Temozolomide

Celecoxib (Pfizer, Täby, Sweden), dimethyl-PGE₂ (dmPGE₂), cyclophosphamide (given as the active metabolite 4-hydroxycyclophosphamide), temozolomide, doxycycline, XAV-939, salinomycin (all from Sigma-Aldrich, Solna, Sweden), O6-BG, Wnt-C59 and LGK974 (Cayman Chemical, Ann Arbor, MI, USA) were dissolved in dimethyl sulfoxide (Sigma-Aldrich). Cisplatin, irinotecan, doxorubicin and vincristine were purchased from the local pharmacy (Apoteket AB, Sweden) and diluted according to manufactures instructions. G007-LK was a kind gift from Dr Krauss, University of Oslo, Norway14 (link)27 (link). The mTOR inhibitors rapamycin (Sirolimus, LC Laboratories, Woodburn, MA, USA) and CCI-779 (TemSirolimus, a kind gift from Wyeth Pew River, NY, USA) were dissolved in 99.5% ethanol. LiCl was dissolved in H₂O. All inhibitors/activators were further diluted in OptiMEM (Gibco BRL, Sundbyberg, Sweden) to the desired in vitro concentration. temozolomide for the in vivo studies was supplied by the local pharmacy (Apoteket AB). For in vivo use of Celecoxib and temozolomide in the D283 xenograft study, the stock was prepared as a suspension in a vehicle fluid consisting of 0.5% methylcellulose (w/v; Sigma-Aldrich) and 0.1% Tween 80 (v/v; Sigma-Aldrich) in sterile water. For the LS174T xenograft study, temozolomide was dissolved in NaCl and doxycycline in water.

Cell viability was monitored using cell counting kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). Briefly, 5 × 10³ cells/well were plated into 96-well microplates. Next, the absorptions of the cells were measured at different indicated time points (1, 2, 3, 4, 5 and 6 days after seeding into plates) using a Benchmark Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. To investigate the inhibitory effect of temozolomide and radiation in cells, a series of concentrations of temozolomide (Sigma, St. Louis, MO, USA, 0, 10, 20, 40, 80 μM) or radiation intensity (0, 0.5, 1, 2, 4 Gy) were added to the cells.

U87MG and U118MG PTEN mutant and LN229 PTEN wild type (wt) glioblastoma cell lines as well as SW1783 PTEN mutant grade III astrocytoma cell line, were cultured in Dulbeco's Modified Eagle's Medium (DMEM) supplemented with 10 % inactive fetal bovine serum (FBSi, Gibco Invitrogen), at 37 ºC in a humidified 5% CO₂ atmosphere.
Patient-derived Glioma Stem Cell TG1 was obtained as described previously [41 (link)] and maintained in DMEM/F12 plus N2, G5 and B27 (Invitrogen) at 37 ºC in a humidified 5% CO₂ atmosphere.
PARP inhibitors PJ34 (Alexis Biochemicals) and AZD2281/olaparib (Deltaclon) were used. Olaparib was dissolved in DMSO and PJ34 was dissolved in water. Both were stored at −20ºC. Cells were treated with 10 μM olaparib or 10 to 20 μM PJ34 during 24, 48 or 72 hours.
Temozolomide (T2577-25MG Sigma-Aldrich, St Louis, USA) was dissolved in DMSO and stored at −20ºC. Cells were treated with 100 μM Temozolomide during 24, 48 or 72 hours.