Gentargeting des Onkogens HER-2 und des Wachstumsfaktors VEGF über Intronribozyme und Untersuchungen zur HER-2-abhängigen Resistenz gegenüber Taxol und Viscumin

Cancer is becoming a more and more important disease in our population in the last years. Cancer is the second often cause of death in Germany (statistics of the German cancer register - published in 2006). Even more important becomes the development of new options in the treatment of cancer. The HER-2-receptor is a tyrosinkinasis-receptor. It is an important marker for agressive cancer growth in different tumors and corresponds with an increased potential in metastasis and increased resistance to cytostatic agents. The influence of HER-2 on the chemoresistance of ovarian cancer is controversionally discucced. This dissertation investigates the influence of a reduced level of HER-2 on the proliferation of the ovarian cancer cells SKOV-3 and their chemoresitance to the cytostatic agents Taxol and Viscumin. The HER-2 receptor was inhibited either by specific ribozymes or by the HER-2 antibody Herceptin. Ribozymes are katalytic RNA molecules that specifically cut the target RNA and lead to an inhibition of the protein biosynthesis. It is shown that the proliferation of SKOV-3 cells in adhesion and softagar cultures drops corresponding to the HER-2 level of the cells. An additive effect between ribozymes and Herceptin could not be shown. A reduced level of HER-2 resulted in a reduced chemosensitivity of the cells. An additive effect of ribozymes and Herceptin on the chemosensitivity of ovarian cancer cells could not be observed. Further this dissertation examined the efficiency of intronribozymes. Therefore the expression of the oncogene HER-2 in SKOV-3 ovarian cancer cells and the expression of the growth facotr VEGF in U87 glioblastoma cells was inhibited by specific intronribozymes. In SKOV-3 cells intronribozymes lead to a reduced level of HER-2. This effect was demonstrated in FACS-analysis and ELISA. Likewise intronribozymes were capable of reducing the VEGF expression in U87 glioblastoma cells. This effect was secured by northern blot analysis and ELISA. Using intronribozymes that were inactivated by mutation in the catalytic center resulted in a reduced VEGF protein level without influencing the VEGF RNA level. Intronribozymes showed an equally effective and specific ability as exonribozymes in the inhibition of the protein biosynthesis of a specific protein. Intronribozymes seemed to have an additional antisense effect. This dissertation supplies evidence that intronribozymes are a specific, effective and promising genetargeting strategy. Further this dissertation shows that the proliferation and chemosensitivity of SKOV-3 cells is depending on the HER-2 level. The HER-2 level of SKOV-3 cells could be equally efficiently downregulated by ribozymes and the HER-2 antibody Herceptin.