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 - 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 - 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 - Karl, I. A1 - Jossberger-Werner, M. A1 - Schmidt, N. A1 - Horn, S. A1 - Goebeler, M. A1 - Leverkus, M. A1 - Wajant, H. A1 - Giner, T. T1 - TRAF2 inhibits TRAIL- and CD95L-induced apoptosis and necroptosis JF - Cell Death & Disease N2 - The relevance of the adaptor protein TNF receptor-associated factor 2 (TRAF2) for signal transduction of the death receptor tumour necrosis factor receptor1 (TNFR1) is well-established. The role of TRAF2 for signalling by CD95 and the TNF-related apoptosis inducing ligand (TRAIL) DRs, however, is only poorly understood. Here, we observed that knockdown (KD) of TRAF2 sensitised keratinocytes for TRAIL- and CD95L-induced apoptosis. Interestingly, while cell death was fully blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) in control cells, TRAF2-depleted keratinocytes were only partly rescued from TRAIL- and CD95L-induced cell death. In line with the idea that the only partially protective effect of zVAD-fmk on TRAIL- and CD95L-treated TRAF2-depleted keratinocytes is due to the induction of necroptosis, combined treatment with zVAD-fmk and the receptor interacting protein 1 (RIP1) inhibitor necrostatin-1 fully rescued these cells. To better understand the impact of TRAF2 levels on RIP1- and RIP3-dependent necroptosis and RIP3-independent apoptosis, we performed experiments in HeLa cells that lack endogenous RIP3 and HeLa cells stably transfected with RIP3. HeLa cells, in which necroptosis has no role, were markedly sensitised to TRAIL-induced caspase-dependent apoptosis by TRAF2 KD. In RIP3-expressing HeLa transfectants, however, KD of TRAF2 also strongly sensitised for TRAIL-induced necroptosis. Noteworthy, priming of keratinocytes with soluble TWEAK, which depletes the cytosolic pool of TRAF2-containing protein complexes, resulted in strong sensitisation for TRAIL-induced necroptosis but had only a very limited effect on TRAIL-induced apoptosis. The necroptotic TRAIL response was not dependent on endogenously produced TNF and TNFR signalling, since blocking TNF by TNFR2-Fc or anti-TNFα had no effect on necroptosis induction. Taken together, we identified TRAF2 not only as a negative regulator of DR-induced apoptosis but in particular also as an antagonist of TRAIL- and CD95L-induced necroptosis. KW - Nekrose KW - Apoptosis Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119166 SN - 2041-4889 VL - 5 ER - TY - JOUR A1 - Walter, T. A1 - Collenburg, L. A1 - Japtok, L. A1 - Kleuser, B. A1 - Schneider-Schaulies, S. A1 - Müller, N. A1 - Becam, J. A1 - Schubert-Unkmeir, A. A1 - Kong, J. N. A1 - Bieberich, E. A1 - Seibel, J. T1 - Incorporation and visualization of azido-functionalized N-oleoyl serinol in Jurkat cells, mouse brain astrocytes, 3T3 fibroblasts and human brain microvascular endothelial cells JF - Chemical Communications N2 - The synthesis and biological evaluation of azido-N-oleoyl serinol is reported. It mimicks biofunctional lipid ceramides and has shown to be capable of click reactions for cell membrane imaging in Jurkat and human brain microvascular endothelial cells. KW - Ceramide KW - Apoptosis KW - Golgi KW - N-oleoyl serinol KW - Jurkat cells Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191263 VL - 52 IS - 55 ER - TY - JOUR A1 - Kirschmer, Nadine A1 - Bandleon, Sandra A1 - von Ehrlich-Treuenstätt, Viktor A1 - Hartmann, Sonja A1 - Schaaf, Alice A1 - Lamprecht, Anna-Karina A1 - Miranda-Laferte, Erick A1 - Langsenlehner, Tanja A1 - Ritter, Oliver A1 - Eder, Petra T1 - TRPC4α and TRPC4β Similarly Affect Neonatal Cardiomyocyte Survival during Chronic GPCR Stimulation JF - PLoS ONE N2 - The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca\(^{2+}\) component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na\(^{+}\) and Ca\(^{2+}\) influx. Gαq protein-coupled receptor (GPCR) stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca\(^{2+}\) influx which has been regarded as ideal Ca\(^{2+}\) source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT) activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca\(^{2+}\) signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca\(^{2+}\) transients in neonatal rat cardiomyocytes (NRCs) showed that TRPC4α and TRPC4β affected Ca\(^{2+}\) cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca\(^{2+}\) transient amplitude at baseline and TRPC4β increasing the Ca\(^{2+}\) peak during angiotensin II (Ang II) stimulation. NRCs infected with TRPC4β (Ad-C4β) also responded with a sustained Ca\(^{2+}\) influx when treated with Ang II under non-pacing conditions. Consistent with the Ca\(^{2+}\) data, NRCs infected with TRPC4α (Ad-C4α) showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not translated into an increased propensity towards hypertrophy but rather less hypertrophy during GPCR stimulation. Further analyses revealed that, although hypertrophy was preserved in Ad-C4α NRCs and even attenuated in Ad-C4β NRCs, cardiomyocytes had an increased apoptosis rate and thus were less viable after chronic GPCR stimulation. These findings suggest that TRPC4α and TRPC4β differentially affect Ca\(^{2+}\) signals, calcineurin/NFAT signaling and hypertrophy but similarly impair cardiomyocyte viability during GPCR stimulation. KW - Apoptosis KW - calcineurin signaling cascade KW - small interfering RNAs KW - G protein coupled receptors KW - hyperexpression techniques KW - heart KW - adenoviruses KW - cardiac pacing Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-178539 VL - 11 IS - 12 ER - TY - JOUR A1 - Silwedel, Christine A1 - Haarmann, Axel A1 - Fehrholz, Markus A1 - Claus, Heike A1 - Speer, Christian P. A1 - Glaser, Kirsten T1 - More than just inflammation: Ureaplasma species induce apoptosis in human brain microvascular endothelial cells JF - Journal of Neuroinflammation N2 - Background Ureaplasma species (spp.) are commonly regarded as low-virulent commensals but may cause invasive diseases in immunocompromised adults and in neonates, including neonatal meningitis. The interactions of Ureaplasma spp. with host defense mechanisms are poorly understood. This study addressed Ureaplasma-driven cell death, concentrating on apoptosis as well as inflammatory cell death. Methods Human brain microvascular endothelial cells (HBMEC) were exposed to Ureaplasma (U.) urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). Resulting numbers of dead cells as well as mRNA levels and enzyme activity of key agents in programmed cell death were assessed by flow cytometry, RNA sequencing, and qRT-PCR, respectively. xCELLigence data were used for real-time monitoring of changes in cell adhesion properties. Results Both Ureaplasma isolates induced cell death (p < 0.05, vs. broth). Furthermore, Ureaplasma spp. enhanced mRNA levels for genes in apoptosis, including caspase 3 (Up3 p < 0.05, vs. broth), caspase 7 (p < 0.01), and caspase 9 (Up3 p < 0.01). Caspase 3 activity was increased upon Uu8 exposure (p < 0.01). Vice versa, Ureaplasma isolates downregulated mRNA levels for proteins involved in inflammatory cell death, namely caspase 1 (Uu8 p < 0.01, Up3 p < 0.001), caspase 4 (Uu8 p < 0.05, Up3 p < 0.01), NOD-like receptor pyrin domain-containing 3 (Uu8 p < 0.05), and receptor-interacting protein kinase 3 (p < 0.05). Conclusions By inducing apoptosis in HBMEC as main constituents of the blood-brain barrier, Ureaplasma spp. may provoke barrier breakdown. Simultaneous suppression of inflammatory cell death may additionally attenuate host defense strategies. Ultimate consequence could be invasive and long-term CNS infections by Ureaplasma spp. KW - Ureaplasma urealyticum KW - Ureaplasma parvum KW - Neuroinflammation KW - Meningitis KW - Caspase KW - Apoptosis KW - HBMEC Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200711 VL - 16 ER -