Cell culture and tumor samples
GBM cell lines, T98G and U87MG, were maintained at 37°C in 5% CO2, in α-MEM medium supplemented with 15% heat-inactivated fetal bovine serum (FBS) and DMEM medium supplemented with 10% FBS, respectively. Cells were cultured up to 80% confluence and were harvested after trypsin treatment. Cells were then washed twice in PBS, pH 7.4, and counted in a Neubauer chamber. An aliquot of the cell pellet containing ~ 1x107 cells was frozen at -80°C for further protein extraction. For thapsigargin treatment, cells were cultured in 35 mm plates with 1 μM thapsigargin in medium for 24 h. The use of human material in this study was approved by the National Bioethics Commission of Brazil and by the Ethics Committee of the Medical School of Sao Paulo, University of Sao Paulo. Written consent was obtained from patients authorizing the use of their tissues in the present investigation.
Cell proliferation and doubling time assay
Cells were counted in a Neubauer chamber. Briefly, 2 × 105 cells of each line were plated onto 100 mm2 plates. Every 24 h the cells were treated with trypsin, centrifuged and resuspended in PBS for counting after dilution in a Turkey’s solution. Two counts were performed for each lineage, in triplicate. For doubling time analysis, cells were plated onto 6-well plates in triplicate at a concentration of 5 × 104 cells/well in DMEM. After 24, 48, 72 and 96 h, cells were collected after trypsinization and counted in a Neubauer chamber. Doubling time (in h) was calculated using the following formula = h × ln(2)/ln(c2/c1), where c is the number of cells at each time of collection and ln is a neperian logarithm. (Roth V, 2006 http://www.doubling-time.com/compute.php).
Cell migration assay
Transwell assays were done using 24-well plate inserts with pores 8 μm in diameter (Greiner Bio-One), in which 2.5 × 104 cells/mL (500μL/well) were plated on the top of the insert in serum-free medium containing 1 μM thapsigargin. The bottom of the pit was covered with 500 μL of DMEM and α-MEM with 10 or 15% fetal bovine serum (FBS), respectively, for the U87MG and T98G cell lines. Cells were allowed to migrate toward to the bottom chamber containing medium with FBS for 18 h. The cells on the upper membrane surface were removed with a cotton tip, and the migratory cells attached to the lower membrane surface were fixed in 4% paraformaldehyde and stained with 0.2% crystal violet solution. The lower membrane surface was photographed in ten randomly selected fields under 100x microscope magnification and the cells were counted in each field using the Image J software (NIH, Bethesda, MD). The number of migratory cells was obtained by triplicate analysis of three individual wells.
T98G and U87MG cell pellets of were thawed at 4°C and resuspended in lysis solution with 7.7 M urea, 2.2 M thiourea, 4% CHAPS and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO), submitted to three cycles of 5 minutes in an ultrasound bath (UltraSonic Clear 750, UNIQUE) for complete pellet resuspension and centrifuged at 20,000 x g for 30 min at 4°C. Protein concentration was determined by the method of Bradford .
Two-dimensional gel electrophoresis
The first dimensional is electric focusing (IEF) was done on the IPGphor system (Ettan IPGphor I GE Healthcare, Psicataway, NJ) using Immobiline Dry IPG strips (7 cm, pH 3-10 non-linear; GE Healthcare, Uppsala, Sweden) and 300 μg of protein extract were applied to IPG strips. IEF was started at 500 V and the voltage was gradually increased to 5000 V until completing 40,000 volt-h and the procedure ended at 8000 V for 30 minutes. After IEF, the gel strips were equilibrated in buffer I for reduction (6 M urea; 50 mM Tris–HCl, pH 8.8, 2% SDS, 30% glycerol, and 1% DTT), and buffer II for alkylation (1% DTT was replaced with 2.5% iodoacetamide); each sample was gently shaken for 15 minutes at room temperature (22°C). The second dimensional separation was done on 12.5% SDS-polyacrylamide gels (10 cm × 10 cm × 1 mm) using Hoofer SE 600 vertical chambers. Gels were stained with colloidal Comassie blue-G250 and images were captured by transmission scanning (Image Scanner, Pharmacia-Biotech, Sweden) using Magic Scan software (GE-Healthcare). Gel images were analyzed with the Image Master software, v.7.0 (GE-Healthcare). Replicates of 12 gels (triplicate of gels for each cell line) were obtained, and statistical analysis was carried out using Anova followed by the Student’s t-test for the selection of protein spots ( Additional file 2: Table S2).
Trypsin digestion and mass spectrometry analysis
Selected protein spots were manually excised from the gels. SDS and Coomassie blue were removed by successive changes of 50% acetonitrile in 0.1 M of ammonium bicarbonate, pH 7.8, following dehydration in neat acetonitrile and drying in Speed Vac (Savant, New York, NY, USA). Proteins were then digested with 0.5 μg of modified trypsin (Promega Corp., Madison, WI, USA) in 0.1 M ammonium bicarbonate for 18 h at 37°C. The reaction was stopped by the addition of 1 μL of neat formic acid. Tryptic peptides were extracted by passive elution and desalted in micro-tips filled with reverse-phase resin (POROS R2, Perceptive Biosystems, Foster City, CA, USA), previously equilibrated in 0.2% formic acid. The sample was desalted by two washes of 150 μL of 0.2% formic acid. Peptides were eluted in 30 μL of 60% methanol/5% formic acid solution, concentrated in Speed Vac and resuspended in α-cyano-4-hydroxycinnamic acid matrix solution (5 mg/mL). Two to five micro liters of each sample were loaded in MALDI target and analyzed with a MALDI-TOF-TOF mass spectrometer (Axima Performance – Kratos-Shimadzu, Manchester, UK).
PMF and CID-MS/MS spectra from 2 DE selected protein spots were submitted to protein identification by searching against the Swiss-Prot database version 57.2 filtered for Homo sapiens (total of 20,402 human sequences) using MASCOT version 2.2.04. The database search parameters were as follows: trypsin hydrolysis, one missing cleavage was allowed, fixed modification for carbamidomethyl-Cys and variable modification for methionine oxidation. The mass tolerance for precursor ions was 1.2 Da and mass tolerance for fragment ions was set at 0.8 Da. Protein identification was supported by MS/MS analysis of individual ions using CID-MS/MS. Proteins were identified on the basis of at least two unique peptides or one peptide if its score was higher than 35 (p < 0.05) or the amino acid sequence was consistently covered by a series of b and y type ion fragments ( Additional file 2: Table S2 and pride XML compatible file as Additional file 3).
The differentially expressed proteins were submitted to GO:: Term Finder software  and functional classification for biological processes was performed by gene ontology ( Additional file 1).
Western blotting was used to verify the expression of GRP78 in the protein extract from U87MG and T98G cell lines before and after thapsigargin. Samples were submitted to SDS-PAGE (12% polyacrylamide gels) and transferred to Hybond PVDF membranes (Amersham Biosciences) for one h at 35 V. A PVDF membrane was blocked with 5% dry non-fat milk, followed by incubation with goat polyclonal anti-GRP78 antibody (Santa Cruz Biotechnology). After TBS-T washing, PVDF membranes were incubated with donkey anti-goat immunoglobulin-peroxidase conjugate (Sigma Chemical) for 1 h at room temperature (22°C). The immune complexes were visualized by enhanced chemiluminescence using an ECL kit (GE Healthcare), and detected using Image Quant LAS 4000 mini (GE-Healthcare, USA).
Quantification of glucose and lactate under normoxic cell culture conditions
U87MG and T98G cell lines were seeded onto six well plates and incubated for 24 and 48 h with 3.5 × 104 cells (10 cm2/well) containing 2 mL of culture medium (4 g/L glucose, 2.6 g/L HEPES, 3.6 g/L NaHCO3, 1% penicillin/streptomycin, 1% L-glutamine, 10% FBS). In order to measure glucose consumption and lactate formation by U87MG and T98G cells, 3.5 × 104 cells were incubated in six-well plates. After a stabilization period, the plates were washed using phosphate buffered saline (PBS) and the medium was replaced with 2 mL of phenol-red-free minimum medium containing glucose, fetal calf serum (FCS) and L-glutamine. The assays were performed 24 and 48 h after incubation, along with cell counting. For glucose and lactate quantification, 200 μL supernatant samples were collected in micro tubes and frozen until the time for assay. Glucose quantification was performed using an enzymatic colorimetric assay based on the D-glucose oxidase method (Latest Diagnostic, São Paulo, Brazil). The color intensity was measured at 520 nm. The D-Lactate Assay Kit (Latest Diagnostic, São Paulo, Brazil) was used to measure D-lactate which is specifically oxidized by D-lactate oxidase and generates proportional color (λmax = 550 nm). The protocols were developed according to manufacturer instructions. Data were reported as glucose and lactate in μg/cell and represent the mean ± SEM of 6 determinations, and were analyzed by the Student’s t test.
Total RNA extraction and cDNA synthesis
Total RNA was extracted from tissues using RNeasy Mini Kit (Qiagen Inc, Hilden, Germany). RNA quantification and purification was evaluated by measuring absorbance and A260/A280 ratios in the range of 1.8-2.0 were considered satisfactory for purity standards. Denaturing agarose gel electrophoresis was used to assess the quality of the samples. Synthesis of cDNA was performed by reverse transcription from 1 μg total RNA, previously treated with 1 unit of DNase I (FPLC-pure, GE Healthcare, Piscataway, NJ) using random and oligo(dT) primers, an RNase inhibitor and Superscript III (Invitrogen Inc, Carlsbad, CA), following the manufacturer’s recommendations. The resulting cDNA was then treated with 1 unit of RNase H (GE Healthcare), diluted with TE buffer, and stored at -20°C until later use.
Quantitative real time (qRT-PCR)
The expression level of GRP78 was determined by qRT-PCR using the SYBR Green approach, in duplicate. Quantitative data were normalized relative to the internal housekeeping genes hypoxanthine phosphoribosyltransferase 1 (HPRT), beta-glucuronidase (GUSB) and TATA-box binding protein (TBP). The geometric mean of the three genes was used for relative expression analysis. Primer sequences were as follows (5′-3′): GRP78 F: GGTGACCTGGTACTGCTTGATGT, GRP78 R: TCCTTGGAATCAGTTTGGTCAT, HPRT F: TGAGGATTTGGAAAGGGTGT, HPRT R: GAGCACACAGAGGGCTACAA, GUSB F: AAAATACGTGGTTGGAGAGCTCATT, GUSB R: CCGAGTGAAGATCCCCTTTTTA, TBP F: AGGATAAGAGAGCCACGAACCA and TBP R: CTTGCTGCCAGTCTGGACTGT synthesized by IDT (Integrated DNA Technologies, Coralville, IA). Sybr Green I amplification mixtures (12 μl) contained 3 μl of cDNA, 6 μl of 2x Power Sybr Green I Master Mix (Applied Bios stems, Foster City, CA), and forward and reverse primers. Reactions were run on an ABI 7500 Real-Time PCR System (Applied Bios stems). The cycle conditions comprised incubation at 50°C for 2 min to activate UNG, initial denaturation at 95°C for 10 min, and 40 cycles at 95°C for 15 sec, and 60°C for 1 min. The minimum concentration of primers was determined by the lowest threshold cycle (Ct) and maximum amplification efficiency while minimizing nonspecific amplification (at a final concentration of 200 nM for GRP78, HPRT and TBP, and of 400 nM for GUSB). Analysis of DNA melting curves demonstrated a single peak for the whole set of primers. Standard curves were analyzed for all genes to check the efficiency of amplification of each gene. Additionally, agarose gel electrophoresis was employed to check the size of the amplified PCR product. GRP78 relative expression levels were analyzed in 10 non-neoplastic brain tissues (NN), 10 grade I astrocytomas (AGI) and 20 grade IV astrocytomas (AGIV). The 2-ΔΔCT equation was applied to calculate the relative expression of tumor samples versus the mean of non-neoplastic tissues where ΔCt = mean Ct GRP78 – Ct geometric mean of housekeeping genes and ΔΔCt = ΔCt tumor – mean ΔCt non-neoplastic tissues .
For immunohistochemical detection of GRP78, tissue sections were routinely processed and subjected to antigen retrieval. Briefly, slides were immersed in 10 mM citrate buffer, pH 6.0 and incubated at 122°C for 3 min using an electric pressure cooker (BioCare Medical Walnut Creek, CA). Specimens were then blocked and further incubated with a mouse monoclonal antibody raised against human GRP78 (goat polyclonal, N-20, Santa Cruz – sc-1050) at a final dilution of 1:200 at 16-20°C for 16 h. The reaction was developed using a Novolink commercial kit (Novocastra, New Castle, UK) at room temperature using diaminobenzidine, and Harris hematoxylin for nuclear staining. All prepared slides were independently analyzed by two pathologists, and the positive reaction was quantitated for GRP78 as the percentage of positive cytoplasm cells: zero (0), when no positivity was detected; 1, when up to 25% of positive cells were present; 2, for 26-50% of positive cells; 3, for 51-75% of positive cells, and 4, for over 76% of positive cells.
The statistical analysis of GRP78 expression by qRT-PCR in AGI, AGIV samples and NN tissues was performed by one-way ANOVA. Post hoc Tukey’s tests were applied to compare the differences between the non-neoplastic group and each tumor group Differences were considered to be statistically significant at p < 0.05.