Supplementary MaterialsFIGURE S1: High temozolomide (TMZ) concentrations significantly reduce glioblastoma cell line viability. time-dependent manner. High TMZ concentrations with IC50 DPG was able to induce U87MG (C) and T98G (D) cell viability reduction in incubation times (24, 48, 72, and 96 h) lower than those observed when used alone in a dose- and time-dependent manner. The graphic shows the standard deviation of three independent experiments. Statistics were performed in a two-tailed 0.05. All experiments were performed in triplicate and were repeated at least twice. Graphics are representative of one of three independent experiments. Image_2.TIF (202K) GUID:?8EFEF4DB-3125-4168-836E-E14292855A18 FIGURE S3: Dipotassium glycyrrhizinate (DPG) down-regulates and and mRNA levels in U87MG (= 0.02 and = 0.003, respectively) and (B) T98G (= 0.03 and = 0.008, respectively) compared to untreated cell lines using 18S reference. DPG decreases IRAK2 and TRAF6 mRNA levels in (C) U87MG-pcDNA3.3-miR146a and (D) T98G-pcDNA3.3-miR146a (= 0.03 and = 0.04, respectively) compared to untreated pcDNA3.3-miR146a cells using 18S reference. Data represent means and standard deviations of a representative experiment performed in triplicate. Statistics were performed in a two-tailed 0.05. Image_3.TIF (87K) GUID:?3EF01B3F-93B5-4F50-9F08-952F216C1207 Abstract It has been shown that nuclear factor kappa-B (NF-B) is constitutively activated in glioblastoma (GBM), suggesting that the pathway could be a therapeutic target. Glycyrrhetic acid (GA), a compound isolated from licorice (and and and by inducing DNA fragmentation and oxidative stress (Hibasami et al., 2005, 2006; Sivasakthivel et al., 2008). Both nuclear factor kappa B (NF-B) and tumor necrosis factor- (TNF-) are the key factors involved in cancer-related inflammation (Mantovani et al., 2008). NF-B mediates the transactivation of genes encoding inflammatory cytokines (e.g., TNF-), anti-apoptotic factors (e.g., and inhibition. Additionally, we suggested that DPG might be used for combinational therapy in GBM along with TMZ and we also provided information that brain tumor stem cells are targeted by DPG-mediating inhibition. Materials and Methods Reagents Dulbeccos Modified Eagles Medium (DMEM) high glucose and fetal calf serum (FCS) were obtained from Cultilab, Campinas, S?o Paulo, Brazil. DPG [chemical abstracts service (CAS) number 68797-35-3] and TMZ (CAS number 85622-93-1) were obtained from Verdi Cosmticos LTDA (Joanpolis, S?o Paulo, Brazil) and Sigma (Schering Plough Temodal?), respectively. Dehydroxymethylepoxyquinomicin (DHMEQ) was synthesized as previously described (Suzuki et al., 2004). It was dissolved in dimethyl CD46 sulfoxide (DMSO) (Synth, Diadema, S?o Paulo, Brazil) to prepare a 10 mg/ml stock solution. For single Tasisulam sodium and Tasisulam sodium combinatorial cell line treatments, TMZ was diluted in DMEM to prepare a 2,000 M stock solution. All treatment assays were performed in the presence of 10% FCS. Cell Lines U87MG and T98G cell lines were gently donated by Dr. Adriana da Silva Santos Duarte from Hemocenter, State University of Campinas, Campinas, S?o Paulo, Brazil. Both were cultured in DMEM supplemented with 10% FCS and 1% streptomycin/penicillin (Cultilab, Campinas, S?o Paulo, Brazil). For all experiments, 1 106 cells/ml were seeded and grown for 48C72 h before experimental treatments. Cells were maintained at a 37C, 5% CO2 environment and were passaged by Trypsin 0.25% (Cultilab) every 3C4 days. Cells were fed every 2C3 days and used for the experiments until the seventh passage after thawing. MTT Assay Cell viability was determined by MTT assay using DPG concentrations based on a previous publication in a murine macrophage-like cell line, RAW264.7, a human intestinal colorectal adenocarcinoma cell line, Caco2, and human colon carcinoma cell line HT29 with Tasisulam sodium 300 M (Vitali et al., 2013). Briefly, cells were seeded in 96-well flat-bottom plates (0.2 106 cells/plate), and 16.