Cellular accumulation and DNA interaction studies of antitumor metal complexes

Die unzureichende Wirkung platin-basierter Krebstherapeutika in einer Vielzahl solider Tumore, die schweren Nebenwirkungen und das Problem von erworbener und intrinsischer Resistenz machen die Entwicklung von Arzneimitteln mit einem anderen Wirkmechanismus notwendig. Ein Schwerpunkt dieser Arbeit war daher die Untersuchung der Wirkungsweise des Ruthenium-Komplexes KP1019, der bereits in einer klinischen Phase I – Studie erfolgversprechende Resultate erzielte. Basierend auf der Hypothese einer Aktivierung durch Reduktion wurde der Einfluss des intrazellulären Antioxidans Ascorbinsäure (Vitamin C) untersucht. Ascorbinsäure verstärkte die Aktivität von KP1019, was sich durch höhere Zytotoxizität in der humanen Darmkrebszelllinie SW480, den humanen Gebärmutterkarzinomzellen KB-3-1 und der mehrfach resistenten Subzelllinie KBC-1 gezeigt hat. Darüberhinaus konnte in weiteren Versuchen unter dem Einfluss von Ascorbinsäure eine verstärkte Reaktivität von KP1019 mit dem Modell-Biomolekül Plasmid-DNA mittels EMSA nachgewiesen werden. Um die erhöhte Aktivität erklären zu können, wurde zunächst die zelluläre Akkumulation untersucht und der Ruthenium-Gehalt der Zellen mittels ICP-MS bestimmt. Dabei zeigte sich, dass Ascorbinsäure die intrazelluläre Ruthenium-Konzentration im Vergleich zu Zellen, die nur mit KP1019 behandelt wurden, nicht veränderte. Bei der Inkubation mit KP1019 kommt es zur Bildung reaktiver Sauerstoffspezies (ROS), die bei der zytotoxischen Wirkung eine Rolle spielen. Die Frage, ob die ROS-Bildung durch Ascorbinsäure beeinflusst wird und damit zur Erklärung der erhöhten zytotoxischen Wirkung von KP1019 beitragen kann, wurde durch den DCFA-DH Assay mit anschließender FACS Messung untersucht. Es zeigte sich, dass der Gehalt an durch KP1019 gebildete ROS durch Ko-Inkubation mit Ascorbinsäure dosisabhängig abnimmt und somit als Ursache der verstärkten Aktivität von KP1019 ausgeschlossen werden kann. Basierend auf den Resultaten wird angenommen, dass eine direkte Wechselwirkung zwischen KP1019 und Ascorbinsäure und die daraus resultierende Reduktion des Ruthenium-Zentrums für die verstärkte Reaktivität verantwortlich ist. Eine weitere Aufgabenstellung war die Untersuchung biologischer Effekte nicht-klassischer Platinverbindungen. Hierzu wurden drei Paare cis- und trans- konfigurierter Platin(II)-Acetonoxim-Komplexe und ein Paar Platin(II)-Pentanonoxim-Komplexe ausgewählt, bei denen die trans-Komplexe deutlich zytotoxischer als ihre cis-konfigurierten Analoga wirken und vor allem in der cisplatin-resistenten Zelllinie SW480 aktiver als Cisplatin sind. Um die stärkere Aktivität erklären zu können, wurde zunächst die zelluläre Akkumulation gemessen. Dabei konnte eine erhöhte zelluläre Anreicherung im Falle der vier trans-Platin(II)-Komplexe im Vergleich zu den entsprechenden cis-Komplexen sowie zu Cisplatin und Transplatin nachgewiesen werden. Aufgrund der Annahme dass DNA, wie bei Cisplatin, das zelluläre Target ist, wurde die Anbindung an die DNA in Zellen untersucht und mittels ICP-MS gemessen. Auch hier zeigten die trans-Komplexe stärkere Effekte, die in höheren rb-Werten (Platin pro Phosphor) resultierte. Untersuchungen des genotoxischen Potenzials der trans-Platin(II)-Komplexe und die Fähigkeit der Verbindungen, Quervernetzungen zu verursachen, wurden mit Hilfe des Comet Assays durchgeführt und zeigten eine von Cisplatin deutlich abweichende Wirkung auf die DNA. Zusätzliche Informationen über die Art der DNA-Wechselwirkung brachten zellfreie Experimente mit Plasmid-DNA und der Nukleobase GMP, die in Übereinstimmung mit den zuvor erwähnten Versuchen eindeutige Unterschiede zu Cisplatin belegen. Die Abweichungen der DNA-Wechselwirkung der neuen trans-Platin(II)-Komplexe und Cisplatin lassen auf andersartige Wirkmechanismen schließen. Zusammenfassend ist zu erwähnen, dass die vorliegenden Studien der zellulären Akkumulation von metall-basierten Krebstherapeutika und deren Wechselwirkung mit Biomolekülen zum Verständnis des Wirkmechanismus beitragen. Der Fokus der klinischen Weiterentwicklung liegt auf Verbindungen, die anders wirken als derzeit verwendete Medikamente. Solche Wirkstoffe könnten es ermöglichen, Resistenz-Phänomene zu überwinden, oder ein geringeres Risiko ernster Nebenwirkungen aufweisen.The limitations of clinically used platinum-based anticancer drugs in a variety of solid tumors, severe side effects and the problem of acquired and intrinsic resistance necessitate the development of anticancer therapeutics with a different mode of action. Hence, within this PhD thesis the ruthenium based investigational anticancer drug KP1019 which showed promising results in a phase I clinical trial was investigated with regard to its mode of action. Based on the ‘activation by reduction’ hypothesis, the influence of the intracellular antioxidant ascorbic acid (vitamin C) was studied. Ascorbic acid enhanced the activity of KP1019 which was shown by higher cytotoxicity in the human colon carcinoma cell line SW480, human cervical carcinoma KB-3-1 cells, and the multidrug-resistant subline KBC-1. Moreover it was demonstrated by means of EMSA that ascorbic acid enhanced interaction of KP1019 with the model biomolecule plasmid DNA. To explain increased activity, cellular accumulation was investigated and ruthenium contents were measured by ICP-MS. Those studies revealed that ascorbic acid did not alter the intracellular ruthenium concentration compared to KP1019-only treated cells. During incubation with KP1019 intracellular reactive oxygen species (ROS) are produced. To elucidate whether generation of ROS is influenced by ascorbic acid and therefore may contribute to the enhanced cytotoxic potential of KP1019, ROS were analyzed by the DCFA-DH assay and subsequent FACS measurements. It was evinced that KP1019-induced ROS were reduced by ascorbic acid in a dose-dependent manner and thus can be excluded as a reason for increased activity of KP1019. Based on the obtained results, it is assumed that the direct interaction of KP1019 and ascorbic acid, resulting in reduction of the ruthenium center, is responsible for the enhanced reactivity. A further scope was investigation of non-classic platinum drugs with regard to their biological behavior. Therefore three pairs of cis and trans configured platinum(II)-acetone oxime complexes and one pair of 3-pentanone oxime platinum(II) complexes, which displayed higher cytotoxicity for the trans configured complexes than their cis configured counterparts and proved more cytotoxic than cisplatin in the inherently cisplatin resistant cell line SW480, were selected. To explain the higher activity, cellular accumulation was determined, revealing enhanced concentrations in cells treated with the four trans platinum(II) complexes compared to the respective cis complexes as well as cisplatin and transplatin. Based on the assumption that DNA is the critical cellular target as it is with cisplatin, binding to DNA was investigated by in vitro DNA metalation experiments and determined by ICP-MS measurement. Again, the trans complexes showed stronger effects resulting in higher rb values (platinum per phosphorus). Studies on the genotoxic potential of the trans platinum(II) complexes as well as their ability to induce cross-links were conducted by means of the Comet Assay and showed a distinct impact on the DNA compared to cisplatin. Additional information about the type of DNA interaction were obtained by cell free experiments with plasmid DNA and the nucleobase GMP which, in accordance with the other experiments, also indicated differences compared to cisplatin. The variations in DNA interactions between the novel trans platinum(II) complexes and cisplatin give reason to think of a different mode of action. Concluding, the presented investigations of cellular accumulation and interaction with biomolecules of metal based anticancer drugs contribute to the understanding of the mode of action of those complexes. The emphasis for further clinical development is on substances that act differently from currently used drugs because such complexes may overcome resistance phenomena or bear a lower risk of severe side effects

Garud: Role of Narratives in Sustaining Organizational Innovation

S ustaining innovation is a vital yet difficult task. Innovation requires the coordinated efforts of many actors to facilitate (1) the recombination of ideas to generate novelty, (2) real-time problem solving, and (3) linkages between present innovation efforts with past experiences and future aspirations. We propose that innovation narratives are cultural mechanisms that address these coordination requirements by enabling translation. Specifically, innovation narratives are powerful mechanisms for translating ideas across the organization so that they are comprehensible and appear legitimate to others. Narratives also enable people to translate emergent situations that are ambiguous or equivocal so as to promote real-time problem solving. With their accumulation, innovation narratives provide a generative memory for organizations that enable people to translate ideas accumulated from particular instances of past innovation to inform current and future efforts

The Collective Construction of Work Group Moods.

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Knowing where you stand: Physical isolation, perceived respect, and organizational identification among virtual employees

T his research investigates the relationship between virtual employees' degree of physical isolation and their perceived respect in the organization. Respect is an identity-based status perception that reflects the extent to which one is included and valued as a member of the organization. We hypothesize that the degree of physical isolation is negatively associated with virtual employees' perceived respect and that this relationship explains the lower organizational identification among more physically isolated virtual employees. In two field studies using survey methods, we find that perceived respect is negatively associated with the degree of physical isolation, and respect mediates the relationship between physical isolation and organizational identification. These effects hold for shorter-and longer-tenured employees alike. Our research contributes to the virtual work literature by drawing attention to physical isolation and the important but neglected role of status perceptions in shaping virtual employees' organizational identification. We also contribute to the literature on perceived respect by demonstrating how respect is affected by the physical context of work

Estratégias de promoção de um ambiente de trabalho seguro para a prevenção de queimaduras

Objetivou-se conhecer a viso dos trabalhadores sobre as estratgias de segurana durante a realizao de suas atividades laborais antes da ocorrncia do acidente. Estudo de abordagem qualitativa, exploratrio e descritivo, com cinco trabalhadores, maiores de 18 anos, que sofreram acidentes de trabalho por queimadura, internados em um centro de tratamento de queimaduras, de junho a outubro de 2012. Utilizou-se entrevista aberta, cujas informaes foram submetidas anlise de contedo. A pesquisa recebeu aprovao do comit de tica da instituio. Os sujeitos elencaram o treinamento para a realizao das atividades; adeso dos trabalhadores s rotinas e normas de segurana; cuidado interpessoal; barreiras para eliminao dos riscos; fiscalizao por rgos competentes e a presena de profissionais de sade do trabalhador no local de trabalho como estratgias para a promoo de um ambiente seguro. Embora os sujeitos percebessem essas estratgias como importantes para a preservao da sua sade, as mesmas no foram efetivas para a preveno dos acidentes de trabalho por queimaduras. Os resultados mostram a necessidade de investimentos na preveno de agravos sade dos trabalhadores nos ambientes de trabalho.Descritores: Condies de Trabalho; Sade do Trabalhador; Segurana; Queimadura

Estratégias de promoção de um ambiente de trabalho seguro para a prevenção de queimaduras

Objetivou-se conhecer a visão dos trabalhadores sobre as estratégias de segurança durante a realização de suas atividades laborais antes da ocorrência do acidente. Estudo de abordagem qualitativa, exploratório e descritivo, com cinco trabalhadores, maiores de 18 anos, que sofreram acidentes de trabalho por queimadura, internados em um centro de tratamento de queimaduras, de junho a outubro de 2012. Utilizou-se entrevista aberta, cujas informações foram submetidas à análise de conteúdo. A pesquisa recebeu aprovação do comitê de ética da instituição. Os sujeitos elencaram o treinamento para a realização das atividades; adesão dos trabalhadores às rotinas e normas de segurança; cuidado interpessoal; “barreiras” para eliminação dos riscos; fiscalização por órgãos competentes e a presença de profissionais de saúde do trabalhador no local de trabalho como estratégias para a promoção de um ambiente seguro. Embora os sujeitos percebessem essas estratégias como importantes para a preservação da sua saúde, as mesmas não foram efetivas para a prevenção dos acidentes de trabalho por queimaduras. Os resultados mostram a necessidade de investimentos na prevenção de agravos à saúde dos trabalhadores nos ambientes de trabalho. Descritores: Condições de Trabalho; Saúde do Trabalhador; Segurança; Queimadura.&nbsp

Reabilitação e retorno ao trabalho após queimaduras ocupacionais

Objetivou-se conhecer os aspectos que favorecem ou dificultam a reabilitao e o retorno ao trabalho de indivduos que sofreram acidente laboral por queimaduras. Trata-se de um estudo de abordagem qualitativa, exploratrio e descritivo com dois adultos internados em um Centro de Referncia em Assistncia a Queimados do Sul do Brasil. A coleta dos dados foi realizada em outubro de 2012, por meio de entrevista semiestruturada. Os dados foram analisados conforme anlise de contedo, e elencou-se duas categorias: a) queimadura: obstculos para o retorno vida laboral e, b) a vida aps a queimadura: retomando processos interrompidos. Os participantes aps as queimaduras passaram por mudanas fsicas, estticas e emocionais, e estas podem ter impactado negativo no retorno ao trabalho e na rotina de vida. O apoio de familiares e amigos, motivao, vontade de vencer e desejo de voltar ao trabalho foram apontados como aspectos facilitadores para o retorno ao trabalho aps acidente por queimadura. Concluiu-se que importante que os profissionais de enfermagem invistam em aes que contribuam na reabilitao fsica, esttica e emocional dos trabalhadores que sofreram queimaduras ocupacionais

Reabilitação e retorno ao trabalho após queimaduras ocupacionais

Objetivou-se conhecer os aspectos que favorecem ou dificultam a reabilitação e o retorno ao trabalho de indivíduos que sofreram acidente laboral por queimaduras. Trata-se de um estudo de abordagem qualitativa, exploratório e descritivo com dois adultos internados em um Centro de Referência em Assistência a Queimados do Sul do Brasil. A coleta dos dados foi realizada em outubro de 2012, por meio de entrevista semiestruturada. Os dados foram analisados conforme análise de conteúdo, e elencou-se duas categorias: a) queimadura: obstáculos para o retorno à vida laboral e, b) a vida após a queimadura: retomando processos interrompidos. Os participantes após as queimaduras passaram por mudanças físicas, estéticas e emocionais, e estas podem ter impactado negativo no retorno ao trabalho e na rotina de vida. O apoio de familiares e amigos, motivação, vontade de vencer e desejo de voltar ao trabalho foram apontados como aspectos facilitadores para o retorno ao trabalho após acidente por queimadura. Concluiu-se que é importante que os profissionais de enfermagem invistam em ações que contribuam na reabilitação física, estética e emocional dos trabalhadores que sofreram queimaduras ocupacionais.&nbsp

Functional microRNA generated from a cytoplasmic RNA virus

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that play a pivotal role in the regulation of posttranscriptional gene expression in a wide range of eukaryotic organisms. Although DNA viruses have been shown to encode miRNAs and exploit the cellular RNA silencing machinery as a convenient way to regulate viral and host gene expression, it is generally believed that this pathway is not available to RNA viruses that replicate in the cytoplasm of the cell because miRNA biogenesis is initiated in the nucleus. In fact, among the >200 viral miRNAs that have been experimentally verified so far, none is derived from an RNA virus. Here, we show that a cytoplasmic RNA virus can indeed encode and produce a functional miRNA. We introduced a heterologous miRNA-precursor stem-loop sequence element into the RNA genome of the flavivirus tick-borne encephalitis virus, and this led to the production of a functional miRNA during viral infection without impairing viral RNA replication. These findings demonstrate that miRNA biogenesis can be used by cytoplasmic RNA viruses to produce regulatory molecules for the modulation of the transcriptome