Human ovarian cancer cell line, HO8910, was kindly provided by Dr Qixiang Shao of Jiangsu University (Zhenjiang, China). The HO8910 cells were derived from ascites of ovarian adenocarcinoma patients. The cells were maintained for no longer than 3 months as described previously (15).

EGFR, phospho-EGFR (Tyr992), AKT, phospho-AKT (Ser473), FOXA2, and acetyl-histone H3K9 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). α-tubulin and secondary antibodies were procured from Bioworld Technology (Shanghai, China). OCT4, SOX2, p21^Cip1, cyclin D1, and histone H3 antibodies were obtained from Abcam (Cambridge, MA, USA). The EGFR inhibitor AG1478, AKT inhibitor A6730, and HDAC inhibitor TSA were procured from Sigma (St. Louis, MO, USA). TSA and A6730 were dissolved in ethanol as stocks (6.6 mM) and dimethyl sulfoxide as stocks (20 mM), respectively.

In TSA experiments, HO8910 cells were incubated with 100, 200, and 400 nM TSA and harvested at 48 h. When the expression of differentiation-related genes was examined, cells were harvested at 24 h. For cotreatment of TSA and EGFR pathway inhibitors, cells were incubated with 200 nM TSA in presence of AG1478 (10 µM) or A6730 (20 µM) for 24 h.

The morphological changes in TSA-treated cells were observed during a 72-h time course with a light microscope (Olympus, Tokyo, Japan), and images were obtained via a CCD camera (Olympus).

Histones were prepared using the bioepitope nuclear and cytoplasmic extraction kit (Bioworld Technology) following the manufacturer's protocol. Total cellular proteins were isolated directly from cultures in 100-mm Petri dishes after being washed with ice-cold PBS and the addition of 200 µl Cell and Tissue Protein Extraction reagent (Kangchen Biotech, Shanghai, China), containing protease inhibitor and phosphatase inhibitor cocktails (Roche). Antibodies against OCT4 (1:5,000), SOX2 (1:5,000), FOXA2 (1:1,000), cyclin D1 (1:10,000), p21^Cip1 (1:3,000), EGFR (1:1,000), p-EGFR (1:600), AKT (1:1,000), p-AKT (1:800), α-tubulin (1:1,000), and histone H3 (1:2,000) were used for western blot analysis, which was performed as described previously (16).

The cell proliferation assays were performed using CCK-8 and EdU. Briefly, HO8910 cells (5×10³) were seeded in 96-well plates, and then treated with TSA for 24, 48, and 72 h. For CCK-8 analysis, the cells were incubated with 1/10 volume of CCK-8 for 2 h. The plates were then read at 450 nm with a Bio-Rad model 680 microplate reader (Richmond, CA, USA). The cell proliferation rate = OD_experiment/OD_control×100%. For EdU assay, HO8910 cells were treated with 200 nM TSA for 48 h, and then incubated with EdU (50 mM) for 2 h, after which the nuclei were stained with DAPI. The images were photographed by an Olympus inverted microscope system.

The effect of TSA on HO8910 cell cycle phase distribution was determined by flow cytometry. HO8910 cells were fixed in 70% ethanol for 30 min at 4°C. The cells were then incubated with propidium iodide (50 µg/ml) for 30 min at 37°C, after which the fluorescence was measured with a flow cytometer (BD Biosciences, Heidelberg, Germany).

Data are presented as the mean ± SEM. Statistical significance was calculated by Student's t-test or ANOVA, and values of P<0.05 were considered statistically significant.

To elucidate the effect of TSA on ovarian cancer cell differentiation, we examined the morphology of HO8910 cells treated with or without TSA. Microscopic observation of control cells revealed round, stellate-like appearance and growth in clusters. Unlike the morphology of control, a majority of TSA-treated cells exhibited spindle shape and much longer, fine, tapering processes (Fig. 1A). Treatment with TSA also caused a decrease in cell density (Fig. 1A), which suggests that TSA may impede proliferation of HO8910 cells. Consistent with this prediction, we found that TSA treatment caused a notable reduction in cell proliferation rate compared with the control in a dose- and time-dependent manner (Fig. 1B and C). The suppressive effect of TSA on cell proliferation was further confirmed by 5-ethynyl-2′-deoxyuridine (EdU) assay (Fig. 1D).

To identify the mechanism of action of TSA in decreasing cell proliferation, we undertook cell cycle analysis with propidium iodide to label DNA. Table I reveals that treatment with TSA for 48 h led to an accumulation of HO8910 cells in the G₀/G₁ phase (60.2 vs. 82.8%, P<0.05) and a concomitant decrease in the S-phase cell fraction (26.7 vs. 3.8%, P<0.01). The TSA-induced cell cycle arrest was further confirmed by examining the expression of cell cycle regulatory protein cyclin D1 in HO8910 cells. Western blot analysis indicated that TSA treatment exhibited decrease of cyclin D1 levels and increase of p21^Cip1 protein (Fig. 1E). These data suggest that TSA induces cell differentiation and proliferation inhibition of HO8910 cells.

To verify the role of TSA in HO8910 cell differentiation, two groups of genes including FOXA2 as differentiation marker gene, and SOX2 and OCT4 as pluripotency marker genes were evaluated quantitatively. FOXA2 gene was evaluated because it plays important roles in regulating the expression of genes involved in cell differentiation in a number of different organs (17), and is essential for differentiation and development of glands in mouse uterus (18). A dose-dependent upregulation of FOXA2 protein and H3K9 acetylation was observed in TSA-treated cells compared with the controls (Fig. 2). In contrast, the expression of SOX2 and OCT4 proteins was notably restrained by TSA (Fig. 2). This molecular evidence confirms that TSA actually induces the differentiation of HO8910 cells.

Since EGFR expression strongly correlates with tumor resistance to cytotoxic agents (19), EGFR could be a potential target for anticancer therapy. To decipher whether TSA transactivates EGFR in HO8910 cells, we first examined phosphorylation of EGFR and AKT in response to TSA. Our results indicated that TSA caused a time- and dose-dependent increase in phosphorylation of EGFR (Tyr992) and AKT (Ser473) (Fig. 3A and B). The TSA-induced phosphorylation of EGFR and AKT occurred at 15 min and peaked at 30 min (Fig. 3A). The observed changes in EGFR and AKT phosphorylation were reversed in HO8910 cells in response to EGFR inhibitor AG1478 and AKT inhibitor A6730 (Fig. 3C). These data suggest that TSA activates EGFR phosphorylation and subsequent activation of downstream AKT signaling.

The above data indicate that TSA induces ovarian cancer cell differentiation prompted us to test whether the EGFR/AKT pathway was involved in this process. HO8910 cells treated with either AG1478 or A6730 markedly promoted TSA-induced cell differentiation with morphological changes (Fig. 4A). To determine the mechanism by which inhibition of the EGFR/AKT pathway enhances cell differentiation, we tested the expression of differentiation-related genes in HO8910 cells. As expected, combination of TSA and AG1478 or A6730 drastically reduced the levels of SOX2 and OCT4 and increased the expression of FOXA2 in the cells (Fig. 4B). Both inhibitors also caused an increase in global levels of H3K9 acetylation (Fig. 4B).

Next, we tested the effect of TSA on cell cycle regulation when EGFR/AKT signaling pathway was inhibited. Treatment with AG1478 or A6730 decreased the levels of cyclin D1 and increased the expression of p21^Cip1 in HO8910 cells in response to TSA (Fig. 4C). Furthermore, combination treatment with TSA and AG1478 or A6730 led to an accumulation of cells in G₀/G₁ phase in comparison to TSA alone (Table II). Collectively, our data suggest that TSA plays a role in cell differentiation and that blockage of EGFR/AKT signaling pathway enhances TSA-induced differentiation.