced growth arrest of Caco-2 cells is accompanied by inhibition of polyamine biosynthesis as well as activation of polyamine catabolism [75]. One aim of our work was to further characterize molecular events leading to the observed modulation of polyamine metabolism.

3.1.1 Mitogen-activated protein kinases



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