The HepG2 liver cancer cell line (cat. no. KCB 200507YJ) was purchased from the cell bank of the Type Culture Collection of the Chinese Academy of Sciences. The HepG2 cells were authenticated by short tandem repeat DNA profiling analysis and tested to be free of mycoplasma and other contaminants by the vendor. The cells were maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (ScienCell Research Laboratories, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and grown in a humidified atmosphere with 5% CO₂ at 37°C. The cells were treated with SAHA (Abcam) or mock-treated with DMSO (Sigma-Aldrich; Merck KGaA), as indicated.

Cell proliferation was monitored using an xCELLigence Real-Time Cell Analysis instrument (xCELLigence DP System; ACEA Biosciences, Inc.), which can continuously monitor live cell proliferation, morphology and viability with a label-free assay. The experimental procedures used were as previously described (21,22). Briefly, HepG2 cells were seeded at a density of 1×10⁴ cells/well in a 16-well µl plate (E-plate 16), cultured in complete medium supplemented with various doses of SAHA (0, 0.1, 1, 6, 12, 25, 50 and 100 µM), and placed at 37°C in a humidified incubator containing 5% CO₂. The effects of the various doses of SAHA on the proliferation of HepG2 cells were continuously monitored for 90 h. The cell index is a dimensionless parameter that is translated from the electrical impedance measured to denote cell proliferation.

HepG2 cells (1×10⁶) were seeded onto 10-cm diameter dishes and treated with various doses of SAHA (0, 1, 6 and 12 µM) for 48 h. The cells were harvested and subjected to an apoptosis assay using an annexin V/propidium iodide (PI) cell apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd.), according to the manufacturer's instructions. Briefly, the cells were washed with cold phosphate-buffered saline (PBS). After centrifugation at 110 g for 5 min, the cell pellet was resuspended in 1 ml PBS (final concentration ~1–5×10⁵ cells/ml). After washing with 1X annexin V binding solution, the cells were stained with 5 µl of fluorescein isothiocyanate (FITC)-conjugated annexin V and 10 µl PI staining solution at room temperature for 20 min. After washing once with the 1X annexin V binding solution, the labeled cells (1×10⁴ cells) were detected immediately by a flow cytometer (FACSCalibur™; BD Biosciences). The data were analyzed by BD CellQuest Pro software (version 1.0; BD Biosciences). The apoptotic rate was defined as the percentage of FITC-annexin V single positive cells (early apoptotic cells) among total cells.

HepG2 cells (1×10⁶) were seeded onto 10-cm diameter dishes and treated at various doses of SAHA (0, 1, 6 and 12 µM) for 48 h. The cells were then lysed with 0.2 ml of radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.). Total protein samples (60 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gels), transferred to a polyvinylidene difluoride membrane and blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 60 min at room temperature. The membrane was incubated overnight at 4°C with the following primary antibodies: acH4 (1:1,000), acH4K5 (1:5,000), acH4K12 (1:1,000), GRP78 (1:1,500), PERK (1:1,500), phosphorylated (p)-PERK (1:1,500), ATF4 (1:1,500) and CHOP (1:1,500). After washing with TBST buffer, the membrane was incubated with the peroxidase-conjugated anti-rabbit secondary antibody (1:4,000; Abcam; cat. no. ab205718) for 90 min at room temperature. Following treatment with enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.), the Image Lab software (version 4.1; Bio-Rad Laboratories, Inc.) was used to quantitate the band intensities. The antibodies against β-actin (cat. no. ab8227), GRP78 (cat. no. ab21685), ATF4 (cat. no. ab184909), CHOP (cat. no. ab10444), PERK (cat. no. ab65142), acH4K5 (cat. no. ab51997) and acH4K12 (cat. no. ab46983) were purchased from Abcam. The antibody against acH4 (cat. no. 39925) was purchased from Active Motif, Inc. the antibody against p-PERK (cat. no. AF4499) was purchased from Affinity Biosciences.

Total RNA was isolated from HepG2 cells using a TRIzol RNA isolation kit (Thermo Fisher Scientific, Inc.) and the purity and concentration of the RNA samples were determined by a DN2000 ultramicro nucleic acid analyzer (Thermo Fisher Scientific, Inc.). The total RNA was reverse transcribed into cDNA using a First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc), according to the manufacturer's instructions. RT-qPCR reactions were performed as follows: 25°C for 10 min, 48°C for 60 min and 95°C for 5 min. The qPCR was performed using SYBR green mix from Thermo Fisher Scientific, Inc. The reaction conditions included an initial pre-denaturation step at 95°C for 30 sec, followed by 40 cycles of thermal steps consisting of 95°C for 5 sec and 60°C for 30 sec. β-Actin was used as an internal control. The fold-change was calculated by the 2^−∆∆Cq method (23). The primer sequences used for each gene are shown in Table I.

HepG2 cells (1×10⁶) were seeded onto 10-cm diameter dishes and were mock-treated with DMSO (0 µM SAHA) or treated with 6 µM SAHA for 36 h. Following treatment, the cells were subjected to a ChIP assay using a ChIP kit (Sigma-Aldrich; Merck KGaA), according to the manufacturer's instructions. The HepG2 cells were cross-linked with 1% formaldehyde and lysed with 600 µl of radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.), and the lysates were sonicated to fragment the DNA into lengths between 200–1,000 base pairs. Immunoprecipitation was performed with the following antibodies: Anti-acH4 (1:50; Active Motif, Inc.; cat. no. 39925), rabbit IgG (1:50; Abcam; cat. no. ab205718) and anti-RNA Polymerase II (1:50; Abcam; cat. no. ab51462) at 4°C overnight. Protein G-sepharose beads were used for immunoprecipitation, and the immunoprecipitates were washed and eluted, according to the manufacturer's protocol. The immunoprecipitated DNA was recovered by reversing the cross-linking, and were purified and dissolved in distilled water. A corresponding sample handled without the addition of any antibody served as an input control. The ChIP DNA and input DNA were analyzed by qPCR. The abundance of the immunoprecipitated target DNA was expressed as a percentage of the input chromatin DNA. The primers used for detecting the promoter regions of the GRP78, ATF4 and CHOP genes are listed in Table I.

SPSS version 20 statistical software (IBM Corp.) was used for statistical analysis. Data were expressed as the mean ± SD. The one-way analysis of variance method was used for the multivariate comparison, and the least significant difference method was used as a post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Firstly, the effect of SAHA on the proliferation of HepG2 cells was determined by real-time cellular analysis. This involved live monitoring of cell proliferation in a label-free manner. The electrical impedance of adherent cells was detected by the electrode at the bottom of an E-plate 16, and the cell index is a dimensionless parameter that was translated from the measured electrical impedance to denote cell proliferation. After HepG2 cells were incubated for 10 h to allow cell adhesion, SAHA was added at various doses in the culture medium to treat cells. This resulted in a rapid decrease in HepG2 cell proliferation at 10 h (Fig. 1), which was probably due to the transient effect of SAHA vehicle DMSO on cell proliferation. Compared with the mock-treated group (0 µM SAHA), treatment with SAHA at concentrations >1 µM resulted in a notable inhibitory effect on the proliferation of HepG2 cells (Fig. 1). Furthermore, SAHA at 6 µM resulted in significantly (P<0.01) decreased normalized cell index, by >70%, at 50 h after treatment. A significant (P<0.01) suppression of proliferation was observed in HepG2 cells treated with higher doses of SAHA (12, 25 and 50 µM) for longer periods of time. Thus, the antiproliferative effect of SAHA on HepG2 cells occurs in a dose- and time-dependent manner.

The effect of SAHA on apoptosis in HepG2 cells was investigated by annexin V-FITC and PI staining. Following treatment with SAHA for 48 h, the apoptotic rates were significantly elevated in HepG2 cells treated with 1, 6 or 12 µM SAHA, compared with the untreated cells (Fig. 2). While the control group (0 µM SAHA) had an average apoptotic rate of ~1%, 12 µM SAHA led to an increased apoptotic cell population to ~10%. These results indicate that SAHA could significantly promote apoptosis in HepG2 cells in a dose-dependent manner.

Since SAHA was revealed to be a potential inducer of ER stress, the effect of SAHA on ER stress in HepG2 cells was determined by measuring the levels of ER stress-associated molecules, including GRP78, PERK, p-PERK, ATF4 and CHOP. No significant changes were observed in the expression of GRP78 at either the mRNA (Fig. 3A) or protein level (Fig. 3B and C) in cells treated with 1 µM SAHA, whereas cells treated with 6 and 12 µM SAHA had significantly increased levels of GRP78 mRNA and protein compared with control cells (0 µM SAHA). No significant changes in the level of PERK mRNA were observed following treatment with any of the tested doses of SAHA (Fig. 3A). However, the expression of PERK protein was significantly decreased in the HepG2 cells treated with different concentrations of SAHA, while the level of p-PERK protein was significantly increased (Fig. 3B and C), which resulted in an increased ratio of p-PERK/PERK following SAHA treatments (Fig. 3D). Notably, following treatment with SAHA, the mRNA and protein expression levels of ATF4 and CHOP were significantly elevated in a dose-dependent manner (Fig. 3A and C). Moreover, the protein levels of ATF4 and CHOP in the HepG2 cells were significantly upregulated by over 3-fold following treatment with 12 µM SAHA.

The role of SAHA, as an HDACi, in the acetylation of histone H4 was determined by western blot analysis. The protein levels of total acH4, acH4K5 and acH4K12 were detected in HepG2 cells treated at various doses of SAHA for 48 h (Fig. 4). Compared with the control group (0 µM SAHA), the levels of acH4 and acH4K12 were not significantly increased in the HepG2 cells treated with 1 µM SAHA, whereas the level of acH4K5 was significantly increased; these findings were likely due to the varied sensitivities of histone deacetylases to inhibition by SAHA. In cells treated with 6 and 12 µM SAHA, the levels of total acH4, acH4K5 and acH4K12 were all significantly higher than in those treated with 0 and 1 µM SAHA. These results suggest that 6 µM SAHA was sufficient to markedly elevate the acetylation of histone H4.

In order to confirm whether the regulation of transcription of GRP78, ATF4 and CHOP genes by SAHA is mediated by the upregulation of acH4, ChIP assays were conducted. This involved determination and quantitation of the acH4-associated promoter regions of these genes in HepG2 cells (Fig. 5). Following treatment with DMSO (0 µM SAHA) or 6 µM SAHA for 36 h, the HepG2 cells were lysed and immunoprecipitated with specific anti-acH4 antibody. qPCR results demonstrated significant increase in the acH4-associated promoter regions of the GRP78, ATF4 and CHOP genes in HepG2 cells treated with 6 µM SAHA. The promoter regions of ATF4 and CHOP were increased by 2-fold and 3-fold, respectively, the promoter region of GRP78 was enriched by ~4-fold (the output to input ratio increased from ~15 to ~60%). These results confirmed that SAHA induces the expression of ER stress-associated molecules by increasing their transcription, at least partially through enhancing the acetylation of histone H4 in the promoter regions of these genes.