The established malignant glioma cell lines U87, T98G, U373, LN229, and A172 were obtained from the American Type Culture Collection (Manassas, VA). LN18, LNZ428, and LNZ308 were generously provided by Dr. Nicolas de Tribolet. U87, T98G, and U373 were cultured in growth medium composed of minimum essential medium; LN18, LN229, A172, LNZ428, and LNZ308 were cultured in α-minimal essential medium. All growth media contained 10% fetal calf serum, L-glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA) supplemented with sodium pyruvate and nonessential amino acids. Human astrocytes (HA) and human cerebellar astrocytes (HAC) were obtained from ScienCell Research Laboratories (San Diego, CA). Human umbilical vein endothelial cells (HUVEC) were purchased from Cell Applications Inc (San Diego, CA). Astrocytes and HUVEC growth media were obtained from respective vendors.
Soluble human recombinant SuperKillerTRAIL™ (referred as TRAIL in this manuscript) was purchased from Enzo Biochemicals (Enzo Life Sciences, Plymouth Meeting, PA) diluted and stored in KillerTRAIL storage and dilution buffer (Enzo Life Sciences). Caspase inhibitors (Z-IETD-fmk, Z-LEHD-fmk, Z-DEVD-fmk, and Z-VAD-fmk) were purchased from R & D Systems (Minneapolis, MN). Bortezomib was purchased from Chemie Tek (Indianapolis, IN).
Cell Proliferation and Cytotoxicity Assay
Cells (5 × 103/well) were plated in 96-well microtiter plates (Costar, Cambridge, MA) in 100 μl of growth medium, and after overnight attachment, were exposed for 3 days to a range of concentrations of inhibitors, alone and in combination. Control cells received vehicle alone (DMSO). After the treatment interval, cells were washed in inhibitor-free medium, and the number of viable cells was determined using a colorimetric cell proliferation assay (CellTiter96 Aqueous NonRadioactive Cell Proliferation Assay; Promega, Madison, WI). All studies were conducted in triplicate and repeated at least three times independently. Morphological changes such as cell shrinkage, rounding, and membrane blebbing were evaluated by microscopic inspection of cells. Images were taken using an Olympus FluoView 1000 microscope. Images were assembled using Adobe Photoshop CS2 software (Adobe Systems).
Clonogenic Growth Assay
The effect of different inhibitor concentrations on cell viability was also assessed using a clonogenic assay. For this analysis, 250 cells were plated in six-well trays in growth medium, and after overnight attachment, cells were exposed to selected inhibitor concentrations or vehicle for 1 day. Cells were then washed with inhibitor-free medium and allowed to grow for 2 weeks under inhibitor-free conditions. Cells were then fixed and stained according to the manufacturer's protocol (Hema 3 Manual Staining Systems; Fisher Scientific, Pittsburgh, PA). After staining, six well plates were scanned and images were assembled using Adobe Photoshop CS2 software (Adobe Systems).
Annexin V Apoptosis Assay
Apoptosis induction in control (DMSO-treated) or inhibitor-treated cells was assayed by the detection of membrane externalization of phosphatidylserine with Annexin V-FITC conjugate using an Annexin V assay kit according to the manufacturer's protocol (Invitrogen). In brief, 2 × 105 cells were harvested at various intervals after treatment and washed twice with ice-cold phosphate-buffered saline (PBS) and resuspended in 200 μl of binding buffer. Annexin V-FITC and 1 μg/ml propidium iodide were added and cells were incubated for 15 min in a dark environment. The reaction was stopped by adding 300 μl of 1 × binding buffer, and labeling was analyzed by flow cytometry with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).
Antibodies and Western blot analysis
The following antibodies were used: p21 Waf1/ Cip 1 (#2946), Bim (#2819), Bcl-2 (#2872), Bcl-xL (#2764), DcR3 (#4758), DcR1 (#4756), FLIP (# 3210), XIAP (#2042), Survivin (#2808), FADD (#2782), Mcl-1 (#4572), Bik (#4592), Bid (#2002), Cleaved PARP (#9546), Cleaved Caspase 3 (#9664), Cleaved Caspase 8 (9496), DR3 (#3254), NF-κB p65 (#3034), IκB-α (#4814), phospho- IκB-α -Ser32/36 (#9246), β-Actin (#4970) were from Cell Signaling Technology Inc., (Beverly, MA). DR4 (#IMG-141A) and DR5 (#IMG-120A) were from IMGENEX (San Diego, CA). Western blot analysis was performed as described previously (23
). Scanning densitometry was performed on Western blots using acquisition into Adobe Photoshop (Adobe Systems, Inc., San Jose, CA) followed by image analysis (UN-SCAN-IT gel™, version 6.1, Silk Scientific, Utah, UT).
Four optimal 29mer-pRS-shRNA constructs were obtained from Origene (Rockville, MD). Sequences specific for human p65/NF-κB knockdown: GAT GAA GAC TTC TCC TCC ATT GCG GAC AT (shRNA-1); GCT GTG TTC ACA GAC CTG GCA TCC GTC GA (shRNA-2); CCA CCA TCA AGA TCA ATG GCT ACA CAG GA (shRNA-3); GAT CGT CAC CGG ATT GAG GAG AAA CGT AA (shRNA-4); non-targeting (control) sequences: TGA CCA CCC TGA CCT ACG GCG TGC AGT GC (shRNA-non-target) were used for this study. Glioma cells were seeded in a six-well plate (for Western analysis) or 96 well plate (cell proliferation assay) and maintained in complete media containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2. Transfection of p65/NF-κB shRNA was performed by using FuGene 6 according to the manufacturer's recommendations (Roche Applied Science, Indianapolis, IN). For control experiments, cells were transfected with a nontargeting (scrambled) shRNA construct. Cells were plated in culture plates and reached 70% to 80% confluence before transfection. For six well plates, two μg of p65/NF-κB B shRNA or non-targeting shRNA in 250 μL Opti-MEM medium was mixed with 6 μL of FuGene 6 in 250 μL Opti-MEM medium by vortexing. After the mixture was incubated at room temperature for 20 min, 1.5-mL of culture medium was added to make the total volume up to 2 mL. For 96 well plates, 25 ng of shRNA or non-targeting shRNA in 25 μl Opti-MEM medium was mixed with 0.6 μl of FuGene 6 in 25 μl Opti-MEM medium. After the mixture was incubated at room temperature for 20 min, complete medium was added to make the total volume up to 150 μl. Cell proliferation and Western blot analysis was performed as described above.
NF-kB DNA Binding Activity by ELISA and Electrophoretic mobility-shift (EMSA) assay
To detect p65/NF-kB binding activity, cell nuclear extracts were obtained using Nuclear Extraction kit (Active Motif, Carlsbad, CA). Nuclear extracts (15 μg) were used in each reaction in triplicate. The binding activity was evaluated using TransAM kits for NF-kB family members according to manufacturer's protocol (Active Motif). The absorbance was measured at wavelength of 450 nm by a microplate reader (Gene Mate-UniRead 800).
For NF-κB binding activity, a nonradioactive electrophoresis mobility-shift assay kit was used according to the manufacturer's instructions (Panomics, Fremont, CA). Briefly, malignant human glioma cell lines were incubated with or without inhibitors for the indicated period of time. Equal amounts of nuclear extracts was incubated at 15 °C for 30 minutes with a biotinylated oligonucleotide containing the p65/NF-κB binding site, and then the samples were separated on 6.0% non-denaturing polyacrylamide gel in 0.5 × TBE buffer for 90 minutes at 120 V at 4 °C. To confirm specificity, NFκB DNA binding was competitively blocked by an NFκB cold probe. The gel contents were transferred onto Biodyne B membrane (Pall, Ann Arbor, MI) for 45 minutes at 300 mA. The protein/DNA complexes were visualized after exposure to film.
DiOC6 labeling and detection of mitochondrial membrane depolarization
Mitochondrial membrane depolarization was measured as described (24
). In brief, floating cells were collected, and attached cells were trypsinized and resuspended in PBS. Then the cells were loaded with 50 nM 3′,3′-dihexyloxacarbo-cyanine iodide (DiOC6, Invitrogen) at 37°C for 15 min. The positively charged DiOC6 accumulates in the intact mitochondria, whereas mitochondria with depolarized membranes accumulate less DiOC6. Cells were spun at 3,000 × g
, and rinsed with PBS twice and resuspended in 1 ml of PBS. Fluorescence intensity was detected by flow cytometry (FACScan, Becton-Dickinson) and analyzed with the CellQuest software (Becton Dickinson). Percentage of cells with decreased fluorescence was determined.
Unless otherwise stated, data are expressed as mean ± S.D. The significance of differences between experimental conditions was determined using a two-tailed Student's t test. Differences were considered significant at p values <0.05.