Epigenetic Modifiers as Anticancer Drugs: Effectiveness of Valproic Acid in Neural Crest-derived Tumor Cells

Materials and Methods

Cell culture and treatment. U87MG human glioblastoma and G361 human melanoma cells, obtained from Interlab Cell Line Collection (Genoa, Italy), and SKNMC Askin tumor cells, purchased from the American Type Culture Collection (Rockville, USA) were maintained in RPMI (Sigma, USA), supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin and grown at 37°C in a humidified air with 5% CO₂. VPA (Sigma) was dissolved in the culture medium just before use to obtain final concentrations in the range of 0.5-2.0 mM.

Sulforhodamine (SRB) assay. The SRB assay is based on the ability of SRB dye to bind protein basic amino acid residues. The amount of dye incorporated by the cells indicates the cell number. Cells were plated in 96-well plates (100 μl/well) and treated with VPA in the range 0.5-2.0 mM for 1, 4 and 6 days. At the end of treatment, cell culture medium was removed and RPMI was added (50 μl/well). Cells were fixed using 25 μl/well 50% aqueous trichloroacetic acid for 1 h at 4°C, rinsed with water several times and incubated with 50 μl/well sulforhodamine B solution (0.4%) (Sigma) for 30 min. After rinsing with 1% acetic acid and solubilizing in 10 mM Tris for 5 min, the absorbance of each well was measured in a microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm. The results were expressed as a percentage of that of the controls (untreated cells).

MTT assay. The effect of VPA on cell viability was also evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (11).

Annexin V apoptosis detection assay. Experiments were performed on cells seeded in 6-well plates with slides, at a density of 8×10⁴ cells/well, treated for one day with VPA in the range 0.5-1.0 mM. Samples were prepared using 0.5 μg of Annexin V FITC (Bender MedSystems, Austria) for 15 min at the dark. After incubation, the cells were fixed with paraformaldehyde at 4°C for 30 min and marked with 10 μl (1 μg/ml) of propidium iodide per 100 μl assay buffer. After washing with phosphate-buffered saline (PBS) and water, samples were observed under fluorescence microscope using a dual filter set for FITC and rhodamine. Apoptosis was evaluated by counting FITC-labelled cells in five random fields and expressed as percentage of total cell number in comparison to untreated cells.

Zymography. MMP2 and MMP9 activity was determined by gelatin zymography (12). Cells were seeded and after 18 h were placed in serum-free medium (RPMI) with VPA at 1 or 2 mM, or not (control) for 24 h. After centrifugation (300 × g for 10 min), the supernatant was separated and protein concentration determined using 10 μg per lane, with sample buffer (Tris-HCl 1 M, pH 6.8, sodium dodecyl sulphate (SDS) 2%, glycerol 10%, applied to 10% SDS-polyacrylamide gel containing 1 mg/ml gelatin (Sigma). After electrophoresis, SDS was removed from the gel by washing twice with 2.5% Triton X-100 for 1 h. After a brief rinse, the gel was incubated at 37°C for 18 h in buffer at pH 7.6, containing 100 mM Tris-HCl, 10 mM CaCl₂, 20 mM NaCl. The gel was stained with 1% Coomassie Brilliant Blue R250 for 2 h and then treated with destaining solution (40% methanol, 10% acetic acid, 50% distilled water). The MMP activities, indicated by clear bands of gelatin digestion on a blue background, were quantified by using densitometric image analysis software (Image Master VDS; Pharmacia Biotech, Uppsala, Sweden).

Western blot analysis. To determine HDAC-1, MMP2, MMP9, TIMP1, TIMP2, Bax and Bcl2 protein levels, the cells were plated and treated with VPA as described above for zymography. The cells were detached and were collected by centrifugation at 300 × g for 10 min and pellets were resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 0.5% Triton X-100, 5 μM Na₃VO₄) and sonicated on ice in the presence of protease inhibitors. Protein concentration was determined by the method of Lowry (13). Cell lysates (50 μg of protein per lane) were size fractioned in 10% SDS-polyacrylamide gel, prior to transfer to Hybond TM-C Extra membranes (GE Healthcare, UK) by standard protocols. Membranes were blocked for 2 h with 5% milk in transfer buffer saline (TBS) at RT. The membranes were incubated overnight at 4°C with anti-HDAC-1 (Epigentek, USA), anti-MMP2, anti-MMP9, anti-TIMP1, anti-TIMP2 (all Santa Cruz Biotechnology, USA), anti-Bax or anti-Bcl2 (Sigma) primary antibody dissolved in TBS-5% milk. The membranes were washed twice with TBS-5% milk and were incubated for 1 h with the respective peroxidase-conjugated antibodies. The primary antibodies were diluted 1:500 and the antirabbit peroxidase-conjugated antibody was diluted 1:1000 with 5% milk in TBS-1% Tween. The proteins were detected by luminol (GE Healthcare, UK). Bands were quantified by using a densitometric images analysis software (Image Master VDS). The amount of protein in each lane was the same, as confirmed by loading with actin (Sigma).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Effect of VPA (mM) on HDAC-1 protein levels, as assessed by Western blot in cell lysates of G361, SKNMC and U87MG cells after 24 h of treatment. Densitometric data are expressed as the percentage of treated samples with respect to control (CTR). Each bar represents the mean (±SEM) of three independent experiments; *p<0.05 compared to control.

Invasion assay. Invasion of cells into Matrigel was determined using Boyden chambers (NTG, Italy) with polycarbonate membrane, 8.0-μm pore size (NTG). The filter was coated with 12.5 μg of Matrigel (Sigma). After 2 h, cells treated or not with VPA (1.0-2.0 mM) for 24 h, were seeded into the upper part of each chamber (4×10⁵ cell/well in 800 μl in serum-free RPMI). In the lower part, 200 μl RPMI with 10% FBS were added as chemoattractant. After incubation for 8 h at 37°C, non-migratory cells on the upper surface of the filter were wiped with a cotton swab, while cells migrated on the lower surface of the filter were fixed and stained with toluidine blue. The invasion activity was evaluated by counting cells in five random fields using microscopy at ×100 magnification.

Statistical analysis. Data were expressed as the mean (±SEM). Differences were analyzed by Student's t-test and considered statically significant at p<0.05 between the control and experimental samples.