«MODULATION OF POLYAMINE METABOLISM AS A CHEMOPREVENTIVE STRATEGY OF PHYTOCHEMICALS IN A CELL CULTURE MODEL OF COLORECTAL CANCERS Dissertation zur ...»
Cells recognize and respond to extracellular stimuli by engaging specific intracellular programs, such as the signalling cascade, that leads to activation of the mitogen-activated protein kinases (MAPKs). All eukaryotic cells possess multiple MAPK pathways, which coordinately regulate diverse intracellular activities comprising gene expression, mitosis, survival and apoptosis, and differentiation. MAPKs are generally expressed in all cell types, yet their functions to regulate specific responses differ from cell type to cell type. To
date, five distinct groups of MAPKs have been characterized in mammals:
Extracellular signal-regulated kinase (ERK) 1/2, the p38 kinase (p38α, β, γ and δ), c-Jun N-terminal kinase (JNK) 1, 2, 3 and ERK3, ERK4, ERK5, which follow
the same principle of phosphorylation and activation cascades (Reviewed in ). Given the role of MAPKs in many critical responses reqired for cellular homeostasis, it is not surprising, that loss of fine control of MAPK regulation resulting from mutation or changes in expression of proteins regulating MAPK signalling contribute to cancer and thus the modulation of MAPK pathways presents an important anti-cancer strategy . MAPK p38 for example has recently gained attention as a tumor suppressor, as it, upon activation, induced terminal differentiation in rhabdomyosarcoma cells . Similarly, deletion of a p38-inhibitory phosphatase blocked Hras1- and erbB2-induced carcinogenesis in vivo, whereas inhibition of p38 promoted tumor formation . Other studies have evaluated p38 activity in response to chemotherapy, as diverse chemotherapeutic agents stimulate apoptosis in a p38-dependent manner [121;122].
In several studies, resveratrol was also shown to mediate multiple functions by modulating MAPK pathways (Reviewed in MANUSCRIPT V and ). In MANUSCRIPT III we could show as well that incubation with resveratrol causes phosphorylation, and thus activation, of p38 MAPK in colon cancer cells.
Furthermore, combination of resveratrol with an inhibitor of p38 MAPK leads to an inhibition of resveratrol-induced SSAT activation both in Caco-2 and HCTcells. Consequently, an activation of MAPK cascade by resveratrol can be assumed in our system. But, while the activation of p38 plays a crucial role in resveratrol-induced SSAT-activation, an involvement in ceramide-mediated actions, and thus inhibition of ODC-activity (MANUSCRIPT II), is discussed controversially [123-125] and requires further investigations.
3.1.2 Peroxisome-proliferator activated receptor γ
The PPARγ is a nuclear receptor that controls the expression of a large array of genes involved in adipocyte differentiation , lipid metabolism , insulin sensitivity , inflammation  and artherogenesis . There is much evidence that PPARγ plays another crucial role in carcinogenesis as it was shown to affect cell growth, differentiation and apoptosis in several malignant cell lines [131-133]. Additionally, an association between loss-of-function mutations of PPARγ with the development of colorectal cancer in humans was DISCUSSION documented . According to the current findings, an essential role for PPARγ in enhancing SSAT enzyme activity is assumed . In fact, we could show that, in contrast to Caco-2-wild type and Caco-2-empty vector cells, resveratrol failed to increase SSAT activity in Caco-2-dnPPARγ cells.
Here our results point out another important role of PPARγ in resveratrolmediated actions whereas resveratrol-dependent PPARγ activation seem to be mediated at least partly by an activation of the ligand binding domain (LBD/ AF2) because a Gal4-PPARγ chimeric receptor was activated by resveratrol at a concentration of 100 µmol/L, and this concentration was sufficient to induce SSAT as well. In addition, our results suggest that activation of PPARγ by resveratrol is due to kinase activation, leading to phosphorylation-dependent activation of PPARγ coactivators like PGC-1α . Coactivators all interact with a similar surface of the activated ligand binding domain of the receptors and have been suggested to mediate their transcriptional activity . It is well established that, in addition to transcription factors, coactivators can also be targets of multiple signal transduction pathways in response to different stimuli . Puigserver et al.  could show that PGC-1α is activated through p38 MAPK. The mechanism by which p38 activates PGC-1α is not yet clear, but it is suggested that p38 MAPK–mediated phosphorylation counteracts repressor effects, possibly by encouraging the release of a repressor from PGC-1α .
Upon activation, PGC-1α docks on PPARγ and thus can modulate its transcriptional activity . In addition to PGC-1α, resveratrol further leads to an activation of SIRT1, a member of the silent information regulator 2 (Sir2) families of proteins (sirtuins; ref. ). SIRT1 is mainly linked to negative regulation of gene expression as a cofactor through protein deacetylation .
However, there is evidence that SIRT1 can act positively and negatively to control gene expression as a cofactor for PGC-1α. These opposite effects could possibly be due to the recruitment of a different set of coactivators and corepressors through PGC-1α/SIRT1 . This could further be an explanation for the repressive effects of SIRT1 on PPARγ in white fat where PGC-1α is very low .
We were further interested, whether PPARγ plays another crucial role in resveratrol-induced ODC inhibition. But here, in contrast to SSAT induction,
PPARγ activation seems not to play a critical role as no differences could be observed when PPARγ mediated functions are suppressed.
3.1.3 Sphingolipid metabolism Until the late 1970s lipids were primarily thought to serve as inert structural components of cellular membranes, but in recent years it has become more evident that lipids also act as signalling molecules to regulate fundamental cellular responses, such as cell death and differentiation, proliferation and certain types of inflammation [145-147]. One important class of membrane lipids acting as signaling molecules are the sphingolipids, which include ceramides and sphingosine. The generation of ceramide is either triggered by the action of sphingomyelinases, which hydrolyze the plasma membrane component sphingomyelin to yield ceramide and phosphorylcholine, or by de novo synthesis which is initiated by condensation of serine and palmitoyl-CoA catalyzed by SPT . Various biological responses have been attributed to ceramide including cell growth inhibition and induction of apoptosis [149-151].
Furthermore, several chemotherapeutic agents have been shown to act, at least in part, by increasing tumor cell ceramide via de novo synthesis .
Ceramide can be metabolized by glycosylation, acetylation, or by catabolism to sphingosine, which can then be phosphorylated to the anti-apoptotic sphingosine-1-phosphate. Particularly the cellular balance between ceramide and sphingosine-1-phosphate seems to be crucial for a cell’s decision to either undergo apoptosis or proliferate, two events which are implicated in tumor development and growth . Thus, pharmacological manipulation of sphingolipid metabolism to enhance tumor cell ceramide offers a novel approach to cancer chemoprevention and therapy.
Interestingly, we could shown, that the antiproliferative effects of resveratrol closely correlate with a dramatic increase of endogenous ceramide levels.
Similar effects could be observed in a metastatic breast cancer cell model, when ceramide levels increased ~5- and 10-fold after treatment with resveratrol
Figure 7: Ceramide metabolism Major synthetic and metabolic pathways for ceramide. Ceramide can be produced via a de novo biosynthetic pathway which is initiated by condensation of serine and palmitoyl-CoA catalyzed by serine palmitoyltransferase as well as by sphingomyelinase-mediated hydrolysis of sphingomyelin (Adapted from ).
32 and 64 µmol/L, respectively, in comparison with untreated cells . To further examine the cellular activities of ceramide, we worked with the exogenous cell-permeant ceramide-analogs N-acetylsphingosine (C2Ceramide) and N-hexanoylsphingosine (C6-Ceramide). And actually, treatment with C2- or C6-ceramide caused distinct growth inhibition in our colorectal cancer cell model which seems to be the mechanistic explanation for observed growth inhibitory effects of resveratrol.
Nearly 70% of human cancers are associated with the activation of protooncogene c-myc , a transcription factor that directly regulates the expression of ODC by binding to specific CAGGTG sequence in the gene promoter  Based on our earlier findings that resveratrol regulates the expression of both c-myc and ODC genes , together with the results from Flamigni et al.  who demonstrated a reduction of c-myc and ODC expression in leukemia cells after ceramide-treatment, we tried to identify a possible involvement of ceramide synthesis in the regulatory pathway in colorectal cancer cells. We measured c-myc as well as ODC expression and DISCUSSION activity after treatment with C6-ceramide and with resveratrol in combination with the specific serine palmitoyltransferase inhibitors L-cycloserine and myriocin. While C6-ceramide led to an obvious decrease of both c-myc and ODC protein levels, L-cycloserine and myriocin but not sphingomyelinaseinhibitor manumycin conspicuously counteracted the inhibitory effects of resveratrol. These data suggest that the induction of ceramide de novo biosynthesis but not hydrolysis of sphingomyelin is involved in resveratrolmediated inhibition of ODC.
3.1.4 Summary and conclusion
In summary, our data confirm our earlier studies showing that resveratrolmediated growth inhibition of colorectal cancer cells seems to involve modulation of polyamine metabolism. Here we further show that transcription factor PPARγ acts as a p38-dependent target in resveratrol-induced activation of polyamine catabolism (MANUSCRIPT I). On the other hand we could demonstrate that the induction of de novo ceramide biosynthesis plays a crucial role in the inhibition of polyamine biosynthesis (MANUSCRIPT II) (For summary see Figure 8). Besides a decrease in cell proliferation, the observed reduction of cell growth is probably due to an induction of apoptosis, as Wolter et al.  showed an obvious increase of caspase-3-activity in resveratrol-treated cells.
Recent studies further indicate that the activation of catabolic SSAT is related to an induction of programmed cell death . These aspects of resveratrolaction require further investigations.
The identification of increased polyamine concentrations in a variety of cancer tissues has led to the design and development of inhibitors of polyamine metabolism as a new strategy for therapeutic or preventative interventions. The best-known inhibitor of polyamine biosynthesis is α-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (See introduction). The failure of ODC monotherapy in vivo may at least partly also be due to inhibitory effects on catabolic SSAT activity, as DFMO effectively prevented SSAT
Figure 8: Possible Mechanisms of Resveratrol action Further details are described in text induced growth inhibition in a prostate cancer model while typically inhibiting cell growth rather than preventing cell growth inhibition . Although much emphasis in the past has been on the biosynthetic pathway of polyamine metabolism, considerable interest has recently been generated with regard to the catabolic pathways, maintaining a properly balanced ratio of polyamines in cells. This suggestion may well explain the increased efficacy of combined chemopreventive therapy with non-steroidal anti-inflammatory drugs (NSAIDS) in animal models, as these agents recently have been shown, among others, to induce SSAT gene expression . In this context resveratrol could show great potential in the chemoprevention and therapy of colorectal cancers, by simultaneously leading to SSAT activation as well as ODC inhibition.
Ursolic acid is a pentacyclic triterpenoid, with anti-cancer and anti-inflammatory properties. Attempts to show favourable effects in vitro have led to the identification of multiple direct targets for this compound. Inhibition of cell cycle progression in the G1 phase for example was known to be associated with a marked decrease in the protein expression of cyclins and their activating partners the cyclin-dependent kinases and with a concomintant induction of cell cycle inhibitors in miscellaneous cancer cell lines [83;84]. Unfortunately, little research is done in colon cancer cells thereby mainly focusing apoptotic mechanisms.
3.2.1 Cell cycle regulation
Each phase of the cell cycle is governed by a wide spectrum of protein families.
Many of these proteins are synthesized and activated in a precise phase of the cell cycle. In the past decade, the critical role that cell cycle regulation plays in cancer development has been clearly established [159-161]. Indeed, the list of cell cycle perturbations involved in tumor progression has dramatically increased in the last years [159;162] and unscheduled cell proliferation has become one of the chief causes described for malignancy and carcinogenesis .