We used five glioma tumor initiating cell lines (GBM1A [15 (link)], QNS120, GBM612, GBM965, and QNS108 [16 (link)]) that were derived from intraoperative tissue samples of patients with GBM (Table 1 ). Patients were newly diagnosed and had not received prior treatment. Cells were cultured as described previously [16 (link),17 (link)].
aMT stock solution (800013; Fagron, St Paul, MN, USA) was dissolved in 15% propylene glycol (81172-1L, Sigma-Aldrich, St. Louis, MO, USA) in PBS, as previously described [13 (link),18 (link)]. Cells were treated with the vehicle or with aMT (1.0, 1.5, 3.0, 5.0 mM). We treated cells with agomelatine (S1243; Selleck Chemicals, Houston, TX, USA) or tasimelteon (S4281; Selleck Chemicals) (0.1, 0.25, 0.5, 1.0 mM) dissolved in dimethyl sulfoxide (D8418-500ML; Sigma-Aldrich) for 48 h. We also treated cells with temozolomide (TMZ) (S1237, Selleck Chemicals) for 48 h (0.7 and 3.0 mM), either alone or after a 48-h pretreatment with melatonin or the vehicle. For radiation treatment, cells pretreated with aMT or the vehicle for 48 h were exposed to radiation doses of 0.5, 1.0, or 1.5 Gy (XRad160 biological irradiator; Precision X-Ray, Inc., St. Louis, North Branford, CT, USA).
aMT stock solution (800013; Fagron, St Paul, MN, USA) was dissolved in 15% propylene glycol (81172-1L, Sigma-Aldrich, St. Louis, MO, USA) in PBS, as previously described [13 (link),18 (link)]. Cells were treated with the vehicle or with aMT (1.0, 1.5, 3.0, 5.0 mM). We treated cells with agomelatine (S1243; Selleck Chemicals, Houston, TX, USA) or tasimelteon (S4281; Selleck Chemicals) (0.1, 0.25, 0.5, 1.0 mM) dissolved in dimethyl sulfoxide (D8418-500ML; Sigma-Aldrich) for 48 h. We also treated cells with temozolomide (TMZ) (S1237, Selleck Chemicals) for 48 h (0.7 and 3.0 mM), either alone or after a 48-h pretreatment with melatonin or the vehicle. For radiation treatment, cells pretreated with aMT or the vehicle for 48 h were exposed to radiation doses of 0.5, 1.0, or 1.5 Gy (XRad160 biological irradiator; Precision X-Ray, Inc., St. Louis, North Branford, CT, USA).