gms | German Medical Science

Objective: Glioblastoma multiforme (GBM) is the most common primary brain tumor. Its malignancy is determined by a complex interaction of different cell types and the microenvironment. Tumor inducing stem cells might cause these tumors and contribute to their prominent therapy resistance. Interestingly, cytokines as glial cell line-derived neurotrophic factor (GNDF) involved in the regulation of cell proliferation, differentiation and neuronal survival, are highly expressed in GBM. Targeting GDNF in combination with cytotoxic biomolecules such as nitric oxide (NO) might be a strategy to improve treatment efficacy. The nitric oxide donor JS-K releases NO and selectively induces cell death in GBM cells. The aim of this study was to investigate the effect of GDNF on the expression stem cell and progenitor marker genes and its influence on JS-K-induced cell death in vitro.

Method: The expression of stem cell and progenitor markers (Nestin, GFAP, betaIII-tubulin, Sox2), GDNF and GFRa1 in the GBM cell line T98G and primary GBM cells (AS) was assessed after stimulation/inhibition of GDNF by immunocytochemistry, PCR and Western blot. Cells were treated with JS-K (0 - 25 µM), GDNF (10 ng/ml) or anti-GDNF (3 µg/ml) alone or in combination (4 - 72 h). Cytotoxic effects were analyzed by MTT assay. Akt/PI3K activation after treatment was determined by Western blot. Statistical analysis was performed by student’s t-test.

Results: Stem and progenitor markers (Nestin, GFAP, betaIII-tubulin), GDNF and GFRa1 are strongly expressed in primary AS and to a lesser degree in T89G cells. Their expression is independent of GDNF signaling. JS-K decreased viability in a time- and dose-dependent manner (≥ 5 µM) in both cell lines. However, T98G recovered significantly after 72 h. The combination of JS-K with GDNF induced higher survival and resistance compared to JS-K alone, whereas JS-K treatment accompanied by GDNF inhibition enhanced the cytotoxic effect of JS-K and abolished the capability of T89G cells to recover after NO treatment. Western Blot analysis suggests that these effects are mediated via the Akt/PI3K pathway.

Conclusions: GDNF is one mediator of cell survival and resistance in GBM, especially in GBM cells exhibiting stem cell features. The inhibition of GDNF-signaling in combination with anti-proliferative and cytotoxic biomolecules as NO might be a promising tool to improve the efficacy and outcome in multimodal GBM treatment.