Cells and reagents
BL cell lines Namalwa, Raji, Daudi, Ramos and DLBCL cell line DB were available from American Type Culture Collection. Cells were maintained in RPMI-1640 medium, supplemented with 10% heat-inactivated fetal bovine serum in a humidified atmosphere of 95% air and 5% CO2 at 37°C. VPA (V3640) and temsirolimus (PZ0020) were from Sigma-Aldrich. 3-Methyladenine (189490), and ZVAD-FMK (219007) were from Merck & Co. Inc. Bafilomycin A1 (sc-201550) was from Santa Cruz Biotechnology.
Fresh BL cells were extracted from the lymph node and bone marrow of patients. CD34+ cells were isolated from human cord blood using CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec Inc.). The study was approved by the Institutional Review Board and informed consent was obtained in accordance with the Declaration of Helsinki.
MTT reduction assay
To assess growth inhibition, cells were treated with VPA, temsirolimus, either alone or in combination, in a 96-well plate. After 48 h, 0.1 mg MTT (Sigma-Aldrich, M2003) was added to each well. The samples were incubated at 37°C for 4 hours and the absorbance was measured at 490 nm by spectrophotometry.
Flow cytometric assay
To assess the distribution of nuclear DNA content, cells were collected, washed in PBS and fixed overnight in 75% ethanol at -20°C, treated with 1% RNaseA (Merck, 70856–3) for at least 15 minutes at 37°C and stained with 50 μg/ml propidium iodide. Cell apoptosis was analyzed using ApoAlert ANX-V-FITC Apoptosis Kit (Clontech Laboratories, Inc., 630110). Cell autophagy was analyzed using rabbit anti-human LC3 (Cell signaling, CST4108) as the primary antibody and DyLight 405 labeled anti-rabbit antibody (KPL, KPL072-08-15-06) as the secondary antibody. The flow cytometry data were collected by a FACSCalibur machine (Becton Dickinson) and analyzed by FlowJo software.
Determination of the synergistic effect of VPA-temsirolimus combination was performed using the isobologram of Steel-Peckham . Based on dose–response curves of the two agents, three isoeffect curves were constructed. The area surrounded by the isoeffect curves was referred as the envelope of additivity. When the data points fell to the left of the envelope, that is, the combined effect was caused by lower doses of the two agents than was predicted, the combination was regarded as having a synergistic effect. The synergistic effect was further confirmed by the combination index (CI) method described by Chou and Talalay (CalcuSyn software, Biosoft). When at least 80% of CI values for a combination were less than one, the drug combination was considered to be synergistic.
Small-interfering RNA (siRNA) transfection
Namalwa cells were transfected with ATG5, HDAC1, HDAC3 siGENOME SMARTpool or Non-Targeting pool as a negative control using DharmaFECT2 transfection reagent (Dharmacon) following the manufacturer’s instruction.
Cells were lysed in 200 μl lysis buffer (0.5M Tris–HCl, pH 6.8, 2 mM EDTA, 10% glycerol, 2% SDS and 5% β-mercaptoethanol). Protein extracts (20 μg) were electrophoresed on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat dried milk in Tris buffered saline and incubated for 2 hours at room temperature with appropriate primary antibody, followed by horseradish peroxidase-conjugated secondary antibody. The immunocomplexes were visualized using chemiluminescence phototope-horseradish peroxidase kit. Antibodies against LC3-I/II (4108), phosphorylated MTOR (p-MTOR) (2971), MTOR (2972), phosphorylated 4E binding protein-1 (p-4EBP1) (9456), 4EBP1 (9644), phosphorylated P70 ribosomal S6 kinase (p-P70S6K) (9205), P70S6K (2708), HDAC3 (3949), HDAC4 (5392), phosphorylated AKT (p-AKT) (4060), AKT (9272), ACTB (4970), c-caspase-3 (9664), c-PARP (9541) and chemiluminescence phototope-horseradish peroxidase kit (7003) were obtained from Cell Signaling. Antibodies against BECN1 (ab51031), MYC (ab28842), HDAC1 (ab150399) and HDAC2 (ab32117) were from Abcam. Anti-P62 antibody (BML-PW9860) was from Enzo Life Sciences, Inc. Horseradish peroxidase-conjugated goat anti-mouse-IgG (sc-2005) and goat anti-rabbit-IgG (sc-2004) antibodies were from Santa Cruz Biotechnology. ACTB was used to ensure equivalent protein loading.
Enzyme-linked immunosorbent assay
Enzymatic activity of HDAC1 and HDAC3 in lymphoma cells were quantified by enzyme-linked immunosorbent assay using nonisotopic HDAC (BioVision, K331-100) colorimetric kits according to manufacturer’s instructions.
Transmission electron microscopy
Cells and tissue samples were fixed overnight in 2% glutaraldehyde at 4°C, washed in 0.1M cacodylate buffer, postfixed in 1% osmium tetroxide for 1 hour at 4°C, dehydrated in graded ethanol and embedded in Epon 812 (TAAB Laboratories). Ultrathin sections were prepared, collected on copper grids, stained with uranyl acetate and lead citrate, and examined on electron microscopy (Philips CM120). Ultrastructural studies were focused on double membrane-bound autophagic vesicles named autophagosomes, a gold standard for autophagy.
Immunohistochemistry and immunofluorescence
Immunohistochemistry was performed on 5μm-paraffin sections with an indirect immunoperoxidase method using antibodies against CDKN1A and MYC. Immunofluorescence was performed on methanol-fixed cells using anti-BECN1 and anti-P62 as primary antibodies, and diaminotriazinylaminofluorescein-labeled donkey anti-rabbit-IgG antibodies (Abcam, ab6800) as the second antibody.
Nude mice (5-6-week-old) were obtained from Shanghai Laboratory Animal Center and injected subcutaneously with 7×106 Namalwa cells into the right flank. Treatments (10 mice per group) were started after tumor became about 0.5 cm × 0.5 cm in surface (day 0). The control group received dimethyl sulfoxide, while the other three groups received for 21 days oral VPA (0.4%w/v in the drinking water daily), intraperitoneal temsirolimus (5 mg/kg every other day), or in combination, respectively. Tumor volumes were calculated as 0.5 × a × b2, where ‘a’ is the length and ‘b’ is the width.
Terminal deoxytransferase-catalyzed DNA-nick-end labeling (TUNEL) assay
In situ cell apoptosis was confirmed by detection of fragmented DNA, using TUNEL assay, on 5 μm-paraffin sections, using DeadEnd Colorimetric TUNEL System (Promega Corporation, G7360) according to the manufacturer’s instruction. The tissue section of the same murine xenograft model co-treated with bortezomib and SAHA was referred as a positive control, as previously described by our study .
All assays were set up in triplicate and the results were expressed as the mean±S.D. of data obtained from three separate experiments. T-test was applied to compare two normally distributed groups and Bonferroni to perform multiple comparison. P<0.05 was considered statistically significant. All statistical analyses were evaluated using Statistical Package for the Social Sciences (SPSS) 13.0 software (SPSS Inc.).