The GLC-82 cells, grown to 70% of confluency on 60 mm tissue culture dishes (Falcon, Becton-Dickinson, Lincoln Park, NJ, USA), were transfected using the Calcium phosphate precipitation method employing a plasmid DNA mixture including 10 µg of plasmid pCMV-delta R8.9, 2 µg of plasmid p-VSV-G, 10 µg of mission shRNA plasmid shHDAC2, (Sigma-Aldrich St. Louis USA). In the mixture, an equal volume of 2x HBS (280 mm NaCl, 10 mM KCl, 1.5 mm Na₂HPO₄, 12 mM dextrose, 50 mM Hepes) pH 7.4 was added. After 20 min at room temperature the solution was added dropwise to the GLC-82 plate. The plates were incubated at 37˚C overnight. This was performed to generate GLCshHDAC2 cell line. The efficiency of transfection was estimated by fluorescence microscopy, controlling the transfection in a plate cloned with a p-GFP (Green Fluorescent Protein) control. At 24 h after transfection, the cells were maintained in a minimum volume of RPMI-3% FBS in order to concentrate a viral titer.

Supernatants harvested 48 and 72 h post-transfection were filtered (0.45 µm Whatman), and stored at −80˚C until use. GLC-82 cells were plated on six-well plates (Falcon, Becton-Dickinson, Lincoln Park, NJ, USA) with 2 × 10⁵ cell/well, 12 h before infection. Subsequently 2 ml of lentivirus supernatant containing 6 μg/ml polybrene (Sigma St. Louis USA) were added to the cells and were submitted to “Spinoculation”, centrifuged at 1800 rpm for 40 minutes at a temperature of 32˚C, after 6 h of incubation cells were infected with a second inoculum of lentivirus supernatant at 37˚C over night. The efficiency of the infection was estimated treating the cells with puromycin (2 µg/ml) (Bio-Australis Smithfield Australia), and by western blotting analysis using antibodies against the silenced proteins (see results). In another set of experiments, an empty lentivirus infection was used to evaluate the role of infection in the cell proliferation as well as in the pathway transduction. Data obtained from this experiment were evaluated respect to the data obtained using the control GLC-82 wild type not treated cells.

GLCshHDAC2 cell line, was cultured in RPMI 1640 MEDIUM (Sigma St. Louis U.S.A.) supplemented with 10% FBS, penicillin 100 U/ml, streptomycin 100 μg/ml, and fungizone 25 μg/ml (Sigma St. Louis U.S.A.). Lung cancer cells were maintained at 37˚C in a humidified 5% CO₂ atmosphere then were split 1:2 at confluence, usually weekly, and finally they were plated in a 100-mm polystyrene dishes (Falcon, Becton-Dickinson, Lincoln Park, NJ, USA). In all experiments, we used cell lines at a passage earlier than the 10^th. Simvastatin (40 mg) was dissolved in 2 mL of di-methyl-sulfoxide (DMSO, 100%) and brought to a final volume of 10 mL in the free culture medium (stock solution, 0.1 M). This stock solution was diluted 1:10 (final solution, 0.01 M) and stored at −20˚C. During our study, 30 µL of the final solution (30 μM) were added to each plate. When GLCshHDAC2 cells were a 50% of confluence, they were exposed to hydrogen peroxide (0.5 mM) (Sigma St. Louis U.S.A) for 2 h and then treated with 30 μM simvastatin for 24 h. The medium was not changed after treatment. The solvent employed to dissolve these drugs was used as a control. After that period, the medium was removed for TNF-a, IL-6 and IL-8 evaluation (see later) and cells were processed for protein extraction and immunoblotting.

Cell viability was assessed by light microscopy using trypan blue; cell numbers were evaluated by direct counting, performed using a Burker chamber. Cell proliferation was investigated by Methylthiazolyldiphenyl-tetrazolium (MTT) bromide assay, based on the conversion by mitochondrial dehydrogenases of the substrate containing a tetrazolium ring into blue formazan, detectable spectrophotometrically. The level of blue formazan was used as an indirect index of cell density (Mosmann, 1983). Briefly, cells (100 μl/well) were seeded at a density of 1 × 10⁴ cells/ml into 96 well plates (Falcon, Becton-Dickinson, Lincoln Park, NJ, USA). Before and after treatment with H₂O₂ and Simvastatin, cell viability was assessed on a daily basis by adding 10 μl of filter sterilised MTT (Sigma-aldrich Saint Louis U.S.A), 5 mg/ml in PBS, for 2 hours at 37˚C. The medium was then removed and cells were solubilized with acidified isopropanol (Sigma- aldrich Saint Louis U.S.A). After complete solubilization, presence of blue formazan was evaluated spectrophotometrically at a wavelength of 490 and 655 nm. The optical density (O.D.) was calculated as the difference between the absorbance at the reference wavelength and that at the test wavelength. Percent viability was calculated as (O.D. of drug-treated sample/control O.D.) ×100. All experiments were carried out in quadruplicate.

After treatment with hydrogen peroxide (0.5 mM) and simvastatin (30 μM), cells were centrifuged at 1000 rpm for 5 min, the supernatant was withdrawn and the cell pellet washed two times with 2 mL of the PBS. Cells were fixed by incubation with 1 mL of 70% ethanol for 1 h at −20˚C. After several washes the cells were then resuspended in 1 mL of staining solution (Propidium Iodide 500 µg/ml, RNAsi 50 µg/ml, NP40 0.1%) and incubated in the dark for 1 h at 37˚C. Subsequently the cells were washed three times with 2 mL of 1× PBS solution and analyzed through FACScan (Becton Dickinson, San Jose, CA) flow cytometer.

Following treatment, cells were lysed for Western blotting in radioimmuno precipitation assay (RIPA) buffer, as previously described [29] . Nuclear extracts were obtained using the NE-PER cell fractionation kit (Thermo Scientific, Rockford, IL, USA). Briefly, whole cell lysates or nuclear proteins were then separated on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia, Little Chalfont, UK). Immunoblotting was performed using the monoclonal antibodies listed below. Antibody binding was visualized by enhanced chemiluminescence (ECL-Plus; Amersham Pharmacia); intensities of experimental bands were analyzed by computer-assisted densitometry and expressed as arbitrary units, as previously described [30] . These experiments were performed in triplicate. All primary antibodies were used in a 1:1000 dilution in 5% milk/TBS-T unless indicated otherwise. Primary antibody was incubated overnight at 4˚C on a rotating wheel. The primary antibodies used were: Anti-p-Erk, Anti-NFkB p65, Anti-TRADD, Anti-MyD88, Anti-TRAF6, Anti-TRAF2, Anti-RECK, Anti-MMP-9, Anti-MMP-2, Anti-HDAC2, Anti-Ƴ-Tubulin, Anti-Actin (Santacruz, USA). All secondary antibodies were used in a 1:2000 dilution in 5% milk/TBS-T and incubated with the blot for 2 h at room temperature. The secondary antibodies used were: Goat anti-mouse IgG-HRP conjugated, Goat anti-rabbit IgG-HRP conjugated, Donkey anti-goat IgG-HRP conjugated (Santacruz, USA).

The amount of TNF-α, IL-6 and IL-8 released in the supernatant of treated cells was determined by an ELISA assay (Quantikine, R & D system) according to the manufacturer instructions.

All data are expressed as mean ± SEM. Statistical evaluation of the results was performed by Anova. Differences identified by Anova were pointed by unpaired Student’s t-test. The threshold of statistical significance was set at P < 0.05.

Figure 1(a) lane 1 shows the expression of HDA2 in GLC-82 cells (wild type) by western blotting analysis. In Figure 1(a) lane 2 after infection with lentivirus containing the shHDAC2, is highlighted a decreasing in HDCA2 level. The densitometric analysis of the targets in A (Figure 1(b)) demonstrates that almost the 70% of HDA2 result decreased.

MTT assay in GLCshHDAC2 cells, show that H₂O₂ pretreatment induced proliferation

(of about 30% more) of those cells respect to the control without H₂O₂ pretreatment (Figure 2(a)). Further 24 hours simvastatin treatment significantly decreased (P < 0.01) the cells count prominently in respect to H₂O₂ pretreatment (Figure 2(b)).

As shown in Table 1, in GLCshHDAC2 cells G0/G1 phase increase, while decrease S and G2/M-phases respect to GLC-82 wild type. In both cells line simvastatin (30 µM) induced a significant increase in G0/G1 phase, and a decrease in S and G2/M-phase respect to control cells (P < 0.01). In contrast, H₂O₂ (0.5 mM) induced a significant decrease of G0/G1 and a significant increase in S and G2/M phase (P < 0.01) compared to controls; these effects were significantly reverted by simvastatin treatment (P < 0.01).

As shown in Figure 3 in the GLCshHDAC2 cell line, the pretreatment with H₂O₂ followed by simvastatin action induced a significant increase in a) ERK1/2 phosphorylation

pretreatment. Followed by a significant decrease of b) RECK expression (P < 0.01) a significant increase (P < 0.01) of both c) MMP-2 and d) MMP-9 expression (Figure 3(c) and Figure 3(d)). Data represent the mean ± SEM of three independent experiments (lane 2 and 4). *P < 0.01 vs. control; #P < 0.01 vs. H₂O₂ (lane 1 and 3). In Figure 4, under the same condition decribed above, it show an increase of a) NF-κB expression, b) MyD88, c) TRAF-6, d) TRADD and e) TRAF-2 (lane 2 and 4). All of these effects were significantly (P < 0.01) reverted by simvastatin (30 µM) treatment.

ELISA assay in GLCshHDAC2 cells under H₂O₂ (0.5 mM) show a significantly increase (P < 0.01) of TNF-α, IL-6 and IL-8 secretion in cell surnatant media, these effects were

significantly countered (P < 0.01) by a 24 h treatment with simvastatin (30 mM) (Figures 5(a)-(c)).