Figure 2

Evaluation of the MEK inhibitor Trametinib as part of a combination therapy approach with either chemo- or radiotherapy. (A) Effects on cell viability following treatment with Temozolomide (TMZ; at 1, 10 and 100 µM) and Trametinib (at 0.3, 3 and 30 nM) examined by MTT assay after 120 hours treatment. As three concentrations per drug were used, a total of nine distinct combinations could be examined, each combination is represented by a data point of unique colour/shape. Normalized isobolograms were calculated using the CompuSyn software tool. The connecting line represents additivity. Data points located below the diagonal line indicate a synergistic drug-drug interaction and data points above the diagonal line indicate an antagonistic drug-drug interaction. Data are obtained from three independent experiments performed in triplicate and summarized in Table 1. (B) Effect of combination of Trametinib and Temozolomide on the cell number of GB cell lines. The total viable cell number was measured using a cell counter after 120 hours of incubation of A172 (left) and U87MG (right) with 1, 10 and 100 μM Temozolomide in the presence or absence of 30 nM Trametinib as indicated. The control cells were treated with DMSO. The cell number ratio, normalised to controls, is defined as the ratio of cell number in treated population to cell number in the respective control. (C) Effect of Trametinib and Temozolomide combination on induction of apoptosis in GB cell lines. Flow cytometric analysis of Propidium-iodide stained nuclei after 120 hours of treatment with 1, 10 and 100 μM Temozolomide in the presence or absence of 30 nM Trametinib in the GB cell lines A172 (left) and U87MG (right). Control cells were treated with DMSO. DNA fragmentation was taken as readout of apoptosis and treatment induced DNA fragmentation (specific apoptosis) was calculated as described in the methods section. (D) Schematic representation of the drug-irradiation schedules. For combination treatment of Trametinib and Irradiation, Trametinib was administered prior to (<) or after (>) irradiation with 2 Gray in the respective sets as indicated. (E) Effect of Trametinib in combination with irradiation on the cell number of GB cell lines. A172 (left) and U87MG (right) cells were treated with Trametinib, irradiation, or both in differently scheduled combinations as shown in C. Controls were treated with DMSO. The cell number was detected by cell counter after 120 hours always following the last fraction of irradiation. Depicted is the calculated ratio of the respective treatment to the respective control, i.e. control was defined as 1 for all three treatment sets. (F) Effect of Trametinib and irradiation combination on induction of apoptosis in GB cell lines. Flow cytometric analysis of Propidium-iodide stained nuclei of A172 (left) and U87MG (right) cells after treatment with Trametinib, irradiation, or both in differently scheduled combinations as indicated in (C). Controls were treated with DMSO. In (B) mean and +SD of three independent experiments performed in sextuplicate are shown, in (C,E,F) mean and +SD of three independent experiments performed in triplicate are shown. Statistical significance was assessed by ANOVA (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001).