Phytonutriments and chemoprevention

Die Chemoprävention von Krebs ist die Verwendung von Arznei- oder anderen Mitteln um das Krebsrisiko zu vermindern. Da Krebs weltweit zu einem immer bedeutenderen Problem wird, bemüht man sich, neue chemopräventive Substanzen zu finden. Oligomere Procyanidine (OPC, Trimere bis zu Decamere) sind eine Unterklasse der Polyphenole, deren antimikrobielle, antivirale, antidiabetische und kardioprotektive Wirkung schon gezeigt werden konnte. Diese Arbeit beschäftigt sich mit der Fähigkeit der OPCs aus spezifischen Quellen in kolorektalen Krebszelllinien Apoptose zu induzieren. OPCs wurden aus den Pressrückständen von Äpfeln und Quitten mit einer Mischung aus Azeton und demineralisiertem Wasser (60:40) extrahiert. Nach flüssig-flüssig Extraktionen mit Cyclohexan und Ethylazetat wurden die Extrakte mittels einer Sephadex LH-20 Säule mit einem Methanol/Wasser Gradienten fraktioniert. Die Fraktionen wurden mit RP-HPLC, gekoppelt mit einem UV-VIS Detektor, analysiert. Fraktionen die zum Großteil OPCs enthielten wurden zu einem OPC angereicherten Extrakt vereint. Nach der Gehaltsbestimmung (Vanillin und Folin-Assay), wurde der Extrakt an den kolorektalen Krebszelllinien SW480 und SW 620 getestet um das Ausmaß der Apoptose-Induktion festzustellen. Der OPC angereicherte Quitten-Extrakt zeige hohe Aktivität in beiden Zelllinien (71% und 67% apoptotische Zellen im Gegensatz zu 11% bzw 9% Apoptoserate bei unbehandelten Zellen). Der Effekt des OPC angereicherten Apfel-Extraktes auf die Zelllinie SW480 war relativ gering (35%), für die Zelllinie SW620 jedoch höher (58%). Zusätzlich zu den Zelltests wurde versucht Monomere und Oligomere mittels Dünnschichtchromatographie zu isolieren. Isolierte Substanzen könnten an Zellkulturen getestet werden um ihre Effekte und mögliche Synergien aufzuzeigen. Die DC-Trennung ergab keine zufriedenstellenden Ergebnisse. Zusammenfassend lässt sich sagen, dass OPCs erhebliche Effekte auf Krebszellen haben und möglicherweise in der Prävention oder der Behandlung von gastrointestinalen Tumoren von Nutzen sein können.Cancer chemoprevention is the use of pharmaceuticals or other agents to reduce cancer risk. As cancer is worldwide becoming a more and more serious problem a lot of effort is taken to find new chemopreventive substances. Oligomeric procyanidins (OPCs, trimers to decamers) are a subclass of polyphenols that have already proven to have antimicrobial, antiviral, antidiabetic and cardioprotective properties. This work concentrates on the ability of OPCs from specific sources to induce apoptosis in colorectal cancer cell lines. OPCs were extracted from apple and quince pomace with a mixture of acetone and demineralised water (60:40). After liquid-liquid extractions with cyclohexane and ethyl acetate, the extracts were fractionated on Sephadex LH-20 columns with a methanol/water elution gradient. The fractions were analysed by reversed phase HPLC coupled with an UV-VIS detector. Fractions containing major amounts of OPCs were united to the enriched OPC extract. After quantification (vanillin and Folin assay) the extract was tested on colorectal cancer cell lines SW480 and SW620 to assess the induction of apoptosis. The OPC enriched quince extract showed high activity in both cell lines (71% and 67% of apoptotic cells in contrast to 11%/9% apoptosis rate of untreated cells). The effect of the OPC enriched apple extract on the cell line SW480 was relatively low (35%) but higher for the cell line SW620 (58%). Additionally to the tests on cell lines it was tried to separate monomers to oligomers from the extracts by thin layer chromatography. Isolated substances could be tested on cell cultures for effects and possible synergies. No satisfying results could be obtained by TLC-separation. As a conclusion, OPCs have relevant effects on cancer cells and are likely to be of aid in the prevention or the treatment of tumours of the gastrointestinal tract

"What's (the) Matter?", A Show on Elementary Particle Physics with 28 Demonstration Experiments

We present the screenplay of a physics show on particle physics, by the Physikshow of Bonn University. The show is addressed at non-physicists aged 14+ and communicates basic concepts of elementary particle physics including the discovery of the Higgs boson in an entertaining fashion. It is also demonstrates a successful outreach activity heavily relying on the university physics students. This paper is addressed at anybody interested in particle physics and/or show physics. This paper is also addressed at fellow physicists working in outreach, maybe the experiments and our choice of simple explanations will be helpful. Furthermore, we are very interested in related activities elsewhere, in particular also demonstration experiments relevant to particle physics, as often little of this work is published. Our show involves 28 live demonstration experiments. These are presented in an extensive appendix, including photos and technical details. The show is set up as a quest, where 2 students from Bonn with the aid of a caretaker travel back in time to understand the fundamental nature of matter. They visit Rutherford and Geiger in Manchester around 1911, who recount their famous experiment on the nucleus and show how particle detectors work. They travel forward in time to meet Lawrence at Berkeley around 1950, teaching them about the how and why of accelerators. Next, they visit Wu at DESY, Hamburg, around 1980, who explains the strong force. They end up in the LHC tunnel at CERN, Geneva, Switzerland in 2012. Two experimentalists tell them about colliders and our heroes watch live as the Higgs boson is produced and decays. The show was presented in English at Oxford University and University College London, as well as Padua University and ICTP Trieste. It was 1st performed in German at the Deutsche Museum, Bonn (5/'14). The show has eleven speaking parts and involves in total 20 people.Comment: 113 pages, 88 figures. An up to date version of the paper with high resolution pictures can be found at http://www.th.physik.uni-bonn.de/People/dreiner/Downloads/. In v2 the acknowledgements and a citation are correcte

TERT promoter mutation and chromosome 6 loss define a high-risk subtype of ependymoma evolving from posterior fossa subependymoma

Subependymomas are benign tumors characteristically encountered in the posterior fossa of adults that show distinct epigenetic profiles assigned to the molecular group "subependymoma, posterior fossa" (PFSE) of the recently established DNA methylation-based classification of central nervous system tumors. In contrast, most posterior fossa ependymomas exhibit a more aggressive biological behavior and are allocated to the molecular subgroups PFA or PFB. A subset of ependymomas shows epigenetic similarities with subependymomas, but the precise biology of these tumors and their potential relationships remain unknown. We therefore set out to characterize epigenetic traits, mutational profiles, and clinical outcomes of 50 posterior fossa ependymal tumors of the PFSE group. On histo-morphology, these tumors comprised 12 ependymomas, 14 subependymomas and 24 tumors with mixed ependymoma-subependymoma morphology. Mixed ependymoma-subependymoma tumors varied in their extent of ependymoma differentiation (2-95%) but consistently exhibited global epigenetic profiles of the PFSE group. Selective methylome analysis of microdissected tumor components revealed CpG signatures in mixed tumors that coalesce with their pure counterparts. Loss of chr6 (20/50 cases), as well as TERT mutations (21/50 cases), were frequent events enriched in tumors with pure ependymoma morphology (p < 0.001) and confined to areas with ependymoma differentiation in mixed tumors. Clinically, pure ependymoma phenotype, chr6 loss, and TERT mutations were associated with shorter progression-free survival (each p < 0.001). In conclusion, our results suggest that subependymomas may acquire genetic and epigenetic changes throughout tumor evolution giving rise to subclones with ependymoma morphology (resulting in mixed tumors) that eventually overpopulate the subependymoma component (pure PFSE ependymomas)

Bioactivity profiling of physiological resveratrol metabolites

Resveratrol wird nach oraler Aufnahme in den menschlichen Körper fast vollständig metabolisiert. Diese Dissertation untersuchte die Bioaktivität der gebildeten Metabolite Resveratrol-3-sulfat, Resveratrol-disulfate, Resveratrol-3-glukuronid und Resveratrol-4’-glukuronid. Zellkultursysteme für Transporter-abhängige Aufnahme, Krebszellwachstum und Topoisomerase II Hemmung, Arteriosklerose, Immunabwehr und Entzündung wurden etabliert. Resveratrol-3-sulfat wurde nur über den Transporter OATP1B3 aufgenommen und Resveratrol-3,4`-disulfat über OATP1B1 und OATP1B3 (12.4 - 400 µM). Glukuronide zeigten keinen OATP-abhängigen Transport. Zusätzliche Zytotoxizitätstests in der Brustkrebs-Zelllinie ZR-75-1 mit dem Disulfat (10 - 400 µM) zeigten eine Reduktion der lebenden Zellen auf ca. 60%, während vergleichbare Konzentrationen von Resveratrol die lebenden Zellen auf unter 20% reduzierten. In Darmkrebszellen zeigten das 3-Sulfat, Disulfat und das 3-Glukuronid eine Topoisomerase II-Hemmung zwischen 100 und 200 µM, während an 4‘-Position konjugierte Derivate keinen Effekt hatten. Die geringe Zytotoxizität des 3-Sulfates in den Darmkrebszellen CaCo2 und HT29 spiegelte sich auch in dessen, verglichen mit Resveratrol, viermal geringerer Aufnahme in die Zellen wieder. In einem zellbasierten Versuch zu Immunmodulation und Entzündung in aktivierten Makrophagen konnten speziell die sulfatierten Konjugate eine Verminderung von Chemokinen und Zytokinen (IL-8, MIP-1β, MCP-1, IL-6, TNF-α) bei 1 µM und eine Hochregulierung von Sirt1 mRNA durch glukuronierte Substanzen bei 1 µM zeigen. Metabolite zeigten im Gegensatz zu Resveratrol (≥30 µM) keine Bioaktivität im Bezug auf enotheliale Stickstoffmonoxid Synthase, Stickstoffmonoxid (NO) Freisetzung oder intrazellulären reaktiven Sauerstoffspezies (ROS). Nach oraler Aufnahme einer hohen (5 g) und einer niedrigen (50 mg) Dosis Resveratrol konnte kein Einfluss auf die antioxidative Kapazität und ROS in humanem Plasma festgestellt werden, aber die Enzymaktivität von SOD und CAT in Erythrozyten wurde nach Aufnahme der höheren Dosis verringert, im Vergleich zur niedrigeren Dosis. Zusammenfassend kann festgestellt werden, dass physiologisch gebildete Metabolite bioaktiv sind und zu den positiven Effekten von Resveratrol auf die Gesundheit beitragen könnten.Resveratrol is extensively metabolized in the human body after oral ingestion. This thesis aimed at the bioactivity profiling of the most abundant physiological sulfated and glucuronated metabolites Resveratrol-3-sulfate, Resveratrol-disulfates, Resveratrol-3-glucuronide and Resveratrol-4’-glucuronide. For this purpose, in vitro cell culture models were established for uptake, cancer cell growth and topoisomerase II inhibition, atherosclerosis, immunity and inflammation. Uptake experiments (12.4 - 400 µM) showed that Resveratrol-3-sulfate was exclusively transported by OATP1B3 and Resveratrol-3,4’-disulfate was transported into cells by OATP1B1 and OATP1B3. Glucuronides showed no OATP-dependent transport. Additional cytotoxicity tests in the breast cancer cell line ZR-75-1 showed only a maximum reduction of viable cells to about 60% after incubation with the disulfate (10 - 400 µM) whereas comparable concentrations of Resveratrol decreased viable cells to less than 20%. In colon cancer cells, Resveratrol, Resveratrol-3-sulfate, Resveratrol-disulfates and Resveratrol-3-glucuronide showed inhibition of Topoisomerase II at concentrations between 100 and 200 µM, whereas the 4’ conjugated compounds did not. The minor cytotoxic effect of Resveratrol-3-sulfate in colon cancer cell lines CaCo2 and HT29 was in accordance with its four times lower uptake into cells as compared to Resveratrol. In cell based assays of immune-modulation and inflammation in activated macrophages, especially sulfated conjugates (Resveratrol-3-sulfate, Resveratrol-disulfates) showed alleviating effects on chemokine and cytokine release at 1 µM (IL-8, MIP-1β, MCP-1, IL-6, TNF-α) and glucuronated compounds up-regulated Sirt1 mRNA at 1 µM. Resveratrol metabolites showed no bioactivity regarding endothelial nitric oxide synthase, NO release or intracellular ROS, whereas the parent compound did (≥ 30 µM). After oral intake of high (5 g) and low (50 mg) single dose Resveratrol, no effect on plasma antioxidant capacity and plasma ROS could be determined in humans, but enzyme activity of detoxifying SOD and CAT in erythrocytes was blunted after the higher dose as compared to the lower dose. In conclusion, physiologically formed metabolites exert bioactivity and could contribute to the health effects of the parent compound in vivo

The <em>glmS</em> ribozyme is an antibacterial target : Mode of action analysis, investigation of potential metabolite analogs and characterization of <em>glmS</em> ribozyme variants of human pathogens

Bacteria increasingly develop resistances to the known antibiotics, this threatens human health. Thus, novel antibiotics are urgently needed. The currently used antibiotics mainly target five bacterial processes and are therefore limited in the target structures they address. This problem calls for the development of antibiotics that address new targets. Riboswitches are RNA elements mostly found in the non-coding region of bacterial mRNA molecules. Several classes of riboswitches regulate the expression of essential bacterial genes by the recognition of their cognate metabolites. Compounds that exhibit at least part of their activity through the action as metabolite analogs of riboswitches corroborate the suitability of riboswitches as antibacterial targets. The glmS riboswitch constitutes a unique class of riboswitches that regulate the expression of the essential glmS gene by a self-cleavage mechanism, resulting in the degradation of the glmS mRNA. This mechanism is induced by the metabolite glucosamine 6-phosphate (GlcN6P) and magnesium ions, consequently, the glmS riboswitch is also a co-factor dependent ribozyme. The thesis at hand deals with the analysis of different aspects regarding glmS ribozymes from different bacteria. The investigation of potential modulators of glmS ribozyme activity is displayed in section 3.1 and revealed insights into the interaction of the glmS ribozyme RNA with putative metabolite analogs. This section analyses the activity of compounds that originate from an in silico prediction based on the 3D shape of the ribozyme’s natural metabolite, GlcN6P. Section 3.2 studies the potential of the glmS ribozyme as antibacterial target. The investigation of the intracellular mode of action of the metabolite analog carba-glucosamine (CGlcN) is displayed. It is demonstrated that the antibacterial activity of CGlcN is based on the in vivo activation of the glmS ribozyme, presumably by carba-glucosamine 6-phosphate (CGlcN6P). Apart from the analysis of potential glmS ribozyme activators, different glmS ribozyme variants and their properties were studied. The analysis and characterization of putative novel glmS ribozyme sequences and the differential recognition of already known glmS ribozyme activators by those validated glmS ribozymes is displayed in section 3.3. In a similar manner as described for the in silico predicted GlcN6P analogs (section 3.1) putative glmS ribozyme activators, originating from a focused library based on the scaffold of an already described activator, are analyzed regarding their potential activation of glmS ribozyme variants (section 3.3) in section 3.4. The mode of action of the in section 3.4 identfied novel glmS ribozyme activator was subsequently analyzed utilizing the same approach as described in section 3.2. This study underlines the potential of riboswitches as antibacterial targets and furthermore represents the first proven study on ribozymes as antibacterial targets