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Cell division consists of two consecutive processes, mainly characterized by DNA replication and segregation of replicated chromosomes into two separate cells which is divided into two stages, namely mitosis (M), the process of nuclear division and interphase, the periode between two mitosis phases. The stages of mitosis consist of prophase, metaphase, anaphase and telophase while the interphase is subdivided in G1, S and G2 phases (reviewed in [35]) (Figure 3). Each phase is characterized by distinct cellular processes that are required for proper cell division and is regulated by the activation of cyclins, which bind to cyclin-dependent kinases (CDKs) to induce cell-cycle progression towards S phase and later to initiate mitosis [36]. Different cyclins are required at different phases of the cell cycle. For the progression from G1 to S the D type cyclins bind to CDK4 and to CDK6 to form an active kinase that phosphorylates retinoblastoma (Rb) protein. In a hypophosphorylated state, Rb inhibits growth by sequestering the E2F transcription factor. Hyperphosphorylation of Rb by cyclin D-CDK4/6 results in the release of E2F, which activates transcription of cyclins for later phase transition as well as proteins required for DNA synthesis [37]. Another G1 cyclin is cyclin E which

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Figure 3: Phases of the cell cycle (adopted from MANUSCRIPT V) associates with CDK2 to regulate progression from G1 into S phase [38]. Cyclin A binds with CDK2 and this complex is required during S phase [39]. In late G2 and early M, cyclin A complexes with CDK1 to promote entry into mitosis which is further regulated by cyclin B in complex with CDK1 [40]. CDK activity can be counteracted by cell cycle inhibitory proteins, called CDK inhibitors (CKI) which bind to CDK alone or to the CDK-cyclin complex. Two distinct families of CKI have been discovered, the INK4 family (p15 (INK4b), p16 (INK4a), p18 (INK4c) and p19 (INK4d)) and the Cip/Kip family (p21 (Waf1, Cip1), p27 (Cip2) and p57 (Kip2)) [41]. In cancer, there are fundamental alterations in the genetic control of cell division, resulting in an unrestrained cell proliferation. This cell cycle dysregulation occurs through mutation of proteins important at different levels of


the cell cycle, like CDKs, cyclins, CDK-activating enzymes, CKI, CDK substrates, and checkpoint proteins [42]. Hence, the control of cell proliferation represents an important preventive stragety in multistep carcinogenesis.

1.2.2 Induction of apoptosis 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 [43]. The signaling events leading to apoptosis can be divided into two distinct pathways, namely the intrinsic and extrinsic pathway (Figure 4). 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. Cytochrom c binds to the cytosolic protein apoptotic protease activating factor-1 (APAF-1) and procaspase-9 to form the “apoptosome”, which leads to activation of caspase-9 and subsequently caspase-3, resulting in apoptosis [44]. This pathway is primarily governed by proteins of the Bcl-2 family, which include anti- and proapoptotic molecules able to differentially affect mitochondrial homeostasis and cytochrome c release [45]. Moreover, other proteins belonging to the inhibitor of apoptosis protein (IAP) family are able to block a common step downstream of mitochondrial cytochrome c release by inhibiting terminal effector caspase-3 and caspase-7, and interfering with caspase-9 activity and processing [46]. 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 [47]. The extrinsic pathway is characterized by ligand fixation to death receptors present on the cell surface.

These death receptors are members of the TNF (tumor necrosis factor) receptor gene superfamily, which share similar, cysteine rich extracellular domains [48].

Ligation of death receptors results in recruitment of adapter

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Figure 4: Apoptotic pathways (adopted from MANUSCRIPT V) molecules such as FADD (Fas-associated death domain), which in turn recruits procaspase-8 to form the death inducing signaling complex (DISC) [49]. DISC releases caspase-8, which activates caspase-3 [50].

Apoptosis is the mechanism used by metazoans to regulate tissue homeostasis through the elimination of redundant or potentially deleterious cells. The disruption of this mechanism is observed in a variety of cancers. Therefore induction of apoptosis is arguably the most potent defense against cancer.

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1.3 Phytochemicals Figure 5: Dietary agents and their major biological active compounds Information has been accumulated indicating that diets rich in vegetables and fruits can reduce the risk of a number of cancers [51]. Phytochemicals (nonnutrient components in the plant-based diet), such as carotenoids, antioxidative vitamins, phenolic compounds, terpenoids, steroids, indoles and fibers, have been considered responsible for risk reduction [52] (Dietary phytochemicals that most often appear to be protective against cancer are summarized in Figure 5).

In this project we were focusing on the anti-carcinogenic properties of two different phytochemicals, namely the polyphenolic Resveratrol and the triterpenoid Ursolic acid which will be introduced in the following section.


1.3.1 Resveratrol

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transduction pathways have been examined to explain these effects [71;72]. We and others provide several lines of evidence, that resveratrol mediates these anti-carcinogenic effects partly through the modulation of polyamine metabolism [73-76].

1.3.2 Ursolic acid

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The polyamines spermidine, spermine as well as their precursor putrescine are ubiquitious polycationic metabolites in prokaryotic and eukaryotic cells which play an essential role in cell growth by stabilizing DNA structure [86], influencing membrane functions [87] and cell cycle regulating genes [88]. Increasing concentrations are generally associated with cell proliferation and cell transformations induced by growth factors, carcinogens, viruses and oncogenes [89]. Therefore intracellular polyamine pool size is controlled strictly by the combined action of de novo synthesis, catabolism, uptake and export of polyamines. This regulatory mechanism include reactions catalyzed by the biosynthetic enzymes ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (SAMDC) and the catabolic


spermidine/spermine acetyltransferase (SSAT) and FAD-dependent polyamine oxidase (PAO) [85] (Figure 6). An association of excess polyamine levels with cancer was first reported in the late 1960s, when Russel and Snyder reported high levels of ODC activity in several human cancers [90]. In colorectal cancer tissue polyamine contents are increased 3-4 fold over that found in the equivalent normal colonic tissue [91]. Based on these findings pharmacological or natural inhibitors of polyamine metabolism have been studied in vitro [75;92] and in vivo [93] as new potent therapeutic strategies in cancer treatment and prevention.

1.4.1 Inhibition of polyamine biosynthesis

The recognition that polyamines are required for cell growth and that their metabolic pathway is frequently dysregulated in cancers led to the development of inhibitors for each step of the polyamine biosynthetic pathway. 2difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor, remains the prototypical inhibitor of ODC. DFMO initially competes with ornithine for binding to the active site of ODC and is then decarboxylated by ODC to create a highly reactive intermediate that in turn inhibits ODC activity.

Even though triggering promising effects in vitro, DFMO has been less successful in cancer therapy, resulting in cytostatic rather than cytotoxic effects

in vivo [94]. The limited success of DMFO has been attributed to three factors:

(1) the tumour is able to obtain sufficient polyamines to sustain growth from the pool, since the diet, a major source of polyamines would provide a continuous exogenous supply; (2) intestinal bacteria may represent a potential limited source of polyamines; and (3) polyamines may be effectively reutilized following their release from dead cells, especially those of the gut. Consequently, it should not be surprising, that attaining the goal of reducing high polyamine levels in tissues during cancer development might require targeting more processes in the regulatory mechanism of polyamine metabolism. Studies in experimental models could already show that combinations of two drugs, specifically, combinations of DFMO and non-steroidal anti-inflammatory drugs (NSAIDs) were more effective than single-agent strategies [95;96]. One aspect of the rationale for combining ODC inhibitors and NSAIDs might be that


catabolic SSAT provides another transcriptional target for several NSAIDs [95;97].

Flamigni et al. could demonstrate that ODC expression could be regulated in leukaemia cells through exogenous ceramide-analogs. Ceramides are key compounds in the metabolism of sphingolipids and are emerging as important second messengers for various cellular processes including cell cycle arrest, differentiation and apoptosis.

1.4.2 Induction of polyamine catabolism

ODC is not the only polyamine metabolic gene that is regulated by oncogenes and tumour-suppressor genes as both SSAT and PAO activities were found to be decreased in human solid tumors [98]. In human colon and other gastrointestinal cancers the expression of spermidine/spermine acetyltranferase (SSAT) was described to be negatively regulated by the K-ras oncogene, which is commonly mutated – and, as a result, aberrantly activated [99]. K-ras activates the kinase Raf and suppresses expression of SSAT by inhibition of PPARγ which normally binds to PPARγ response elements in the promoter of the SSAT gene [95]. Peroxisome proliferator-activated receptors are ligandinducible transcription factors belonging to the nuclear hormone receptor superfamily [100;101], which regulate transcription of target genes by heterodimerizing with the retinoid x receptor and binding to PPAR response elements [100;102]. PPARγ is expressed at high levels in colonic epithelial and colon cancer cells [103]. Girnun and Spiegelmann [104] hypothesize that PPARγ is exerting its effects early in the carcinogenic process by suppressing tumor formation. Activation of PPARγ therefore could function as an important molecular target of chemopreventive agents.

It was the discovery that specific antitumor polyamine analogs highly induce SSAT activity in a cell type specific manner that led to an increased interest in polyamine catabolism as a drug target. This ability of SSAT induction was suggested to correlate with the reduction of intracellular polyamines followed by cell growth inhibition and apoptosis [105-107].

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1.5 Aims Interest in the concept and practice of chemoprevention as an approach for the control of cancer has increased greatly in the past few years. 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 cancer. The observed anti-carcinogenic effects may be due to blocking effects on the carcinogenesis stages of initiation, promotion, or progression.

However, the precise underlying molecular mechanisms remain largely unknown. Thus, the aim of our study was to characterize chemopreventive effects of the two phytochemicals resveratrol and ursolic acid in a cell culture model of colorectal cancer.

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2.1 PPARγ as a molecular target of resveratrol-induced modulation of polyamine metabolism (MANUSCRIPT I) Based on our former findings that resveratrol induces cell growth inhibition of colon cancer cells via induction of catabolic SSAT, together with the identification of PPAR response elements in the promoter of the SSAT gene by Babbar et al., the aim of this work was to specify the underlying molecular mechanisms and to identify a possible role of transcription factor PPARγ. First, we determined the effects of resveratrol on cell proliferation and cell counts of the two colorectal cancer cell lines Caco-2 and HCT-116. Both cell lines were incubated with increasing concentrations of resveratrol [30-200 µmol/L] for 24, 48 and 72h. After each time interval, both cell proliferation ELISA (BrdU) and crystal violet staining were done. In HCT-116 cells, a significant time- and dosedependent decrease in cell proliferation and cell counts could be measured.

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