gms | German Medical Science

Objective: The sphingosine-1-phosphate (S1P) pathway is mediated through G-protein coupled receptors signaling to the PI3K/Akt, Rho/ROCK and Ras/ERK/MAPK cascades. Previously it was shown that sphingosine kinase 1 and S1P-receptor 1 expression influence survival of glioma patients. FTY720 (fingolimod) is an S1P analogue with promising effects in glioblastoma. We investigate the antiproliferative effects of FTY720 versus temozolomide (TMZ) in glioblastoma cell lines and investigate the modulation of downstream signaling pathways.

Method: Glioblastoma cell lines A172, G28 and U87 were incubated with 5–50 µM FTY720 or TMZ for 24 to 72 h and proliferation was measured using an MTT assay and the xCELLigence real-time cell analyzer system, an impedance based cell proliferation and viability system. IC50 values for FTY720 were calculated at 72 hours and compared to TMZ effects. Gene expression of downstream pathways was quantified by real-time quantitative PCR.

Results: After 24 h, 10 µM FTY720 reduced viable A172 cells to less than 5% and after 72 to 2.3% of untreated controls. Analogous dose-dependent results were obtained for G28 and U87 cells with viable cells below 2% at 72 h using 50 µM FTY720. IC50 at 72 h of FTY720 incubation was 4.6 µM in A172 cells, 17.3 µM in G28 and 25.2 µM in U87 cells, respectively. Furthermore TMZ did not reduce viable cell counts below 50% in any cell line even at 50 µM. In qPCR analysis we observed the distinct gene expression changes after 72h incubation with FTY720 (n-fold relative to beta-actin and untreated controls) for Akt1, MAPK1, PRKCE, Rac1 and Roc1, respectively: in A172 cells 0.37, 0.33, 0.25, 0.20, 0.23; in G28 cells 0.43, 3.47, 2.90, 2.92, 4.64; and in U87 2.02, 0.79, 2.00, 1.07, 1.34

Conclusions: FTY720 has strong antiproliferative effects on glioblastoma cells at micromolar concentrations and also greatly surpasses TMZ effects at the mRNA level. Therefore, it may be a promising targeted drug in this cancer.