Fachbereich Veterinärmedizin



    Kombinationstherapie von Anti-Tumor-Toxinen und Reinsaponinen am Maus-Tumormodell (2015)

    Mallinckrodt, Benedicta-Leonie von (WE 3)
    Berlin: Mensch und Buch Verlag, 2015 — VIII, 111 Seiten
    ISBN: 978-3-86387-589-3
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000099245
    Institut für Veterinär-Biochemie

    Oertzenweg 19 b
    14163 Berlin
    Tel.+49 30 838 62225 Fax.+49 30 838-62584

    Abstract / Zusammenfassung

    Since several years new strategies in the field of cancer therapies are getting more and more important. Especially targeted toxins are able to eliminate tumor cells by receptor specific binding. The classical chemotherapy is also more harmful for tumor tissues than for normal cells but still causes many severe side effects.
    The new therapeutic toxins are binding to tumor specific surface molecules such as the epidermal growth factor receptor (EGFR), which is highly overexpressed on a number of tumors. The binding subsequently leads to apoptosis of the tumor cells and inhibits the tumor growth. When ribosomeinactivating proteins (RIP’s) such as Dianthin or Saporin get merged with the epidermal growth factor, the fusion proteins DE and SE are formed.
    The release of the internalized RIP’s can be enhanced by the presence of triterpenoid saponins (SO1861, GS16 and SA1641). Therefore the efficacy of the toxic effect can be increased while the dose can be reduced. The first step of this work was to closer characterize DE ad SE. The yield of both RIP’s after identical expression was impressively higher in the case of DE compared to SE (0,375 mg/ml compared to 0,15–0,225 mg/ml).
    In addition DE showed a greater stability after exposure for 30 min at 24°C.
    Comparison of the enzymatic activity demonstrated a higher N-Glycosidase activity of DE towards SE (115,7 towards 79,9 pmol Adenin/pmol toxin/h).
    The investigations of both RIP’s in a circular dichroism spectrometer approved the already known, high homology of both toxins. Both graphs were extremely similar even after adding an equal molar concentration (11,3 nM) of SO1861. The screening of the melting points of both RIP’s showed a difference of only 1°C. In order to use different cell lines in animals, the expression of EGFR was examined and EGFR-positive bands were detected in Western Blot analysis. Furthermore, after injecting the cell lines into the animals, the immunhistochemical staining revealed a brown color, which indicates the presence of the receptor. After characterization of DE and SE and the proof of EGFR expression by the targeted cell lines a more exact analysis of the saponins was conducted.
    The examination of the hemolytic activity of the three tested saponins revealed that no hemolytic activity appears up to a concentration of 15,65 μg/ml. This concentration was far below the one, which was planed to be used for the animal experiments. The application of DE did not affect the hemolytic experiments negatively.
    It should be emphasized that SO1861 in comparison to the other saponins had shown the lowest toxicity. This observation was confirmed in the in vitro examinations with a real-time impedancebased device (xCelligence reader).
    Further in vitro experiments have been performed, in which the toxic effect of the RIP’s was tested together with the effect enhancing qualities of the saponins.
    This was not only conducted with the xCelligence reader but also with an endpoint cytotoxicity assay (MTT). In case of the MTT assay the IC50 of the cells that were treated only with SE was at a concentration of 10 nM whereas in combination with GS16 the concentration was 0,0001 nM. These strong enhancing effects of the saponins were also ascertained in the xCelligence reader and high receptor specificity was visible. The combined application of DE and SE was further investigated in vivo in syngeneic mouse models. After the injection of the tumor cells (1,25 × 105 TSA-EGFR cells) and the therapy (15 μg/injection GS16 in combination with 0,3 μg/injection SE or 30 μg/injection GS16 in combination with 0,1 μg/injection SE, in total 28 days, 8 doses) a regression of over 99 % (p < 0.05) was determined in the treated animals. For the first time a similar tumor model was successfully established and treated in athymic nude mice bearing a tumor of human colon carcinoma cells (2 × 106 cells, HCT116). In this case the regression of the tumor was 96 % (p < 0.05) for the treated animals. After finishing the therapies histological parameters were obtained in order to gather the side effects in the different organs. In case of the untreated animals of the disseminated tumor model lung metastasis was found in 75% of the cases apart from the primary tumor. The results of this work can be regarded as a strong basis for further developments of already existing colon cancer therapies and serve as a foundation stone for preclinical studies with this kind of targeted, saponin-enhanced tumor treatments.