TY - JOUR A1 - Wedel, Steffen A1 - Hudak, Lukasz A1 - Seibel, Jens-Michael A1 - Makarevic, Jasmina A1 - Juengel, Eva A1 - Tsaur, Igor A1 - Waaga-Gasser, Ana A1 - Haferkamp, Axel A1 - Blaheta, Roman A. T1 - Molecular targeting of prostate cancer cells by a triple drug combination down-regulates integrin driven adhesion processes, delays cell cycle progression and interferes with the cdk-cyclin axis JF - BMC Cancer N2 - Background: Single drug use has not achieved satisfactory results in the treatment of prostate cancer, despite application of increasingly widespread targeted therapeutics. In the present study, the combined impact of the mammalian target of rapamycin (mTOR)-inhibitor RAD001, the dual EGFr and VGEFr tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on prostate cancer growth and adhesion in vitro was investigated. Methods: PC-3, DU-145 and LNCaP cells were treated with RAD001, AEE788 or VPA or with a RAD-AEE-VPA combination. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT-assay, flow cytometry and western blotting, respectively. Furthermore, tumor cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins as well as migratory properties of the cells was evaluated, and integrin alpha and beta subtypes were analyzed. Finally, effects of drug treatment on cell signaling pathways were determined. Results: All drugs, separately applied, reduced tumor cell adhesion, migration and growth. A much stronger anticancer effect was evoked by the triple drug combination. Particularly, cdk1, 2 and 4 and cyclin B were reduced, whereas p27 was elevated. In addition, simultaneous application of RAD001, AEE788 and VPA altered the membranous, cytoplasmic and gene expression pattern of various integrin alpha and beta subtypes, reduced integrin-linked kinase (ILK) and deactivated focal adhesion kinase (FAK). Signaling analysis revealed that EGFr and the downstream target Akt, as well as p70S6k was distinctly modified in the presence of the drug combination. Conclusions: Simultaneous targeting of several key proteins in prostate cancer cells provides an advantage over targeting a single pathway. Since strong anti-tumor properties became evident with respect to cell growth and adhesion dynamics, the triple drug combination might provide progress in the treatment of advanced prostate cancer. KW - Growth-factor receptor KW - Mammalian target KW - Radical prostatectomy KW - Up-regulation KW - C-MYC KW - Pathway KW - Expression KW - Activation KW - Inhibition KW - Apoptosis Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-141075 VL - 11 IS - 375 ER - TY - JOUR A1 - Seher, Axel A1 - Nickel, Joachim A1 - Mueller, Thomas D. A1 - Kneitz, Susanne A1 - Gebhardt, Susanne A1 - Meyer ter Vehn, Tobias A1 - Schlunck, Guenther A1 - Sebald, Walter T1 - Gene expression profiling of connective tissue growth factor (CTGF) stimulated primary human tenon fibroblasts reveals an inflammatory and wound healing response in vitro JF - Molecular Vision N2 - Purpose: The biologic relevance of human connective tissue growth factor (hCTGF) for primary human tenon fibroblasts (HTFs) was investigated by RNA expression profiling using affymetrix (TM) oligonucleotide array technology to identify genes that are regulated by hCTGF. Methods: Recombinant hCTGF was expressed in HEK293T cells and purified by affinity and gel chromatography. Specificity and biologic activity of hCTGF was confirmed by biosensor interaction analysis and proliferation assays. For RNA expression profiling HTFs were stimulated with hCTGF for 48h and analyzed using affymetrix (TM) oligonucleotide array technology. Results were validated by real time RT-PCR. Results: hCTGF induces various groups of genes responsible for a wound healing and inflammatory response in HTFs. A new subset of CTGF inducible inflammatory genes was discovered (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1], chemokine [C-X-C motif] ligand 6 [CXCL6], interleukin 6 [IL6], and interleukin 8 [IL8]). We also identified genes that can transmit the known biologic functions initiated by CTGF such as proliferation and extracellular matrix remodelling. Of special interest is a group of genes, e.g., osteoglycin (OGN) and osteomodulin (OMD), which are known to play a key role in osteoblast biology. Conclusions: This study specifies the important role of hCTGF for primary tenon fibroblast function. The RNA expression profile yields new insights into the relevance of hCTGF in influencing biologic processes like wound healing, inflammation, proliferation, and extracellular matrix remodelling in vitro via transcriptional regulation of specific genes. The results suggest that CTGF potentially acts as a modulating factor in inflammatory and wound healing response in fibroblasts of the human eye. KW - Bone morphogenetic protein-2 KW - Smooth-muscle-cells KW - Myofibroblast differentiation KW - TGF-beta KW - CYR61 KW - Proliferation KW - Mechanisms KW - Apoptosis KW - Receptor KW - Cancer Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-140189 VL - 17 IS - 08. Okt ER - TY - THES A1 - Polzien, Lisa T1 - BAD Phosphorylation: A Novel Link between Apoptosis and Cancer T1 - BAD Phosphorylierung: Eine Neue Verbindung zwischen Apoptose und Krebs N2 - BAD (Bcl-2 antagonist of cell death, Bcl-2 associated death promoter) is a pro-apoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD (mBAD), little data are available with respect to phosphorylation of human BAD (hBAD) protein. In this work, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serines 75, 99 and 118 of hBAD (Chapter 3.1). Our results indicate that RAF kinases phosphorylate hBAD in vivo at these established serine residues. RAF-induced phosphorylation of hBAD was not prevented by MEK inhibitors but could be reduced to control levels by use of the RAF inhibitor Sorafenib (BAY 43-9006). Consistently, expression of active RAF suppressed apoptosis induced by hBAD and the inhibition of colony formation caused by hBAD could be prevented by RAF. In addition, using surface plasmon resonance technique we analyzed the direct consequences of hBAD phosphorylation by RAF with respect to complex formation of BAD with 14-3-3 proteins and Bcl-XL. Phosphorylation of hBAD by active RAF promotes 14-3-3 protein association, whereby the phosphoserine 99 represents the major binding site. Furthermore, we demonstrate in this work that hBAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity is dependent on phosphorylation status and interaction with 14-3-3 proteins. Additionally, we show that hBAD pores possess a funnel-shaped geometry that can be entered by ions and non-charged molecules up to 200 Da (Chapter 3.2). Since both lipid binding domains of hBAD (LBD1 and LBD2) are located within the C-terminal region, we investigated this part of the protein with respect to its structural properties (Chapter 3.3). Our results demonstrate that the C-terminus of hBAD possesses an ordered β-sheet structure in aqueous solution that adopts helical disposition upon interaction with lipid membranes. Additionally, we show that the interaction of the C-terminal segment of hBAD with the BH3 domain results in the formation of permanently open pores, whereby the phosphorylation of serine 118 proved to be necessary for effective pore-formation. In contrast, phosphorylation of serine 99 in combination with 14-3-3 association suppresses formation of channels. These results indicate that the C-terminal part of hBAD controls hBAD function by structural transitions, lipid binding and phosphorylation. Using mass spectrometry we identified in this work, besides the established in vivo phosphorylation sites at serines 75, 99 and 118, several novel hBAD phosphorylation sites (serines 25, 32/34, 97, 124 and 134, Chapter 3.1). To further analyze the regulation of hBAD function, we investigated the role of these newly identified phosphorylation sites on BAD-mediated apoptosis. We found that in contrast to the N-terminal phosphorylation sites, the C-terminal serines 124 and 134 act in an anti-apoptotic manner (Chapter 3.4). Our results further indicate that RAF kinases and PAK1 effectively phosphorylate BAD at serine 134. Notably, in the presence of wild type hBAD, co-expression of survival kinases, such as RAF and PAK1, leads to a strongly increased proliferation, whereas substitution of serine 134 by alanine abolishes this process. Furthermore, we identified hBAD serine 134 to be strongly involved in survival signaling in B-RAF-V600E containing tumor cells and found phosphorylation of this residue to be crucial for efficient proliferation in these cells. Collectively, our findings provide new insights into the regulation of hBAD function by phosphorylation and its role in cancer signaling. N2 - BAD (Bcl-2 antagonist of cell death, Bcl-2 associated death promoter) ist ein pro-apoptotisches Mitglied der Bcl-2 Proteinfamilie und wird in Abhängigkeit von Wachstumsfaktoren durch Phosphorylierung reguliert. Obwohl der Identifizierung von Phosphorylierungsstellen in murinem BAD (mBAD) in den vergangenen Jahren viel Aufmerksamkeit gewidmet wurde, ist die Phosphorylierung des humanen BAD (hBAD) Proteins kaum charakterisiert. In der vorliegenden Arbeit wird der quantitative Beitrag unterschiedlicher Kinasen in Bezug auf die Phosphorylierung der etablierten Phosphorylierungsstellen Serin 75, 99 und 118 von hBAD dargestellt (Kapitel 3.1). Unsere Ergebnisse deuten darauf hin, dass RAF-Kinasen hBAD in vivo an diesen etablierten Stellen phosphorylieren. Die RAF-bedingte Phosphorylierung konnte nicht durch MEK-Inhibitoren beeinflusst werden, dagegen bewirkte die Gabe des RAF-Inhibitors Sorafenib (BAY 43-9006) eine Reduktion der Phosphorylierung auf das Niveau der Kontrollproben. Übereinstimmend konnte durch die Expression von aktiven RAF-Kinasen die BAD-induzierte Apoptose sowie die BAD-bedingte Inhibierung der Koloniebildung unterdrückt werden. Zusätzlich verwendeten wir Oberflächen-Plasmon-Resonanz-Spektroskopie um die Auswirkungen der RAF-bedingten BAD-Phosphorylierung auf die Komplexbildung von hBAD mit 14-3-3-Proteinen und Bcl-XL zu analysieren. Dabei wurde festgestellt, dass die Phosphorylierung von hBAD durch aktive RAF-Kinasen die Assoziierung von 14-3-3 begünstigt, wobei Phosphoserin 99 die Hauptbindungsstelle darstellt. Weiterhin gelang der Nachweis, dass hBAD in vitro Poren in Lipid-Doppelschicht-Membranen bilden kann. Wir wiesen nach, dass die Fähigkeit von hBAD Poren zu bilden phosphorylierungsabhängig ist und durch die Interaktion mit 14-3-3-Proteinen beeinflusst wird. Außerdem demonstrieren wir in dieser Arbeit, dass die BAD-Poren eine zylinderförmige Geometrie aufweisen und sowohl für Ionen als auch für ungeladene Moleküle mit einer Größe von bis zu 200 Da zugänglich sind (Kapitel 3.2). Da beide Lipid-Bindungsstellen (LBD1 und LBD2) am C-Terminus des hBAD lokalisiert sind, charakterisierten wir des Weiteren diesen Teil des Proteins in Hinblick auf seinen strukturellen Aufbau (Kapitel 3.3). Unsere Ergebnisse demonstrieren, dass der hBAD-C-Terminus in wässriger Lösung eine geordnete β-Faltblattstruktur aufweist und bei Eintritt in eine Lipidumgebung helikale Elemente ausbildet. Zusätzlich zeigen wir in dieser Arbeit, dass die Interaktion des C-terminalen hBAD-Segments mit der BH3-Domäne zur Ausbildung von permanent offenen Poren führt, wobei die Phosphorylierung an Serin 118 eine Notwendigkeit für effektive Porenbildung darstellt. In Gegensatz dazu bewirkte die Phosphorylierung von Serin 99 in Kombination mit der Assoziierung von 14-3-3-Protein eine Inhibierung der Porenbildung. Diese Ergebnisse weisen darauf hin, dass der C-terminale Teil von hBAD durch strukturelle Veränderungen, Lipidbindung und Phosphorylierung entscheidend die Funktion von hBAD reguliert. Mit Hilfe von Massenspektroskopie konnten wir im Rahmen dieser Arbeit, zusätzlich zu den etablierten Phosphorylierungsstellen Serin 75, 99 und 118, einige neue in vivo Phosphorylierungsstellen von hBAD identifizieren (Serin 25, 32/34, 97, 124 und 134, Kapitel 3.1). Um die Regulierung der Funktion von hBAD weiter zu analysieren, untersuchten wir die Rolle dieser neu identifizierten Phosphorylierungsstellen in Bezug auf die BAD-induzierte Apoptose (Kapitel 3.4). Wir fanden heraus, dass im Gegensatz zu den N-terminalen Phosphorylierungsstellen, die Phosphorylierungsstellen am C-Terminus an der Apoptoseregulation mitwirken. Weiterhin weisen unsere Ergebnisse darauf hin, dass RAF-Kinasen, neben PAK1, an der Phosphorylierung von Serin 134 von hBAD beteiligt sind. Interessanterweise bewirkte die Co-Expression von RAF oder PAK1 mit dem wildtypischen hBAD eine erhebliche Verstärkung der Zellproliferation. Diese verstärkte Proliferation konnte durch einen Serin-zu-Alanin-Austausch in hBAD an der Stelle 134 vollständig verhindert werden. Weiterhin entdeckten wir, dass die Phosphorylierung dieser Stelle in B-RAF-V600E enthaltenden Tumorzellen bei der Regulation der Zellproliferation mitwirkt und für eine effiziente Proliferation entscheidend ist. Zusammenfassend gewähren unsere Ergebnisse neue Einblicke in die Regulierung der Funktion von hBAD durch Phosphorylierung sowie in die Rolle von hBAD bei der Krebsentwicklung. KW - Krebs KW - Apoptosis KW - Bcl-2-Proteinfamilie KW - Raf KW - BH3-only proteins KW - BAD KW - cancer Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-56919 ER - TY - JOUR A1 - Kredel, Markus A1 - Muellenbach, Ralf A1 - Johannes, Amelie A1 - Brederlau, Joerg A1 - Roewer, Norbert A1 - Wunder, Christian T1 - Hepatic effects of lung protective pressure controlled ventilation and a combination of high frequency oscillatory ventilation and extracorporeal lung assist in experimental lung injury N2 - Background: Ventilation with high positive end-expiratory pressure (PEEP) can lead to hepatic dysfunction. The aim of this study was to investigate the hepatic effects of strategies using high airway pressures either in pressure-controlled ventilation (PCV) or in high-frequency oscillatory ventilation (HFOV) combined with an arteriovenous extracorporeal lung assist (ECLA). Material/Methods: Pietrain pigs underwent induction of lung injury by saline lavage. Ventilation was continued for 24 hours either as PCV with tidal volumes of 6 ml/kg and PEEP 3 cmH2O above the lower inflection point of the pressure-volume curve or as HFOV (≥12 Hz) with a mean tracheal airway pressure 3 cmH2O above the lower inflection point combined with arteriovenous ECLA (HFOV+ECLA). Fluids and norepinephrine stabilized the circulation. The indocyanine green plasma disappearance rate, serum bilirubin, aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, glutamate dehydrogenase, lactate dehydrogenase and creatine kinase were determined repeatedly. Finally, liver neutrophils were counted and liver cell apoptosis was assessed by terminal deoxynucleotidyl transferase nick end labeling (TUNEL). Results: Aspartate aminotransferase increased in the PCV group about three-fold and in the HFOV+ECLA group five-fold (p<0.001). Correspondingly, creatine kinase increased about two-fold and four-fold, respectively (p<0.001). Lactate dehydrogenase was increased in the HFOV+ECLA group (p<0.028). The number of neutrophils infiltrating the liver tissue and the apoptotic index were low. Conclusions: High airway pressure PCV and HFOV with ECLA in the treatment of lavage-induced lung injury in pigs did not cause liver dysfunction or damage. The detected elevation of enzymes might be of extrahepatic origin. KW - Neutrophils KW - Lung Injury KW - L-Lactate Dehydrogenase KW - Interactive Ventilatory Support KW - In Situ Nick-End Labeling KW - High-Frequency Ventilation KW - Creatine Kinase KW - Aspartate Aminotransferases KW - Apoptosis KW - Positive-Pressure Respiration Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-70833 ER - TY - JOUR A1 - Fischer, Roman A1 - Maier, Olaf A1 - Siegemund, Martin A1 - Wajant, Harald A1 - Scheurich, Peter A1 - Pfizenmaier, Klaus T1 - A TNF Receptor 2 Selective Agonist Rescues Human Neurons from Oxidative Stress-Induced Cell Death JF - PLoS ONE N2 - Tumor necrosis factor (TNF) plays a dual role in neurodegenerative diseases. Whereas TNF receptor (TNFR) 1 is predominantly associated with neurodegeneration, TNFR2 is involved in tissue regeneration and neuroprotection. Accordingly, the availability of TNFR2-selective agonists could allow the development of new therapeutic treatments of neurodegenerative diseases. We constructed a soluble, human TNFR2 agonist (TNC-scTNF(R2)) by genetic fusion of the trimerization domain of tenascin C to a TNFR2-selective single-chain TNF molecule, which is comprised of three TNF domains connected by short peptide linkers. TNC-scTNFR2 specifically activated TNFR2 and possessed membrane-TNF mimetic activity, resulting in TNFR2 signaling complex formation and activation of downstream signaling pathways. Protection from neurodegeneration was assessed using the human dopaminergic neuronal cell line LUHMES. First we show that TNC-scTNF(R2) interfered with cell death pathways subsequent to H(2)O(2) exposure. Protection from cell death was dependent on TNFR2 activation of the PI3K-PKB/Akt pathway, evident from restoration of H(2)O(2) sensitivity in the presence of PI3K inhibitor LY294002. Second, in an in vitro model of Parkinson disease, TNC-scTNFR(2) rescues neurons after induction of cell death by 6-OHDA. Since TNFR2 is not only promoting anti-apoptotic responses but also plays an important role in tissue regeneration, activation of TNFR2 signaling by TNC-scTNF(R2) appears a promising strategy to ameliorate neurodegenerative processes. KW - Tumor-necrosis-factor KW - Glutamate-induced excitotoxicity KW - Single-chain TNF KW - Kappa-B pathway KW - Parkinsons-disease KW - Signaling complex KW - Activation KW - Apoptosis KW - Dopamine KW - Ligand Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133552 VL - 6 IS - 11 ER -