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Caspase-3-Activity assay Activity of caspase 3 was determined using a fluorometric immunosorbent enzyme assay (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions.

Briefly, cells grown in 6-well plates were incubated with increasing concentrations of ursolic acid for 24 h. Cells were washed with ice-cold PBS harvested in lysis buffer and centrifuged at 4°C for 2 minutes at 13000g. Caspase 3 from cellular lysates is captured by a monoclonal antibody on a coated microtiter plate. Following a washing step, subtrate is added that is cleaved proportionally to the amount of activated caspase 3. Due to proteolytic cleavage of the substrate, free fluorescent 7-amido-4-trifluoromethyl-coumarin (AFC) is generated.

Fluorescence was measured (excitation/emission, 430/535 nm) with the fluorescence microplate reader Tecan SpectraFluor PLUS.

SSAT-Activity Cells were washed twice with cold homogenizing buffer (10mM Tris/HCl, pH 7.5, 2.5 mM DTT, 1mM EDTA), harvested by scraping, disrupted by sonification and centrifuged at

15.000 g at 4°C for 15 min. Sixty microlitre aliquots of the supernatant were incubated with

0.3 µmol/L spermidine, 10 µmol/L Tris/HCl (pH 7.8), and 3,7 kBeq [acetyl-14C]CoA (Hartman Analytic GmbH, Braunschweig, Germany) at 37°C for 10 min. The reaction was terminated by chilling and the addition of 20 µl of 1M NH2OH. Subsequently, samples were centrifuged at 15.000 g for 5 min. Thirty microlitres of the supernatant were spotted onto a Whatman P81 paper disc (2.4 cm in diameter). The paper disc was washed with aqua dest.

and ethanol on a filter, dried and transferred to a vial containing 3 ml of scintillation cocktail (Packard Biosciences, Groningen, The Netherlands). Radioactivity was measured in a liquid scintillation counter (Packard Instruments, Meriden, CT). Controls included samples for measurement of non-enzymatic incorporation of [acetyl-14C]CoA into monoacetlyspermidine.

For all treatments tested the assay was repeated without addition of spermidine, to estimate unspecific acetylation by other enzymes than SSAT. These values were subtracted from the results obtained from the SSAT-activity measurements.

Transfection assay The following plasmids were used for transfection: pcDNA3 (Invitrogen, Carlsbad, CA), as an empty vector for control transfection and the plasmid pcDNA3-PPARγL468A/E471A, a dominant-negative double mutant, that was kindly provided by VK Chatterjee (Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom). These constructs were transfected into subconfluent Caco-2 cells with lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 6 h the cells were fed with fresh medium containing 10% FCS. G418 (400 µg/ml) and culture medium containing 10 % FCS. 24 hours later the cells were fed with medium containing G418 (400 µg/ml) and culture medium was replaced twice a week. G418-resistant colonies were collected and used for further analysis.

Statistics The data are expressed as means ± SE of at least three independent experiments. Analysis of variance (ANOVA) was performed when more than two groups were compared, and when significant, multiple comparisons were performed with the Turkey test. A p value 0.05 was considered to be significant.

RESULTS Effects of ursolic acid on cell growth of colorectal cancer cell lines Caco-2 [10-30 µmol/L], HCT-116 [5-15 µmol/L] and HT29 [5-15 µmol/L] cells were incubated with increasing concentrations of ursolic acid 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. (Figures 1A-D) Effect of ursolic acid on cell cycle regulating proteins To decipher the molecular mechanisms leading to cell growth inhibition we started to measure the expression status of several cell cycle regulating proteins (Figure 2), whereby the most prominent effects could be observed with the cell cycle inhibitors p21WAF1/Cip1 and p27Kip1.

While ursolic acid [10-30 µmol/L] leads to a significant dose-dependent increase of p21 WAF1/Cip1 protein levels already after 24h of incubation (~ 30% at 20 µmol/L, *p0.05), an increase of p27 Kip1 levels could not be observed until 48h of treatment (~ 40% at 20 µmol/L, *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 cyclin dependend kinase 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 [10-30 µmol/L] (~ 40% at 20 µmol/L, *p0,05).

Figure 2

Induction of apoptosis by ursolic acid To evaluate a possible influence of apoptosis induction on the cell growth inhibition of CacoHCT-116 and HT-29 cells, we investigated DNA fragmentation as a marker of programmed cell death. Thereby, ursolic acid [5-30 µmol/L] causes a significant dosedependent increase of DNA-fragments after 24h of incubation [***p0,001 vs. control at 15/30 µmol/L in all cell lines] (Figure 3). 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 dosedependent increase of caspase-3-activity in Caco-2-cells [***p0.001 vs. control] (Figure 4).

Figure 3 Figure 4 Expression of apoptosis regulating proteins 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 [10-30 µmol/L] 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%). This results in a significant increase of the BAX/Bcl-2 protein ratio up to 60% (***p0.001 at 30 µmol/L) 14,15 (Figure 5A) which is generally known to trigger apoptosis. We further measured protein levels of TRAIL 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 [10-30 µmol/l] in a dosedependent manner (*p0.05 at 30 µmol/L) (Figure 5B).

Figure 5

SSAT activation by ursolic acid Next we examined the effects of ursolic acid [20-30 µmol/L] on 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. Ursolic acid leads to a significant increase of SSAT activity [***p0,001 vs. control at 30 µmol/L] in Caco-2wildtype cells after 24 h of incubation. In Caco-2-empty vector cells ursolic acid also significantly increases SSAT-activity [***p0,001 vs. control at 30 µmol/L], whereas no effects could be observed when PPARγ mediated functions are suppressed in Caco-2dnPPARγ mutant cells (Fig 6).

DISCUSSION Interest in the concept and practice of chemoprevention as an approach for the control of cancer has increased greatly in the past few years 16. Multiple natural agents have been shown to be effective for blocking carcinogenesis in certain human cancers and animal models.

Using non-toxic chemical substances therefore is regarded as a promising alternative strategy to therapy for control of human cancers. The observed anti-carcinogenic effects may be due to 17,18 blocking effects on the carcinogenesis stages of initiation, promotion, or progression.

However, the precise underlying molecular mechanisms remain largely unknown. The aim of our study was to characterize chemopreventive effects of ursolic acid in a cell culture model of colorectal cancer. Attempts to show favourable effects in vitro have let to the identification of multiple direct targets for this compound. UA blocked cell cycle progression in the G1 phase for example was shown to be associated with a marked decrease in the protein expression of cyclins and their activating partners the cyclin-dependent kinases 19-22 and with a concomitant induction of p21WAF1/Cip1 in miscellaneous cancer cell lines 23-25. Unfortunately, little reseach was done in colon cancer cells thereby mainly focusing apoptotic mechanisms 26,27. After detecting potent cell growth inhibitory properties of ursolic acid we started to measure the expression status of several cell cycle regulating proteins, wherby the most prominent effects could be observed in the upregulation of cell cycle inhibitors p21WAF1/Cip1 and p27Kip1. Contrary to expectations incubation with Ursolic acid also leads to a conspicious increase of cell cycle progressor cyclin E, which is however consistent with earlier findings and Schneider et al. 29, which could show the same effects after treatment by Wolter et al.

with the polyphenol resveratrol and take this as a result of a cell cycle arrest in the S-phase.

However, cell cycle analysis of ursolic acid treated cells presents a predominant arrest in the G1 phase 30,31. These controversial results will deserve further investigations. Moreover, there are several lines of evidence, that the induction of cyclin E by genotoxic stress, such as ionizing radiation 32 or chemotherapeutic agents 33 could play a functional role in the initiation phase of apoptosis in malignant cells lines, in addition to its reported key regulatory role in the control of the G1 to S-phase transition and the initiation of DNA replication 34. Normally, the intracellular level and activities of p27Kip1 and cyclin E correlate negatively as there exist interdependence regulatory mechanisms. Simultaneous accumulations in both p27Kip1 and cyclin E are known to be characteristic phenotypes in cells derived from mice lacking S-phase kinase associated protein 2 (Skp2) suggesting a possible involvement of protooncogene Skp2 in the regulation of p27Kip1 and cyclin E, which might provide a target of ursolic acid mediated actions.

Two major apoptosis pathways have been identified, the death receptor or extrinsic pathway and the mitochondrial or intrinsic pathway. The mitochondrial pathway is regulated by members of the bcl-2 protein family, which can be divided in pro- and anti-apoptotic groups.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines, which can induce apoptotic cell death by engaging the death receptors DR4 and DR5, while sparing most normal cells. In certain tumor cell lines, TRAIL protein expression could be induced by chemopreventive agents resulting in TRAILmediated apoptosis in an autocrine or paracrine manner 37-39. This suggests that endogenously expressed TRAIL, which we could also observe in our colon cancer model after ursolic acid treatment, may be at least partly responsible for the observed chemopreventive effects. The targeting of pro- and anti-apoptotic members of the Bcl-2 family of proteins, is the focus of 40,41 efforts to modulate the intrinsic pathway of apoptosis and effect tumor death. While ursolic acid leads to an upregulation of proapototic BAX protein levels, the expression of the antiapoptotic Bcl-2 was diminished after 24h of incubation. This results in a significant 42,43 increase of the BAX/Bcl-2 protein ratio which is generally known to trigger apoptosis.

Taken together ursolic acid seems to lead to an activation both of extrinsic and intrinsic signaling pathways both resulting in Caspase-3 activation, followed by DNA fragmentation and programmed cell death.

Ornithine-derived polyamines (putrescine, spermidine, and spermine) are biogenic organic polycations that are present in all living cells. They have pleiotropic effects, with a welldescribed role as major metabolic regulators of cell proliferation and cell death balance.

Intracellular levels of polyamines must be maintained within narrow limits, as a decrease is related to cell growth inhibition, whereas an excess appears to be toxic. A correlation between polyamines and cancer have been extensively studied for decades, pointing out the inhibition of polyamine biosynthetic enzymes ornithinedecarboxylase and sadenosylmethioninedecarboxylase or activation of catabolic enzyme spermidine/spermine 46-48 acetyltransferase (SSAT) as a potential chemopreventive strategy. An association between SSAT induction followed by catabolism of the ubiquitous intracellular polyamines and subsequent apoptotic responses was first reported by Ha et al. 49. Furthermore, Chen et al.

could demonstrate that selective interference of polyamine-analogue induced SSAT prevents apoptotic signaling and apoptosis in human melanoma cells 50. Much on the focus on SSAT has been on the functional level, but the regulation of SSAT gene expression has also been a subject of recent investigations. Several transcription factors have been shown to activate SSAT expression. One regulatory pathway is the peroxisome proliferator (PPARs)-dependent pathway. These ligand-inducible transcription factors belong to the nuclear hormone receptor 51,52 superfamily and occur in three different isotypes termed α, β and γ. We could show that PPARγ is essential for SSAT-activation mediated by ursolic acid, which is in accordance with our recently published data, presenting the same effects after treatment with the polyphenol resveratrol 53, possibly by binding to PPAR response elements, which could be identified on the promotor of the SSAT gene 54.

CONCLUSION 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 modulation of the polyamine metabolism, especially the induction of the catabolic enzyme SSAT via PPARγ-dependent mechanisms thereby seems to present the major molecular target in the induction of programmed cell death.

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