The effects of combination therapy with PI-103 and temozolomide on glioblastoma cell viability.

<p>Shown is the relative cell viability of G35 (A), G38 (B) or G40 (C) glioblastoma stem (left) and differentiated (right) cells after treatment with a combination of 1.8 μM PI-103 and 100 μM temozolomide (TMZ) for the indicated times. Shown are the mean+SD of three independent experiments, each the average of six values. The red bar indicates the statistical value that defines the mean of an additive effect.</p

The effects of the PI3K/mTOR inhibitor PI-103 on glioblastoma cells.

<p>(A) Different glioblastoma stem cells were either left untreated (i.e. exposed to DMSO solvent alone) or treated for 24 hrs and 72 hrs with indicated concentrations of PI-103, after which the cell population's viability was assessed. Untreated controls were defined as 100%. (B) Distinct differentiated glioblastoma cells (derived from stem cells used in A) were either left untreated (exposed to solvent alone) or treated for 24 hrs and 72 hrs with indicated concentrations of PI-103, after which the cell population's viability was assessed. Untreated controls were defined as 100%. (C) Different glioblastoma stem cells were either left untreated (exposed to solvent alone) or treated for 24 hrs with indicated concentrations of PI-103. Protein expression levels and phosphorylation status of Akt and S6 ribosomal protein served as surrogate read-outs for PI3K and mTOR activity, respectively, and were analyzed by Western blotting, GAPDH served as loading control. (D) Distinct differentiated glioblastoma cells were either left untreated (exposed to solvent alone) or treated for 24 hrs with indicated concentrations of PI-103. Protein expression levels and phosphorylation status of Akt and S6 ribosomal protein served as surrogate read-outs for PI3K and mTOR activity, respectively, and were analyzed by Western blotting, GAPDH served as loading control. Shown in A and B is the mean+SD of at least three independent experiments carried out in triplicate, while in C and D a representative result of two independent experiments is depicted. Red numbers indicate the p-value derived from a two-sided Student's <i>t</i>-test.</p

The effects of PI-103 on differentiation.

<p>(A) Scheme of the experimental set-up used to collect data presented in B-D, showing Nestin expression in the G 35, G 38 and G 40 stem cells (left) and GFAP expression in the G 35, G 38 and G 40 differentiated cells (right). (B) 24 and 72 hrs after seeding stem cells onto cell culture-treated plastic in the presence of differentiation medium, the total amount of cell, adherent (a surrogate for differentiation) and in suspension (a surrogate for 'stemness' or non-viability) was counted. (C) The relative amounts of differentiated cells (as defined by adhesion) was determined 72 hrs after initiation of differentiation, either in the presence or absence of 1.8 μM PI-103, i.e. controls were exposed to the solvent only (DMSO). (D) The relative amounts of cells, either stem cells (left) or differentiated cells (right) was determined 72 hrs culturing under appropriate conditions, either in the presence or absence of 1.8 μM PI-103, i.e. controls were exposed to the solvent only (DMSO). Shown are the mean+SD of three independent experiments carried out in triplicate. Red numbers indicate the p-value derived from a two-sided Student's <i>t</i>-test.</p

The effects of combination therapy on differentiated glioblastoma cell viability.

<p>Shown is the relative cell viability of G35 (A), G38 (B) or G40 (C) glioblastoma stem (left) and differentiated (right) cells after treatment with a combination of 1.8 μM PI-103 and 10nM irinotecan for the indicated times. Shown are the mean+SD of three independent experiments, each the average of six values. The red bar indicates the statistical value that defines the mean of an additive effect.</p

The effects of prolonged exposure to 1.8 μM PI-103 on glioblastoma cells.

<p>(A) Different glioblastoma cells, either stem cells (left) or differentiated cells (right) were left untreated (i.e. exposed to DMSO solvent alone) or treated for indicated times with 1.8 μM PI-103. Protein expression levels and phosphorylation status of Akt and S6 ribosomal protein served as surrogate read-outs for PI3K and mTOR activity, respectively, and were analyzed by Western blotting, GAPDH served as loading control. (B) After seeding cells, either untreated (exposed to solvent alone)or treated with 1.8 μM PI-103 were counted every 24 hrs for a total of 120 hrs. (C) Cells were cultured either in the presence or absence of 1.8 μM PI-103 for indicated times, while controls were exposed to the solvent only (DMSO). This was followed by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. Treatment induced DNA fragmentation, a surrogate for apoptosis induction, is shown relative to spontaneous cell death of untreated cells. (D) Cells were cultured either in the presence or absence of 1.8 μM PI-103 for 24 hrs, while controls were exposed to the solvent only (DMSO), and the percentage of cells within the live population that reside in the G_0/1 phase of the cell cycle were determined by FACS analysis of propidium iodide-stained nuclei. Shown in A is a representative result of two independent experiments, while B, C and D depict the mean+SD of three independent experiments carried out in triplicate. Red numbers indicate the p-value derived from a two-sided Student's <i>t</i>-test.</p

The effects of PI-103 on cellular motility.

<p>(A) Differentiated glioblastoma cells (upper panel) or established cell lines (lower panel) were allowed to grow ~70% confluent, upon which they were either left untreated or pretreated with 1.8 μM PI-103 for 1 hr (see box-out for morphological example of 1 hr treatment). Cellular monolayers when then scared with a micropipette tip and the wound was allowed to close for 18 hrs, in the presence or absence of PI-103, i.e. controls were exposed to the solvent only (DMSO). (B) The random motility of glioblastoma cells, both cell lines and freshly differentiated cells, was tracked via timelapse microscopy for 6 hrs in the presence or absence of 1.8 μM PI-103, i.e. controls were exposed to the solvent only (DMSO). Shown in A is a representative result of three independent experiments performed in triplicate, while B depicts the summary of two independent experiments, in each 10 cells were tracked and their average travel distance assessed. Red numbers indicate the p-value derived from a two-sided Student's <i>t</i>-test.</p