Sandra Ulrich

Peroxisome Proliferator–Activated Receptor γ as a Molecular Target of Resveratrol-Induced Modulation of Polyamine Metabolism

Previous results indicate that the polyphenol resveratrol inhibits cell growth of colon carcinoma cells via modulation of polyamine metabolic key enzymes. The aim of this work was to specify the underlying molecular mechanisms and to identify a possible role of transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma). Cell growth was determined by bromodeoxyuridine incorporation and crystal violet staining. Protein levels were examined by Western blot analysis. Spermine/spermidine acetyltransferase (SSAT) activity was determined by a radiochemical assay. PPARgamma ligand-dependent transcriptional activity was measured by a luciferase assay. A dominant-negative PPARgamma mutant was transfected in Caco-2 cells to suppress PPARgamma-mediated functions. Resveratrol inhibits cell growth of both Caco-2 and HCT-116 cells in a dose- and time-dependent manner (P < 0.001). In contrast to Caco-2-wild type cells (P < 0.05), resveratrol failed to increase SSAT activity in dominant-negative PPARgamma cells. PPARgamma involvement was further confirmed via ligand-dependent activation (P < 0.01) as well as by induction of cytokeratin 20 (P < 0.001) after resveratrol treatment. Coincubation with SB203580 abolished SSAT activation significantly in Caco-2 (P < 0.05) and HCT-116 (P < 0.01) cells. The involvement of p38 mitogen-activated protein kinase (MAPK) was further confirmed by a resveratrol-mediated phosphorylation of p38 protein in both cell lines. Resveratrol further increased the expression of PPARgamma coactivator PGC-1alpha (P < 0.05) as well as SIRT1 (P < 0.01) in a dose-dependent manner after 24 hours of incubation. Based on our findings, p38 MAPK and transcription factor PPARgamma can be considered as molecular targets of resveratrol in the regulation of cell proliferation and SSAT activity, respectively, in a cell culture model of colon cancer.

PPARγ is a key target of butyrate-induced caspase-3 activation in the colorectal cancer cell line Caco-2

Background: Butyrate, a potent histone deacetylase inhibitor, belongs to a promising new class of antineoplastic agents with the capacity to induce apoptosis of cancer cells. However, the underlying mechanisms of action have yet not been elucidated. Aim: To further investigate the molecular events involved in butyrate-induced caspase-3 activation in Caco-2 wild-type, empty-vector and dominant-negative PPARγ mutant cells along the signalling pathway. In this context, the involvement and up-regulation of PPARγ was examined. Results: Stimulation of cells with butyrate resulted in increased expression of PPARγ mRNA, protein, and activity as well as phospho-p38 MAPK protein expression and caspase-3 activity. Arsenite, a direct stimulator of p38 MAPK, also led to an increased PPARγ expression, thereby mimicking the effects of butyrate. In contrast, butyrate-mediated up-regulation of PPARγ was counteracted by co-incubation with the p38 MAPK inhibitor SB203580. Treatment of cells with butyrate resulted in both increased caspase-8 and -9 activity and reduced expression of XIAP and survivin. However, butyrate-mediated effects on these apoptosis-regulatory proteins leading to caspase-3 activation were almost completely abolished in Caco-2 dominant-negative PPARγ mutant cells. Conclusions: Our data clearly unveil PPARγ as a key target in the butyrate-induced signalling cascade leading to apoptosis via caspase-3 in Caco-2 cells.

Phytochemicals Resveratrol and Sulforaphane as Potential Agents for Enhancing the Anti-Tumor Activities of Conventional Cancer Therapies

Even though conventional cancer therapies, comprising surgery and chemo- and radiotherapy, play an important role in the treatment of most solid tumours, successful therapeutic outcome is often limited due to high toxicity and related side-effects, as well as the development of multi-drug resistances. Therefore, there is need for new therapeutic strategies not only to obtain higher treatment efficacy, but also for the reduction of toxicity and adverse effects. Emerging evidence suggests that natural compounds with distinct anticarcinogenic activity may be considered as potential agents for enhancing the therapeutic effects of common cancer treatments. By using the examples of resveratrol and sulforaphane this review will summarize the findings of recent investigations focusing this topic so far and the current knowledge of the molecular mechanisms by which these selected phytochemicals may potentiate the anti-tumor effects of different cancer therapies.

Molecular characterization of EP6—A novel imidazo[1,2-a]pyridine based direct 5-lipoxygenase inhibitor

5-Lipoxygenase (5-LO) is a crucial enzyme of the arachidonic acid (AA) cascade and catalyzes the formation of bioactive leukotrienes (LTs) which are involved in inflammatory diseases and allergic reactions. The pathophysiological effects of LTs are considered to be prevented by 5-LO inhibitors. In this study we present cyclohexyl-[6-methyl-2-(4-morpholin-4-yl-phenyl)-imidazo[1,2-a]pyridin-3-yl]-amine (EP6), a novel imidazo[1,2-a]pyridine based compound and its characterization in several in vitro assays. EP6 suppresses 5-LO activity in intact polymorphonuclear leukocytes with an IC(50) value of 0.16μM and exhibits full inhibitory potency in cell free assays (IC(50) value of 0.05μM for purified 5-LO). The efficacy of EP6 was not affected by the redox tone or the concentration of exogenous AA, characteristic drawbacks known for the class of nonredox-type 5-LO inhibitors. Furthermore, EP6 suppressed 5-LO activity independently of the cell stimulus or the activation pathway of 5-LO contrary to what is known for some nonredox-type inhibitors. Using molecular modeling and site-directed mutagenesis studies, we were able to derive a feasible binding region within the C2-like domain of 5-LO that can serve as a new starting point for optimization and development of new 5-LO inhibitors targeting this site. EP6 has promising effects on cell viability of tumor cells without mutagenic activity. Hence the drug may possess potential for intervention with inflammatory and allergic diseases and certain types of cancer including leukemia.

Isothiocyanate sulforaphane inhibits protooncogenic ornithine decarboxylase activity in colorectal cancer cells via induction of the TGF-β/Smad signaling pathway.

SCOPE The objective of this study was to elucidate molecular mechanisms behind the antitumor activities of the isothiocyanate sulforaphane (SFN) in colorectal cancer cells. METHODS AND RESULTS Cell growth was determined by BrdU incorporation and crystal violet staining. Protein levels were examined by Western blot analysis. Ornithine decarboxylase (ODC) activity was assayed radiometrically. Reverse transcriptase-PCR was used for measuring mRNA expression. For reporter gene assays plasmids were transfected into cells via lipofection and luciferase activity was measured luminometrically. Acetyl-histone H3 and H4 chromatin immunoprecipitation (ChIP) assays were performed followed by PCR with TGF-β-receptor II promoter specific primers. We could show that SFN-mediated cell growth inhibition closely correlates with a dose-dependent reduction of protein expression and enzymatic activity of ODC. This effect seems to be due to reduced protein levels and transactivation activity of transcription factor c-myc, a direct regulator of ODC expression, as a consequence of SFN-induced TGF-β/Smad signaling. The coherency of these results was further confirmed by using TGF-β receptor kinase inhibitor SB431542, which largely abolishes inhibitory effects of SFN on both, ODC activity and cell growth. CONCLUSION Since elevated ODC enzyme activity is associated with enhanced tumor development, SFN may be a dietary phytochemical with potential to prevent carcinogenesis.

696 Resveratrol Sensitizes Colorectal Cancer Cells to Oxaliplatin-Induced Cell Death via Intracellular Polyamine Depletion

Background: Microtubule-targeting agents (MTAs) are cancer drugs that act primarily by altering microtubule dynamics and interfering with mitosis. While MTAs are in widespread clinical use, innate and acquired resistance is an ongoing problem. Resistance is thought to stem primarily from drug efflux and expression of alternate tubulin isoforms with MTAresistant microtubule dynamics. We found that the compound T0070907 reduced tumor growth in a murine xenograft model of colon cancer. In human colorectal cancer (CRC) cell lines, T0070907 caused near-total loss of the α and β tubulin proteins, leading to collapse of visible microtubule filaments and eventual cell death. Dramatic tubulin loss is a novel phenotype not reported for any of the standard MTAs. This observation suggests the possible development of MTAs that work by a mechanism less susceptible to escape via expression of alternate tubulin isoforms. As a first step toward determining the molecular mechanisms of tubulin loss, we determined whether T0070907 affects degradation and activity of the microtubule associated proteins that control microtubule polymerization. Methods: The effects of T0070907 on tubulin levels and phosphorylation of the microtubuleassociated protein stathmin/Op18 and its upstream kinase MARK kinase were examined using Western blot in the CRC cell lines HT-29, SW620, and DLD-1. Tubulin protein halflives were measured after addition of the protein synthesis inhibitor cycloheximide. Results: T0070907 caused loss of α and β, but not γ, δ, or e tubulin. The magnitude of the tubulin reduction was much greater than that found when microtubule destabilization leads to tubulin synthesis inhibition. Tubulin half lives decreased from >12h to 6h, suggesting increased degradation. Consistent with this idea, treatments that sequestered tubulin in polymerized microtubules (paclitaxel), leaving the tubulin unavailable for degradation, reduced T0070907-induced tubulin loss. Conversely, the microtubule-destabilizing agents nocodazole and vinblastine increased the rate of tubulin loss. T0070907 did not strongly affect microtubule polymerization In Vitro, suggesting that it does not directly bind to either microtubules or tubulin. However, T0070907 led to increases in MARK kinase activation as well as phosphorylation and inactivation of its downstream target, the microtubulesequestering protein stathmin/Op18. Conclusion: T0070907 promotes tubulin degradation and stathmin inactivation. As knockdown of stathmin in Drosophila leads to a drastic reduction in tubulin levels, we propose that T0070907 reduces tubulin in part by targeting stathmin.

695 Sulforaphane Sensitizes Colorectal Cancer Cells to Oxaliplatin Induced Cell Growth Inhibition: Key Role of p38 MAPK

Background: Microtubule-targeting agents (MTAs) are cancer drugs that act primarily by altering microtubule dynamics and interfering with mitosis. While MTAs are in widespread clinical use, innate and acquired resistance is an ongoing problem. Resistance is thought to stem primarily from drug efflux and expression of alternate tubulin isoforms with MTAresistant microtubule dynamics. We found that the compound T0070907 reduced tumor growth in a murine xenograft model of colon cancer. In human colorectal cancer (CRC) cell lines, T0070907 caused near-total loss of the α and β tubulin proteins, leading to collapse of visible microtubule filaments and eventual cell death. Dramatic tubulin loss is a novel phenotype not reported for any of the standard MTAs. This observation suggests the possible development of MTAs that work by a mechanism less susceptible to escape via expression of alternate tubulin isoforms. As a first step toward determining the molecular mechanisms of tubulin loss, we determined whether T0070907 affects degradation and activity of the microtubule associated proteins that control microtubule polymerization. Methods: The effects of T0070907 on tubulin levels and phosphorylation of the microtubuleassociated protein stathmin/Op18 and its upstream kinase MARK kinase were examined using Western blot in the CRC cell lines HT-29, SW620, and DLD-1. Tubulin protein halflives were measured after addition of the protein synthesis inhibitor cycloheximide. Results: T0070907 caused loss of α and β, but not γ, δ, or e tubulin. The magnitude of the tubulin reduction was much greater than that found when microtubule destabilization leads to tubulin synthesis inhibition. Tubulin half lives decreased from >12h to 6h, suggesting increased degradation. Consistent with this idea, treatments that sequestered tubulin in polymerized microtubules (paclitaxel), leaving the tubulin unavailable for degradation, reduced T0070907-induced tubulin loss. Conversely, the microtubule-destabilizing agents nocodazole and vinblastine increased the rate of tubulin loss. T0070907 did not strongly affect microtubule polymerization In Vitro, suggesting that it does not directly bind to either microtubules or tubulin. However, T0070907 led to increases in MARK kinase activation as well as phosphorylation and inactivation of its downstream target, the microtubulesequestering protein stathmin/Op18. Conclusion: T0070907 promotes tubulin degradation and stathmin inactivation. As knockdown of stathmin in Drosophila leads to a drastic reduction in tubulin levels, we propose that T0070907 reduces tubulin in part by targeting stathmin.