and plated on ultra low adherence 96 well plates at 2000 cells per well for all subsequent drug testing. Alternatively, patient derived dissociated GBM tissues were plated onto laminin selleck chem 1 coated plates. Cell populations were dissociated using Acutase and e panded for 5 10 passages, then plated on flat bottom for drug testing. Confirmation of stem cell marker e pression Primary neurospheres were cytospun onto glass slides. Adherent primary cultures were grown onto Permano chamber slides. Cells were incubated with human Nestin antibody and then with fluorescein labeled secondary antibodies, then stained with DAPI. The cells were visualized under a UV micro scope. Drug testing and survival assay As e plained above, cells were seeded onto either regular or ultra low adherence 96 well plates and incubated for 18 24 hours and then treated with vehicle control or single drugs or drug combinations.

After 96 hours of incubation, Alamar Blue was added directly to the culture medium, and the fluorescence measured at 560 90 after 4 12 hours to determine the number of viable cells. The IC50 was calculated. Prediction of blood brain barrier permeation by active compounds Although ample evidence has demonstrated that drugs of virtually any size or chemotype can enter brain tumor via leaky tumor microvessels, the ability to penetrate the intact blood brain barrier is reasonably hypothe sized to be useful for treating tumor cells infiltrating normal brain tissue along fiber tracts. Hence we estimated the capacity of active anti GBM compounds to cross the BBB.

We used standard software to calculate the Log BB value Log BB 0. 0148 PSA 0. 152 CLogP 0. 139. PSA polar surface area, p octanol water parti tion coefficient. Determination of cell cycle, autophagy, and apoptosis Cell cycle analysis GBM cells were seeded into 10 cm dishes at a density of 1 106, cultured overnight followed by the addition of 3 uM pitavastatin with 24 or 48 hours of incubation. Cells were trypsinized and fi ed in 70% ethanol for 30 minutes, incubated with 25 ug ml propidium iodide and 250 ug ml RNAase in PBS for 1 hour at 50 C. After PI staining, cells were analyzed via flow cytometry, and the percentage of cells in G0 G1, S and G2 phases were calculated by ModFit LT software version 3. 0. Detection of caspase activity Caspase 3 activity was measured with the Invitrogen Enzcheck caspase 3 assay kit 2 according to the man ufactures protocol.

Briefly, 3 106 U118 cell were cul AV-951 tured and pitavastatin, irinotecan or the combination was added to the medium for 12 or 24 hours. Then 106 cells were lysed, DEVD R110 fluorescence substrate was added, and the fluorescence signal was measured and compared with a standard curve. Caspase 3 7 activity was measured by the Apo One caspase3 7 Kit. 20,000 cells were seeded on to 24 well plates, pitavastatin and vehicle were added, followed by incubation and caspase 3 7 activity was measured phase 3 using a fluorescence based substrate. Detection of autophagy markers GFP