The BGC-823 human gastric cancer cell line was purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Science, Chinese Academy of Sciences (Shanghai, China). The MCF-7 human breast cancer and KYSE-510 human esophageal squamous cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were maintained in RPMI 1640 medium containing penicillin (100 U/ml), streptomycin (100 mg/ml) and 10% fetal bovine serum at 37°C in a humidified incubator supplemented with 5% CO₂ in air. Cell morphology was observed with an Olympus IX71 inverted microscope (Olympus Corporation, Tokyo, Japan). TSA was purchased from Beyotime Institute of Biotechnology (Haimen, China) and dissolved in dimethylsulfoxide (6.62 mm) for storage. The TSA was diluted to the appropriate concentrations with phosphate-buffered saline (PBS) prior to use. The primary antibodies used in this study were a rabbit monoclonal antibody to E-cadherin (Cell Signaling Technology, Inc., Danvers, MA, USA), a goat polyclonal antibody to β-catenin (Santa Cruz Biotechnology, Inc., Dalls, TX, USA) and a mouse monoclonal antibody to β-actin (Abmart, Shanghai, China). The secondary antibodies were HRP-conjugated anti-rabbit, mouse or goat secondary antibodies (Cell Signaling Technology, Inc.).

The cells were seeded on 96-well plates and allowed to adhere overnight. Following treatment with TSA for 24 h, the cells were washed with cold PBS and fixed in 4% paraformaldehyde for 15 min, followed by permeabilization in 0.1% Triton X-100 for 5 min. The cells were then blocked with 5% goat serum for 30 min, followed by incubation with primary antibody overnight at 4°C. Following careful washing, the cells were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature, then stained with 3,3′-diaminobenzidine subsequent to additional thorough washing.

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and quantified by spectrophotometric measurement (Molecular Devices, Sunnyvale, CA, USA). RNA (2 μg) was reverse-transcribed to cDNA using the RevertAid first strand cDNA synthesis kit (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. qPCR analysis was performed using SYBR Green-based detection on a StepOnePlus real-time PCR instrument (Applied Biosystems, Inc., Foster City, CA, USA). The relative mRNA expression levels of the genes of interest were normalized to those of GAPDH and calculated using the 2^−ΔΔCt method. The following primers were used: Sense: 5′-GAC CGC ACA CAG CAA GGC GAT-3′ and antisense: 5′-GCT GTC CCG CCG ATT GAG GG-3′ for Vimentin; sense: 5′-TCA GCA GGG CCG GAG ACC TA-3′ and antisense: 5′-TCC ACG GGC CTG TCT CGC TT-3′ for Twist; sense: 5′-CGC CCC CAT ACC AGA ACC TCG-3′ and antisense: 5′-GTC CAG TTG GCA CTC GCC CC-3′ for E-cadherin; sense: 5′-TGG TGC CCA GGG AGA ACC CC-3′ and antisense: 5′-TGT CAC CTG GAG GCA GCC CA-3′ for β-catenin; and sense: 5′-CTG GGC TAC ACT GAG CAC C-3′ and antisense: 5′-AAG TGG TCG TTG AGG GCA ATG-3′ for GAPDH.

To prepare the whole-cell extract, the cells were washed with cold PBS once and harvested by scraping in radioimmunoprecipitation assay lysis buffer. The protein content was determined by the Bradford assay (Beyotime Institute of Biotechnology, Shanghai, China). The extracted proteins were separated by SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Beyotime Institute of Biotechnolgy, Haimen, China). The membranes were first blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline with Tween-20 and then probed with the indicated primary antibodies with gentle agitation at 4°C overnight. Subsequent to washing four times, the membranes were incubated with HRP-conjugated secondary antibodies 1 h. The signals were detected using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific).

A total of 1×10⁶ cells were plated on 6-well plates. When the monolayer reached ~70% confluence, a scrape was inflicted using sterile pipette tips. The wound-healing rate was evaluated by capturing images of the cells after 24 h with an Olympus IX71 inverted microscope (Olympus Corporation).

The cells were seeded on 96-well plates at 10 cells per well and allowed to adhere overnight. Fresh medium either with or without 200 nm TSA was then added to each well. After three days, the numbers of colonies in each well with a diameter >100 μm were counted using an an Olympus IX71 inverted microscope (Olympus Corporation).

The data are presented as the mean ± standard error of the mean. Statistical analyses were performed using Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

TSA is known as a potent HDAC inhibitor, which binds to the zinc-containing catalytic domain of the class I and II HDACs, including HDACs 1 to 10 (11). To determine whether TSA induces EMT in cancer cells, BGC-823 human gastric cancer cells were treated with different concentrations of TSA. Untreated BGC-823 cells exhibited a cobblestone-like epithelial morphology. As shown in Fig. 1A, after 24 h treatment with 200 nm TSA, the cells exhibited a spindle-like shape similar to mesenchymal cell characteristics, but 50 nm TSA did not exert these effects (Fig. 1A). TSA also stimulated mesenchymal-like morphological changes in MCF-7 human breast cancer cells in a concentration-dependent manner (Fig. 1B). Following three days culture with 200 nM TSA, BGC-823 and MCF-7 cells all reached ~100% confluence and the mesenchymal cell morphology disappeared, indicating that 200 nM TSA induced no marked cytotoxicity in BGC-823 or MCF-7 cells (Fig. 1A).

To confirm whether TSA functions as an inducer of EMT, the effect of TSA on the expression levels of mesenchymal and epithelial markers was determined by qPCR. As shown in Fig. 2A, 200 nm TSA treatment significantly upregulated Vimentin and Twist mRNA transcription in BGC-823 cells. In MCF-7 cells, the Vimentin mRNA expression levels were increased but the Twist expression levels were marginally reduced in response to TSA treatment (Fig. 2B). However, the E-cadherin transcription levels were significantly increased in the two cell lines following TSA treatment (Fig. 2A and B). Immunocytochemistry and western blotting assays further confirmed that TSA induced E-cadherin expression (Fig. 2C–E). The KYSE-510 human esophageal squamous cancer cells exhibited no marked morphological change in response to TSA treatment (Fig. 2F); however, E-cadherin expression levels were increased following TSA treatment, indicating that the upregulation of E-cadherin was independent of TSA-induced mesenchymal-like cytoskeleton remolding (Fig. 2G).

The cytoskeletal remodeling during EMT induces spindle-like cancer cell morphology, which facilitates cell mobility. A wound-healing assay was performed to examine the effect of TSA on cancer cell migratory ability. As shown in Fig. 3A and B, 200 nm TSA attenuated the wound-healing process in BGC-823 and MCF-7 cells.

Cells that have undergone EMT have been demonstrated to be the source of cancer stem-like cells (5). High colony-forming efficiency is one of the hallmarks of cancer stem cells. It was determined whether TSA enhances the cancer cell colony-forming ability. Fig. 4 indicates that cancer cell colony formation was significantly inhibited in the TSA treatment groups compared with the control groups. Since BGC-823 cells are able to achieve confluency in 200 nm TSA (Fig. 1A), the reduced colony-forming ability in BGC-823 and MCF-7 was not due to the cytotoxic effect of TSA.

Diverse extra- and intracellular signals, including those of the Wnt/β-catenin signaling pathway, have been reported to induce and/or maintain EMT in various cell types (12). In the present study, β-catenin mRNA expression levels were found to be significantly upregulated in BGC-823 and MCF-7 cells following TSA treatment (Fig. 5A). However, β-catenin protein expression levels in BGC-823 cells were reduced following TSA treatment (Fig. 5B), suggesting that TSA is involved in either translational or post-translational regulation of β-catenin expression.