The human cervical cancer cell line SiHa was obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China), cultured in Dulbecco’s modified Eagle’s medium (DMEM; 4.5 g/l glucose; Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin in a 5% CO₂ incubator at 37°C.

NDGA (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) and the stock solution was diluted to the required concentrations in DMEM prior to being added to the cell culture medium. Final DMSO concentrations were ≤0.2%. Antibodies against Ac-Histone H3 (Lys 9/14) and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against p53 were purchased from Signalway Antibody (Pearland, TX, USA). All other chemicals were of reagent grade.

Cell proliferation was measured by the MTT assay. SiHa cells were seeded into 96-well culture plates at 5×10³ cells per well. Each group consisted of six parallel wells and was subsequently treated with various concentrations of NDGA for 24, 48 and 72 h. The control cells were treated with 2% DMSO in culture medium. Following treatment, the cells were incubated with MTT reagent (0.5 mg/ml) at 37°C for 4 h. The resulting formazan crystals were solubilized by adding 150 μl DMSO to each well. The optical density (OD) at 492 nm was measured and cell viability was determined using the formula: cell viability (%) = (absorbance of the treated wells - absorbance of the blank control wells)/(absorbance of the negative control wells - absorbance of the blank control wells) × 100%. The MTT experiments were repeated at least three times.

The cell cycle was analyzed using flow cytometry. Briefly, cells (1×10⁶) were collected and washed twice in phosphate-buffered saline (PBS), then fixed in 70% alcohol for 30 min at 4°C. After being washed in cold PBS three times, the cells were resuspended in 1 ml of PBS solution with 40 μg of propidium iodide (Sigma) and 100 μg of RNase A (Sigma) for 30 min at 37°C. The samples were then analyzed for their DNA content by fluorescence activated cell sorting (BD Immunocytometry Systems, San Jose, CA, USA). Each experiment was repeated at least three times.

Total RNA was isolated from cells using RNAiso Plus (Takara, Japan) and reverse-transcribed using a reverse transcription system (Promega, Madison, WI, USA) according to the manufacturer’s instructions. PCR amplification of different genes was performed using Easy Taq DNA Polymerase (TransGen Biotech, China). Table I shows the primer sequences used for RT-PCR and the product sizes. GAPDH served as an internal control. The optimal reaction conditions for PCR were determined for each primer pair. Denaturation was at 94°C for 40 sec and annealing at 56°C for 40 sec, followed by elongation at 72°C for 40 sec. PCR products were analyzed by 1.5% agarose/ethidium bromide gel electrophoresis. OD values of mRNA were analyzed with Pro-Gel 4.0 Software and GAPDH was used to normalize mRNA. Each experiment was repeated at least three times.

Nuclear extracts from SiHa cells were prepared using a Nuclear Protein Extraction kit (BestBio, China). The protein concentrations were determined using an Enhanced BCA Protein Assay kit (Beyotime, China). The nuclear proteins (40 μg) were fractionated using sodium dodecyl sulfate-10% polyacrylamide gels for electrophoresis (SDS-PAGE) and the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked at room temperature for 4 h with 5% bovine serum albumin in Tris-buffered saline containing 0.05% Tween-20, 10 mmol/l Tris-HCl pH 7.5 and 150 mmol/l NaCl, and then incubated with the appropriate primary antibodies overnight at 4°C. Horseradish peroxidase-conjugated anti-goat IgG was used as the secondary antibody, and the protein bands were detected using the enhanced chemiluminescence method. The relative protein levels were normalized to β-actin and expressed as percentages of the control. The membrane was incubated for 30 min at 50°C in buffer that contained 2% SDS, 62.5 mmol/l Tris (pH 6.7) and 100 mmol/l 2-mercaptoethanol. The membrane was then washed and incubated with the other primary antibody.

ChIP assays were performed using a ChIP Assay kit (Beyotime) according to the manufacturer’s instructions. In brief, SiHa cells were fixed by 1% formaldehyde for 10 min at 37°C. Following washing with cold PBS, the cells were lysed in SDS lysis buffer and sonicated to shear the DNA to an average fragment size of 600 bp. The lysates were cleared by centrifugation and Ac-Histone H3 (Lys 9/14) antibody was added to the supernatants. Immunoprecipitation from soluble chromatin was conducted overnight at 4°C. Immune complexes were collected with protein A+G agarose/salmon sperm DNA for 60 min at 4°C and then extensively washed. The samples were extracted with elution buffer (1% SDS, 0.1 mol/l NaHCO₃) and heated at 65°C for 4 h to reverse cross links. The DNA was purified and used for amplification of the p21 or p27 promoter DNA. The primers used for p21 were: forward, 5′-ACCAACGCAGGCGAGGGACT-3′; and reverse, 5′-CCGG CTCCACAAGGAACTGA-3′ (12). The primers used for p27 were: forward, 5′-TGGAGAAGCACTGCAGAGAC-3′; and reverse, 5′-GCGTGTCCTCAGAGTTAGCC-3′ (12). The products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Each experiment was repeated at least three times.

The quantitative variables are presented as means ± SD. The difference between two groups was assessed using an independent t-test. The differences among ≥3 groups were compared using one-way ANOVA. P<0.05 was considered to be statistically significant.

The treatment of SiHa cells with various concentrations of NDGA for 24–72 h resulted in cell growth inhibition in a dose- and time-dependent manner (Fig. 1A). A cell cycle assay was performed and it was found that cell cycle progression of SiHa cells was significantly inhibited at the G₁ phase. As shown in Fig. 1B, the SiHa cells showed a higher proportion of cells at the G₁ phase and a decrease in the proportion of cells at the G₂ phase, compared to the control cells. Cell cycle distribution analysis showed that the increase in G₁-phase cells observed in SiHa was significant (P<0.05) and the induction of cell cycle arrest was dose-dependent. These results demonstrated that NDGA substantially inhibited the proliferation of SiHa cells and induced G₁ phase arrest in vitro.

To determine the mechanisms of NDGA-induced cell cycle arrest, the effect of NDGA on the expression of p21, a key mediator of G₁/S transition, was examined. It was found that NDGA treatment for 72 h significantly increased the levels of p21 mRNA (Fig. 1C) by up to nearly 2-fold.

The level of histone H3 acetylation was determined at Lys 9 and 14 in SiHa cells treated with NDGA. Nucleic proteins were isolated from cells treated with NDGA (80 μmol/l) for 72 h. Cells were treated with histone deacetylase inhibitor (HDACi) trichostatin A (TSA) (300 nmol/l) as a positive control. Western blot analysis showed that prior to incubation with NDGA, the level of acetylated histone H3 in SiHa cell was low (Fig. 2A). Incubation with NDGA for 72 h resulted in the accumulation of acetylated histone H3.

The effect of NDGA on the acetylation of histone H3 associated with the p21 gene promoter was examined by ChIP. Chromatin fragments from cells cultured with and without NDGA (80 μmol/l) for 72 h were immunoprecipitated with an antibody against acetylated histone H3 at Lys 9 and Lys 14. DNA from the immunoprecipitated DNA-protein complex was isolated. From this DNA, a 324-bp fragment of the p21 promoter region was amplified. In the control, the acetylated histone H3 was nearly undetectable. After treatment with NDGA, we observed a significant increase in acetylated histone H3 within p21 promoter (Fig. 2B).

To determine the mechanism by which NDGA induced histone H3 acetylation, the effect of NDGA on SMRT transcription was detected. The results showed that NDGA inhibited the SMRT mRNA level in a dose-dependent manner (Fig. 2C).

To determine whether this effect was selective for a limited number of genes, the level of histone H3 acetylation in the p27 gene was examined. The expression of p27 mRNA increased no more than doubled, while the p27 protein level up-regulated significantly (Fig. 3A and B). No change in the levels of histone H3 acetylation was detected following treatment with NDGA (Fig. 3C). The results suggest that the NDGA-induced changes in histone acetylation were localized to specific areas of the chromatin.

The effect of NDGA on the protein level of p53, a tumor suppressor which is also a critical regulator of p21 transcription, was detected. The results showed that NDGA elevated the p53 protein level in a dose-dependent manner (Fig. 4A). The effect of NDGA and TSA on the transcription of HPV16 E6, which induces degradation of p53 was also investigated. As₂O₃ was used as a positive control, as it inhibits the expression of HPV16 E6/E7 (16). The results showed that NDGA significantly inhibited the HPV16 E6 mRNA level in a dose-dependent manner (Fig. 4B and C), whereas TSA had no such effect.