gms | German Medical Science

Objective: FTY720 as immunomodulative drug has also an impact on cell signaling pathways involved in cell proliferation and survival, and several studies suggest an effect on epithelial to mesenchymal transition (EMT) pathways. In this study we investigated the antiproliferative effect of FTY720 on immortalized and primary human glioblastoma cell lines and whether there is a synergistic effect in the combination with Temozolomide (TMZ). In a second step the influence of FTY720 on the gene expression level of EMT related genes was investigated.

Methods: U87, A172 and U251 as well as three primary human glioblastoma cell cultures were plated to 80-90% confluence. FTY720 was administered at a dose of 5 and 10μM alone and in combination with 150μM TMZ for 72 hours. Afterwards cell survival was evaluated by XTT-Assay and automatic cell count and colony forming assays were performed. For gene expression analysis cells were prepared in the same way. After 72 hours RNA was isolated and expression of CDH1, CDH2, CDK1, VIM, SNAI1, SNAI2, ZEB an STAT3 was analyzed by quantitative real-time PCR.

Results: FTY720 had an antiproliferative effect on human glioblastoma cells at 5 and 10μM. At a dose of 10μM FTY720 had a significantly stronger impact on cell survival than TMZ in A127 (p=0.002), U87 (p=0.041), U251 (p=0.002) and all patients derived cell lines (p=0.002, 0.002, 0.015). Combined treatment with FTY720 10μM and TMZ 150μM was significantly more effective than TMZ alone in all cell cultures and the combined treatment had an stronger impact than any single treatment on the survival fraction in U87 (p=0.001), U251 (p=0.001) and one patient derived cell line (p<0.001). In the other cell lines the same trend was observed, however not reaching statistical significance. No significant change of the expression of the EMT-related genes CDH1, CDH2, CDK1, VIM, SNAI1, SNAI2, ZEB an STAT3 was observed.

Conclusion: FTY720 has an antiproliferative effect not only on immortalized but also on primary human glioblastoma cell lines alone and in combination with TMZ. The combination of FTY720 and TMZ in the treatment of glioblastoma cells leads to cell death in micromolar concentration of FTY720 and is significantly more effective than treatment with TMZ alone. Despite recent evidence, no influence on the expression of relevant EMT genes could be observed, suggesting a different mechanism of action of FTY720.