Charakterisierung von in vivo Modellen des humanen nicht-kleinzelligen Lungenkarzinoms zur Therapieoptimierung

Das Bronchialkarzinom ist die häufigste Todesursache bei den Krebserkrankungen und weist eine schlechte Prognose auf. Die Behandlung besteht aus einer Chemotherapie mit platinbasierten Medikamenten, doch der Erfolg ist unbefriedigend. In den letzten Jahren wurden zielgerichtete Therapien gegen Proteine wie den EGFR entwickelt. Klinische Studien zeigten, dass nur Subpopulationen von den Medikamenten Erlotinib und Cetuximab profitieren. Eine bessere (Vor-)Selektion der Patienten ist wünschenswert, um unnötige Behandlungen zu vermeiden. Für diese Analysen bedarf es relevanter präklinischer Modelle. Im Rahmen dieser Arbeit wurden 25 Xenograftmodelle des Lungenkarzinoms vergleichend charakterisiert. Ein Schwerpunkt bestand im Vergleich der Xenografts mit ihren Patiententumoren. Die Analyse der Histologie, der Proliferationsmarker als auch der Genexpressionsprofile fand übereinstimmende Ergebnisse in den Patiententumoren und ihren abgeleiteten Xenografts. Mit Hilfe von mRNA-, Protein- und SNP-Profilen ressistenzassoziierter Marker der Chemotherapie konnte die Bedeutung der Modelle zur Charakterisierung von prädiktiven und prognostischen Markern aufklärt werden. Diese Arbeit untersuchte auch Marker der anti-EGFR-Therapien. mRNA- und Proteinprofile der ERBB-Rezeptoren sowie der Liganden wurden erstellt und stimmten mit publizierten klinischen Daten überein. Genexpressionsstudien in Erlotinib Respondern und Non-Respondern zur Therapieoptimierung identifizierten den Wachstumsfaktor VEGFA als Ziel für eine Kombinationsbehandlung mit dem Angiogeneseinhibitor Bevacizumab. Die Kombination von Bevacizumab mit Erlotinib führte zu einem reduzierten Tumorwachstum. Die Ergebnisse dieser Arbeit machten deutlich, dass die individuellen Tumoreigenschaften in den patientenabgleiteten Xenografts auf Gen- und Proteinebene erhalten bleiben und diese als Modelle zur Markeranalyse sowie zur Therapieoptimierung eingesetzt werden können.Lung cancer is still one of the most frequent cancers worldwide. The treatment option is classical chemotherapy that is based upon the combination of platin-based drugs. But no further improvement seems to be possible. For some years targeted drugs against single proteins like the EGFR were developed. The clinical trials showed that only subpopulations of patients benefit from the treatment. A better selection of patients to avoid treatment would be helpful. Therefore, pre-clinical models that are suitable for analysis and that represent clinical populations of patients are required. In this work 25 patient derived xenografts from lung cancer were intensely studied. First, the xenografts were compared with their corresponding patient tumor. The analysis of the histology and the expression of proliferation and epithelial or mesenchymal markers showed concordance of the patient tumor and the derived xenograft. The gene expression profiles were also maintained. Further analysis should elucidate the relevance of the xenografts as models for the characterisation and validation of predictive and prognostic markers. SNP, mRNA and protein expression profiles of resistance markers for chemotherapy were generated and showed similarities with clinical data. As marker for the anti-EGFR targeted therapies the ERBB receptors and the ligands of the EGFR were analysed. The mRNA and protein expression profiles resemble clinical data sets. An optimisation of the therapy should be achieved with gene expression studies. The vascular endothelial growth factor A was identified for a combination treatment with the anti-angiogenic drug bevacizumab in erlotinib resistant tumors. The combination of erlotinib and bevacizumab reduced the tumor growth in selected models. In summary, the analysis could show that the individual characteristics of the patient tumor were maintained in the xenograft. The models are a reliable tool for studies designed to improve treatment strategies

Response of Patient-Derived Non-Small Cell Lung Cancer Xenografts to Classical and Targeted Therapies Is Not Related to Multidrug Resistance Markers

Tumor cells that are nonsensitive to anticancer drugs frequently have a multidrug resistant (MDR) phenotype. Many studies with cell lines and patient material have been done to investigate the impact of different resistance markers at protein and mRNA level in drug resistance but with contradictory outcome. In the present study, 26 well-characterised patient-derived non-small cell lung cancer xenografts were used. The known chemosensitivity to etoposide, carboplatin, gemcitabine, paclitaxel and erlotinib was compared to the protein and mRNA expression of BCRP, LRP, MDR1, and MRP1. Further, four of these xenografts were short-term treated to analyse possible regulation mechanisms after therapeutic interventions. We found a borderline correlation between the bcrp mRNA expression and the response of xenografts to etoposide. All other constitutive mRNA and protein expression levels were not correlated to any drug response and were not significantly influenced by a short term treatment. The present results indicate that the expression levels of MDR proteins and mRNA investigated do not play an important role in the chemoresistance of NSCLC in the in vivo situation

Synergistic Antitumour Properties of viscumTT in Alveolar Rhabdomyosarcoma

Aqueous mistletoe extracts from the European mistletoe (Viscum album) contain mainly mistletoe lectins and viscotoxins as cytotoxic compounds. Lipophilic triterpene acids, which do not occur in conventional mistletoe preparations, were solubilised with β-cyclodextrins. The combination of an aqueous extract (viscum) and a triterpene-containing extract (TT) recreated a whole mistletoe extract (viscumTT). These extracts were tested on rhabdomyosarcoma in vitro, ex vivo, and in vivo with regard to anticancer effects. Viscum and viscumTT inhibited cell proliferation and induced apoptosis effectively in a dose- dependent manner in vitro and ex vivo, whereas TT showed only moderate inhibitory effects. viscumTT proved to be more effective than the single extracts and displayed a synergistic effect in vitro and a stronger effect in vivo. viscumTT induced apoptosis via the extrinsic and intrinsic pathways, evidenced by the loss of mitochondrial membrane potential and activation of CASP8 and CASP9. CASP10 inhibitor inhibited apoptosis effectively, emphasising the importance of CASP10 in viscumTT-induced apoptosis. Additionally, viscumTT changed the ratio of apoptosis-associated proteins by downregulation of antiapoptotic proteins such as XIAP and BIRC5, thus shifting the balance towards apoptosis. viscumTT effectively reduced tumour volume in patient- derived xenografts in vivo and may be considered a promising substance for rhabdomyosarcoma therapy

FRACIONAMENTO FÍSICO DA MATÉRIA ORGÂNICA DE UM LATOSSOLO VERMELHO DISTRÓFICO TÍPICO PELO MÉTODO DE SONICAÇÃO

A matéria orgânica do solo (MOS) pode ser definida como todo material que contém carbono orgânico do solo (COS), incluindo os microrganismos, vivos e mortos, resíduos de plantas e animais parcialmente decompostos, produtos da sua decomposição e substâncias orgânicas microbiológicas e/ou quimicamente alteradas. O fracionamento físico da MOS é caracterizado pela sua avaliação de forma menos destrutível comparado com o fracionamento químico. É realizada através das alterações na proporção das frações lábeis, como o carbono da fração grosseira, carbono orgânico particulado (COP), bem como, as frações que estejam associadas aos minerais (COAM), as quais podem fornecer informações importantes sobre a sustentabilidade ambiental e sobre a qualidade do solo. Os resultados obtidos deste fracionamento, que consiste na separação das frações da MOS agrupadas em diferentes tamanhos de partícula, são mais diretamente relacionados com frações que realmente existem no solo. Uma das técnicas de fracionamento físico da matéria orgânica é a sonicação. Essa técnica opera através da vibração ultrassônica, onde ocorre a formação de bolhas microscópicas, pela cavitação, o suficiente para produzir pressão e energia para o rompimento de ligações responsáveis pela agregação das partículas. Neste processo ocorre uma máxima dispersão na superfície das partículas do solo, fendas e linhas de fraqueza, com uma mínima alteração da amostra. A energia necessária liberada para essa dispersão promove uma desagregação pelo trabalho, além de calor pelo aquecimento da solução. Neste trabalho, o nível de energia de 522 W ((J mL-1) foi a necessária para dispersar diversos compartimentos da matéria orgânica do solo com maior proporção de carbono (C) associada aos minerais (COAM), e também com menor proporção, associada à fração particulada (COP)

A Natural Combination Extract of Viscum album L. Containing Both Triterpene Acids and Lectins Is Highly Effective against AML In Vivo

Aqueous Viscum album L. extracts are widely used in complementary cancer medicine. Hydrophobic triterpene acids also possess anti-cancer properties, but due to their low solubility they do not occur in significant amounts in aqueous extracts. Using cyclodextrins we solubilised mistletoe triterpenes (mainly oleanolic acid) and investigated the effect of a mistletoe whole plant extract on human acute myeloid leukaemia cells in vitro, ex vivo and in vivo. Single Viscum album L. extracts containing only solubilised triterpene acids (TT) or lectins (viscum) inhibited cell proliferation and induced apoptosis in a dose-dependent manner in vitro and ex vivo. The combination of viscum and TT extracts (viscumTT) enhanced the induction of apoptosis synergistically. The experiments demonstrated that all three extracts are able to induce apoptosis via caspase-8 and -9 dependent pathways with down-regulation of members of the inhibitor of apoptosis and Bcl-2 families of proteins. Finally, the acute myeloid leukaemia mouse model experiment confirmed the therapeutic effectiveness of viscumTT-treatment resulting in significant tumour weight reduction, comparable to the effect in cytarabine-treated mice. These results suggest that the combination viscumTT may have a potential therapeutic value for the treatment AML

Fracionamento físico da matéria orgânica de um latossolo vermelho distrófico típico pelo método de sonicação

Soil organic matter (SOM) can be defined as any soil organic carbon (SOC) material, including living and dead micro-organisms, partially decomposed plant and animal waste, decomposition products and organic microbiological substances and / or chemically altered. The physical fractionation of SOM is characterized by its less destructible evaluation compared to the chemical fractionation. It is performed through changes in the proportion of labile fractions, such as coarse fraction carbon, particulate organic carbon (POC), as well as fractions that are associated with minerals (COAM), which can provide important information on environmental sustainability and soil quality. The results obtained from this fractionation consisting of the separation of the fractions of the MOS grouped in different particle sizes are more directly related to the fractions that exist in the soil. One of the techniques of physical fractionation of organic matter is sonication. This technique operates through ultrasonic vibration, where the formation of microscopic bubbles occurs, through cavitation, produces pressure and energy for the rupture of connections responsible for the aggregation of particles. The maximum dispersion on the surface of soil particles, cracks and lines of weakness, with minimal sample change. The required energy released for this dispersion promotes disaggregation by heating the solution. In this work, the energy level of 522 W (J mL-1) was necessary for the dispersion of the various compartments of soil organic matter with a higher proportion of carbon (C) associated with minerals (COAM) and the lesser proportion associated with the particulate fraction (POC).A matéria orgânica do solo (MOS) pode ser definida como todo material que contém carbono orgânico do solo (COS), incluindo os microrganismos, vivos e mortos, resíduos de plantas e animais parcialmente decompostos, produtos da sua decomposição e substâncias orgânicas microbiológicas e/ou quimicamente alteradas. O fracionamento físico da MOS é caracterizado pela sua avaliação de forma menos destrutível comparado com o fracionamento químico. É realizada através de alterações na proporção das frações lábeis, como o carbono da fração grosseira, carbono orgânico particulado (COP), bem como, as frações que estejam associadas aos minerais (COAM), as quais podem fornecer informações importantes sobre a sustentabilidade ambiental e sobre a qualidade do solo. Os resultados obtidos deste fracionamento, que consiste na separação das frações da MOS agrupadas em diferentes tamanhos de partícula, são mais diretamente relacionados com frações que realmente existem no solo. Uma das ferramentas para que a técnica de fracionamento físico da matéria orgânica seja efetuada é a sonicação. O sonicador opera através da vibração ultrassônica, onde ocorre a formação de bolhas microscópicas, pela cavitação, o suficiente para produzir pressão e energia para o rompimento de ligações responsáveis pela agregação das partículas. Neste processo ocorre uma máxima dispersão na superfície das partículas do solo, fendas e linhas de fraqueza, com uma mínima alteração da amostra. A energia liberada para essa dispersão promove o aumento da temperatura da solução com o aumento do tempo de sonicação. Neste experimento, o nível de energia de 522 W (J mL-1) foi a necessária para dispersar os compartimentos da matéria orgânica do solo com maior proporção de carbono (C) associada aos minerais (COAM), e também com menor proporção, associada à fração particulada (COP)

GADD45A and CDKN1A are involved in apoptosis and cell cycle modulatory effects of viscumTT with further inactivation of the STAT3 pathway

Abstract ViscumTT, a whole mistletoe preparation, has shown synergistic induction of apoptosis in several pediatric tumor entities. High therapeutic potential has previously been observed in Ewing’s sarcoma, rhabdomyosarcoma, ALL and AML. In this study, we analyzed modulatory effects on the cell cycle by viscumTT in three osteosarcoma cell lines with various TP53 statuses. ViscumTT treatment induced G1 arrest in TP53 wild-type and null-mutant cells, but S arrest in TP53 mutant cells. Blockage of G1/S transition was accompanied by down-regulation of the key regulators CDK4, CCND1, CDK2, CCNE, CCNA. However, investigations on the transcriptional level revealed secondary TP53 participation. Cell cycle arrest was predominantly mediated by transcriptionally increased expression of GADD45A and CDKN1A and decreased SKP2 levels. Enhanced CDKN1A and GADD45A expression further played a role in viscumTT-induced apoptosis with involvement of stress-induced MAPK8 and inactivation of MAPK1/3. Furthermore, viscumTT inhibited the pro-survival pathway STAT3 by dephosphorylation of the two sites, Tyr705 and Ser727, by down-regulation of total STAT3 and its direct downstream targets BIRC5 and C-MYC. Moreover, tests of the efficacy of viscumTT in vivo showing reduction of tumor volume confirmed the high therapeutic potential as an anti-tumoral agent for osteosarcoma

Multiple Active Compounds from Viscum album L. Synergistically Converge to Promote Apoptosis in Ewing Sarcoma.

Ewing sarcoma is the second most common bone cancer in children and adolescents, with poor prognosis and outcome in ~70% of initial diagnoses and 10-15% of relapses. Hydrophobic triterpene acids and hydrophilic lectins and viscotoxins from European mistletoe (Viscum album L.) demonstrate anticancer properties, but have not yet been investigated for Ewing sarcoma. Commercial Viscum album L. extracts are aqueous, excluding the insoluble triterpenes. We recreated a total mistletoe effect by combining an aqueous extract (viscum) and a triterpene extract (TT) solubilized with cyclodextrins. Ewing sarcoma cells were treated with viscum, TT and viscumTT in vitro, ex vivo and in vivo. In vitro and ex vivo treatment of Ewing sarcoma cells with viscum inhibited proliferation and induced apoptosis in a dose-dependent fashion, while viscumTT combination treatment generated a synergistic effect. Apoptosis occurred via intrinsic and extrinsic apoptotic pathways, evidenced by activation of both CASP8 and CASP9. We show that viscumTT treatment shifts the balance of apoptotic regulatory proteins towards apoptosis, mainly via CLSPN, MCL1, BIRC5 and XIAP downregulation. ViscumTT also demonstrated strong antitumor activity in a cell line- and patient-derived mouse model, and may be considered an adjuvant therapy option for pediatric patients with Ewing sarcoma

Viscum, TT and viscumTT alter apoptosis-related protein expression.

<p><b>A.</b> Whole-cell protein extracts from TC-71 cells after 24h of treatment with viscum, TT or viscumTT concentrations approximating the IC50 were analyzed using the R&D systems human proteome profiler apoptosis array (n = 1). Bars represent the n-fold change in apoptosis-related protein expression relative to untreated control expression. <b>B.</b> TC-71 and MHH-ES-1 cells were treated with viscum, TT or viscumTT at the concentrations shown for 24h, then expression of the apoptosis-related proteins indicated was examined in whole-cell lysates using western blotting. Representative pictures are shown from 3 independent experiments. Mistletoe lectin (ML) and oleanolic acid (OA) concentrations were used as a measure of viscum and TT active agent extract concentration, respectively.</p