1004P - Effect of Hedgehog signaling pathway on the proliferation of high-grade gliomas

Abstract

High-grade gliomas are the most aggressive brain tumors. A clear understanding of the oncogenesis mechanisms and searching for specific targets among the proteins of signaling pathways involved in oncogenesis are needed to develop more effective therapy. According to current data, the Hedgehog pathway is involved in oncogenesis of glioma. In the case where the signal pathway is activated, GLI transcription factors alter the level of expression of the target genes, thus affecting the processes of proliferation, angiogenesis, chemoresistance, invasive and migratory activity. The aim of this study was to evaluate the effect of the activator (SHH) and inhibitor (cyclopamine) of Hedgehog signaling pathway on the proliferation of human glioma cell lines U87-MG, U251-MG and cells of human astrocytes. Cell proliferation was investigated using xCELLigence system, which allows measurement of the electrical resistance of the gold microelectrodes located on the bottom of 16-well E-plates. It was shown that the SHH ligand increases proliferation of U251-MG cell line and human astrocytes. SHH ligand has no effect on proliferation of U87-MG cell line. Cyclopamine has an inhibitory effect on the proliferation of human glioma cell lines U87-MG and U251-MG, and has no effect on human astrocytes. Mann-Whitney test and Student's t-test with significance level &agr; = 0.05 was used for statistical analysis. Cyclopamine has an inhibitory effect on U251-MG culture, so we can assume that the pathway is active. Proliferation of U87-MG cell line was decreased by adding cyclopamine and adding SHH has no effect. The pathway also is active, but the addition of an activator does not provide additional stimulus. It can be assumed that the signaling pathway is most active. In human astrocytes cyclopamine has no effect on the proliferative activity of cells, and it is increased with the SHH addition. In this case the pathway is inactive, but SHH has an activating effect. Our experiments with cyclopamine and SHH provide additional information for determining the activity of a signaling pathway that will help to develop a quantitative assessment of functioning Hedgehog signaling pathway in the known cell lines and in primary cell cultures.