Clusterin Regulates Drug-Resistance in Melanoma Cells

Clusterin has recently been shown to act as an antiapoptotic protein that confers drug-resistance in models of epithelial tumors. The aim of our work was to provide an insight into a possible role of clusterin in the regulation of drug-resistance in melanoma. In tissue samples, clusterin expression was low in nevi, but high in primary melanoma and melanoma metastases. Clusterin was also strongly expressed in melanoma cell lines, but was barely detectable in cultured melanocytes. To elucidate a possible role of clusterin in drug-resistance of melanoma, clusterin expression was regulated by either plasmid-driven overexpression or by antisense-mediated downregulation. Clusterin overexpression was associated with an increase in drug-resistance, i.e., with an increased survival of melanoma cells in the presence of cytotoxic drugs. In contrast, downregulation of clusterin by 2′-O-(2-methoxy)ethyl (2′MOE)-modified antisense oligonucleotides (AS-ODN) directed against clusterin mRNA significantly reduced drug-resistance, i.e., decreased survival of melanoma cells in the presence of cytotoxic drugs. To evaluate the effects of clusterin-antisense treatment in vivo, we applied an SCID-mouse/human-melanoma xenotransplantation model. Pre-treatment of mice with the 2′MOE-modified clusterin AS-ODN was associated with a significantly improved tumor response to dacarbazine as compared with animals pretreated with a scrambled control oligonucleotide. Taken together, we show that clusterin is strongly expressed in melanoma. Downregulation of clusterin reduces drug-resistance, i.e., reduces melanoma cell survival in response to cytotoxic drugs in vitro and in vivo. Thus, reducing clusterin expression may provide a novel tool to overcome drug-resistance in melanoma

Enzymatic synthesis of antibody-human serum albumin conjugate for targeted drug delivery using tyrosinase from Agaricus bisporus

Highly specific targeted drug delivery devices can be obtained with antibody-human serum albumin (mAb-HSA) conjugates. However, their conventional production involves several reaction steps including chemical modification and activation of both proteins followed by cross-linking often involving toxic chemicals. Here, we describe the enzymatic synthesis of mAb-HSA conjugates for targeted drug delivery devices using tyrosinase from Agaricus bisporus under mild reaction conditions (pH 6.8, 25 [degree]C). Reaction conditions were optimized by using fluorescence labeled HSA to facilitate SDS-PAGE analysis with fluorescence scanning. Enzymatic cross-linking in the presence of natural low molecular weight phenolic compounds (e.g. caffeic acid) resulted in reaction products in the molecular weight range of [similar]216 kDa, corresponding to mAb-HSA conjugates. The composition of the conjugates was confirmed with tryptic digestion followed by LC-MS/MS analysis of the resulting peptide fragments. Successful binding of mAb-HSA conjugates (in contrast to free HSA) to MHC II molecules, located on antigen-presenting cells, was demonstrated by both ELISA and flow cytometry analysis.This work has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL and FWF, DK: Metabolic and Cardiovascular Disease: W1226-B18. We thank Tamara Reiter, Graz University of Technology for technical support with SEC; Exbio from the Czech Republic for providing the mAbs and Britta Obrist, Medical University of Graz and the Austrian Centre of Industrial Biotechnology, for technical assistance with LC-MS/MS analysis

Functionalization of catheters with antimicrobial enzymes

Biofilme, die sich auf Blasenkathetern bilden, führen zu Infektionen, auch Katheter assoziierte Harnwegsinfekte (CAUTI) genannt, die 40 % aller im Krankenhaus auftretenden nosokomialen Infektionen ausmachen und eine Entfernung des Katheters und eine intensive Antibiotikatherapie notwendig machen. Da es sehr schwierig ist, Biofilm- assoziierte Infektionen zu behandeln, ist Vorbeugung der einzig sinnvolle Weg um die Anzahl an CAUTI zu reduzieren. Die Verwendung von Enzymen mit antimikrobiellen Eigenschaften ist eine recht neue Entwicklung auf diesem Gebiet und inkludiert Cellobiose Dehydrogenase (CDH), da es das starke Oxidationsmittel Wasserstoffperoxid (H2O2) produziert. In dieser Arbeit wurde das Potential der CDH für die Verwendung als antimikrobielle und antibiofilm Substanz untersucht. Als erstes wurden die optimalen Bedingungen, die zur maximalen antimikrobiellen Aktivität gegen Bakterien führen etabliert und folglich an Mikroorganismen getestet, die häufig Katheteroberflächen kolonisieren. Die stärksten Effekte konnten gegen den multiresistenten S. aureus und A. baumanii beobachtet werden. Partiell hydrolysierte Exopolysaccharide wurden als mögliches Substrat für die CDH identifiziert wodurch ein sich selbst erhaltendes antimikrobielles System ermöglicht wird. Das Protokoll, das für die kovalente Immobilisierung entwickelt wurde, führte zu einer sehr stabilen antimikrobiellen Oberfläche, wobei das Enzym über einen Zeitraum von 16 Tagen noch immer 20% seiner Ursprungsaktivität behielt. Kurzzeit- und Langzeitinkubationen mit S. aureus führten zu einer 70% Reduktion des bakteriellen Wachstums auf der Oberfläche. Die Beschichtung zeigte keine Anzeichen von Toxizität gegen Säugetierzelllinien auch nicht wenn antimikrobiell wirksame Substratkonzentrationen zugegeben wurden. Die CDH ist daher ein sehr stark antimikrobiell und antibiofilm wirksames Enzym und allen Anforderungen an eine neue antimikrobielle Blasenkatheterbeschichtung entspricht.Biofilms forming on urinary catheters cause infections also known as catheter associated urinary tract infections (CAUTI) that account for 40% of all nosocomial infections occurring in hospitals, causing the removal of the catheter alongside intensive antibiotic treatment. Due to the difficulties faced when treating biofilm-associated infection, prevention is key in reducing the number of CAUTI. The use of enzymes with antimicrobial properties is quite a recent development and among them cellobiose dehydrogenase (CDH) which produces the strong oxidizing agent hydrogen peroxide (H2O2). In this study, the potential of using CDH as an antimicrobial and antibiofilm agent is investigated. First, the conditions under which CDH exerts maximum antimicrobial activity against bacteria were established and then tested against clinical isolates commonly colonizing catheter surfaces. Strong effects against strains such as A. baumanii and the multidrug resistant S. aureus were demonstrated. Partially hydrolyzed exopolysaccharides, a major constituent of the biofilm matrix, were identified as potential substrates for CDH, enabling a self-sustaining antimicrobial system. The covalent immobilization protocol developed yielded a highly stable antimicrobial surface, with an enzyme remaining 20% of its original activity over 16 days. Both short term and long term incubation with S. aureus lead to around 70% reduction of growth on the coated surface. The coating didnt show any signs of toxicity against the mammalian cell lines HEK 239 and RAW 264.7 when incubated with antimicrobial effective substrate concentrations. CDH is a very potent antimicrobial and antibiofilm enzyme fulfilling all requirements for a novel urinary catheter coating.Barbara ThallingerZusammenfassung in deutscher SpracheUniversität für Bodenkultur Wien, Dissertation, 2015OeBB(VLID)193115

Antifouling and Antibacterial Multifunctional Polyzwitterion/Enzyme Coating on Silicone Catheter Material Prepared by Electrostatic Layer-by-Layer Assembly

The formation of bacterial biofilms on indwelling medical devices generally causes high risks for adverse complications such as catheter-associated urinary tract infections. In this work, a strategy for synthesizing innovative coatings of poly­(di­methyl­siloxane) (PDMS) catheter material, using layer-by-layer assembly with three novel functional polymeric building blocks, is reported, i.e., an antifouling copolymer with zwitterionic and quaternary ammonium side groups, a contact biocidal derivative of that polymer with octyl groups, and the antibacterial hydrogen peroxide (H₂O₂) producing enzyme cellobiose dehydrogenase (CDH). CDH oxidizes oligosaccharides by transferring electrons to oxygen, resulting in the production of H₂O₂. The design and synthesis of random copolymers which combine segments that have antifouling properties by zwitterionic groups and can be used for electrostatically driven layer-by-layer (LbL) assembly at the same time were based on the atom-transfer radical polymerization of di­methyl­amino­ethyl meth­acrylate and subsequent partial sulfo­beta­ini­zation with 1,3-propane sultone followed by quaternization with methyl iodide only or octyl bromide and thereafter methyl iodide. The alternating multilayer systems were formed by consecutive adsorption of the novel polycations with up to 50% zwitterionic groups and of poly­(styrene­sulfonate) as the polyanion. Due to its negative charge, enzyme CDH was also firmly embedded as a polyanionic layer in the multilayer system. This LbL coating procedure was first performed on prefunctionalized silicon wafers and studied in detail with ellipsometry as well as contact angle (CA) and zetapotential (ZP) measurements before it was transferred to prefunctionalized PDMS and analyzed by CA and ZP measurements as well as atomic force microscopy. The coatings comprising six layers were stable and yielded a more neutral and hydrophilic surface than did PDMS, the polycation with 50% zwitterionic groups having the largest effect. Enzyme activity was found to be dependent on the depth of embedment in the multilayer coating. Depending on the used polymeric building block, up to a 60% reduction in the amount of adhering bacteria and clear evidence for killed bacteria due to the antimicrobial functionality of the coating could be confirmed. Overall, this work demonstrates the feasibility of an easy to perform and shape-independent method for preparing an antifouling and antimicrobial coating for the significant reduction of biofilm formation and thus reducing the risk of acquiring infections by using urinary catheters