The EC1 human esophageal carcinoma cell line was provided by The Department of Cell Biology, Hong Kong University (Hong Kong, China). This cell line has been demonstrated to be poorly-differentiated squamous cell carcinoma (27). EC1 cells were propagated in monolayer culture in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% inactivated fetal bovine serum (56°C; 30 min; Hyclone Laboratories, Logan, UT, USA), 1×10⁵ U/l penicillin and 100 µg/l streptomycin in a humidified atmosphere with 5% CO₂ at 37°C.

TSA was purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany) and dissolved in dimethyl sulfoxide (DMSO) to produce a 5 mM stock solution, which was stored at −20°C. Control cells were treated with DMSO in parallel during each experiment.

EC1 cell lines were seeded at a density of 5×10⁵ cells/well in 96-well microtiter plates. The cells were incubated at 37°C with 5% CO₂ for 24 h and then were treated with TSA at various concentrations (0.1, 0.3 and 0.5 µM; prepared from a stock solution dissolved in DMSO) for 24, 48 and 72 h. Cells treated with identical concentrations of DMSO were used as controls. A total of 4 h prior to absorbance evaluation, 10 µl Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) solution was added to each well and incubated for 24 h at 37°C. Absorbance was determined at a wavelength of 450 nm for each well using an enzyme-labeling instrument (Multiskan G0; Thermo Fisher Scientific, Inc.). All experiments were performed independently in triplicate.

Following incubation with or without TSA at various concentrations (0.1, 0.3 and 0.5 µM) for 48 h, EC1 cells were harvested using 2.5 g/l trypsin and washed twice with PBS. A total of 1×10⁵ cells were stained with fluorescein isothiocyanate (FITC)-Annexin V/propidium iodide (PI) using an Annexin V-FITC kit (Beckman Coulter, Inc., Brea, CA, USA), according to the manufacturer's protocol. Subsequently, the apoptosis of 1.5×10⁴ stained cells was quantified using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). Each experiment was performed in triplicate. BD CellQuest™ software version 3.0 (BD Biosciences) was used to calculate the proportion of apoptotic cells. Negative staining for Annexin V and PI indicated viable cells; early apoptotic cells were positive for Annexin V and negative for PI, whereas late apoptotic cells were positive for Annexin V and PI.

EC1 cells were cultured on 6-well plates (5×10⁵ cells/well) at 37°C for 24 h prior to infection with H101 Ads at a multiplicity of infection (MOI) of 100, and in the presence or absence of 0.3 µM TSA. The cells and the supernatants were harvested 24, 48 and 72 h following infection, freeze-thawed 3 times and serially diluted. HEK293 cells (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science, Shanghai, China) were seeded at a density of 1×10⁵ (100 µl) cells/well with 2% DMEM in 96-well microtiter plates. Each sample (cells and supernatants) that was diluted serially 10 times with 2% DMEM was added to 96-well microtiter plates at 37°C for 10 days. Each titer was repeated 10 times. The same volume of 2% DMEM was added as a control. Viral titers were calculated by infecting serially diluted virus particles in HEK293 and determined using the limiting dilution method (4) (determination of the 50% infective dose in tissue culture using HEK293 cells).

EC1 cells were seeded at a density of 5×10⁵ cells/well at 37°C in 96-well microtiter plates. Following culture at 37°C for 24 h, cells were incubated with H101 Ads at an MOI of 100 in the presence or absence of 0.3 µM TSA for 24, 48 and 72 h. Cell viability was evaluated using CCK-8. A total of 10 µl of the CCK-8 solution was added to each well and incubated at 37°C for 2 h. Absorbance was determined at a wavelength of 450 nm for each well using an enzyme-labeling instrument (Multiskan G0). All experiments were performed independently in triplicate.

Nu/nu athymic female mice, 4–6 weeks old and weighing 18–22 g, were obtained from Shanghai Laboratory Animal Co. Ltd. (Shanghai, China). All animals were housed in specific pathogen free laminar airflow boxes at a temperature of 25–26°C, with a humidity of 50%, and administered sterile food and water ad libitum. The mice were treated in accordance with the Guide for the Care and Use of Laboratory Animals of Henan Province, China, and experimental procedures were approved by the Medical Ethics Committee of Zhengzhou University (Zhengzhou, China). To obtain xenograft tumors, a 4×10⁶ EC1 cell resuspension (200 µl) was injected subcutaneously into the dorsal right flank of the athymic mice. The animals were monitored for tumor growth every other day. Upon reaching the required mean tumor volume of ~100 mm³ (volume = length × width² × 0.5), a total of 24 mice were randomly assigned to the following 4 groups (6 mice per group): The TSA alone group, the H101 alone group, the TSA and H101 combination treatment group, and the control group. The treatment protocol comprised of TSA (0.3 µmol/l, 200 µl TSA) administered as intratumoral injections 3 days prior to H101 injection. The H101 treatment protocol comprised of 100 µl H101 (1×10⁸ plaque-forming units) administered as intratumoral injections on days 2, 7, 11, 15 and 19. The control group received five injections of 100 µl PBS on days 2, 7, 11, 15 and 19. Tumor size was measured every 7 days using Vernier calipers. The mice from each group were sacrificed by cervical dislocation on day 21.

Tissue sections preserved in 2.5% glutaraldehyde-polyoxymethylene solution at room temperature for 24 h, were dehydrated and embedded in paraffin following routine methods, and sectioned to 4-µm thick. The sections were deparaffinized in xylene followed by treatment with a graded series of ethanol and distilled water, and thorough rinsing with PBS. Following microwave treatment in citrate buffer (pH 6.0), the container was placed in boiled water for 20 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol at room temperature for 10 min. The tissue samples were incubated with a rabbit anti-coxsackie and adenovirus receptor (CAR) monoclonal antibody (dilution, 1:200; catalog no. sc-50462; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Following washing three times with PBS, the tissue samples were incubated for 30 min with the goat anti-rabbit IgG-horseradish peroxidase secondary antibody (dilution, 1:2,000; catalog no. sc-2004; Santa Cruz Biotechnology, Inc.). Antibody binding was subsequently detected using 0.5% 3,3′-diaminobenzidine hydrochloride (DAB; Sigma-Aldrich; Merck Millipore) at room temperature without light for 3 min. The sections were then washed three times with PBS, counterstained with hematoxylin for 15 sec and dehydrated at room temperature. The images were analyzed by Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Tumor tissues from xenografts of the aforementioned mice were lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 100 µg/ml phenylmethylsulfonyl fluoride) for tissue homogenization. After 20 min on ice, the lysates were centrifuged at 20,430 × g at 4°C for 10 min. The supernatants were used as whole cell extracts. Cell lysates (50 µg) were separated using 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were incubated with 5% non-fat dried milk dissolved in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. The membranes were then incubated with a rabbit CAR monoclonal antibody (dilution, 1:200; Santa Cruz Biotechnology, Inc.) at 4°C overnight. After washing three times with 0.1% TBS-T, the tissue samples were incubated with the aforementioned horseradish peroxidase-conjugated IgG secondary antibody for 2 h at room temperature. A Pierce™ enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Inc.) was used to detect the target proteins. The bands were subjected to densitometry for quantitation using the Bio-Rad Quantity One^® software (version 4.6.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Quantitative data were expressed as the mean ± standard deviation. One-way analysis of variance was used to compare significant differences amongst the groups. Two-tailed Student's t-tests were used for comparisons between two groups. Data analyses were performed using SPSS 13.0 software for Windows (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

TSA, an established class I and II HDACI, has been reported to exert numerous antitumor effects by inhibiting cell proliferation and inducing cell apoptosis (24). The present study aimed to examine the ability of TSA to promote the antitumor effects of oncolytic H101, but not to affect cell viability. Therefore, EC1 cells were treated with various concentrations of TSA, and EC1 cell viability and apoptosis were evaluated. As presented in Fig. 1A, the viability of EC1 cells was not significantly inhibited by TSA at doses of 0.1 and 0.3 µM after 72 h of treatment (P=0.542 for 0.1 µM vs. control; P=0.218 for 0.3 µM vs. control). However, the viability of EC1 cells was significantly inhibited at doses >0.5 µM (P<0.001; Fig. 1A). In addition, the proportion of EC1 cells in early apoptosis was not markedly increased at TSA doses <0.3 µM (P=0.773 for 0.1 µM vs. control; P=0.350 for 0.3 µM vs. control; Fig. 1B). These results indicated that doses of ≤0.3 µM TSA did not significantly alter EC1 cell viability.

To examine whether TSA is able to impact H101 replication, end point dilution titrations were performed on HEK293 cells. Following treatment with 0.3 µM TSA for 24, 48 and 72 h, viral titers increased 55.82-fold, 238.84-fold and 527.46-fold in EC1 cells, respectively (Fig. 2A). H101 replication was significantly increased in EC1 cells treated with TSA, compared with the untreated control cells (P=0.002 for TSA 24 h vs. control; P<0.001 for TSA 48 h vs. control; P<0.001 for TSA 72 h vs. control). Subsequently, the antitumor effects of TSA on H101 in EC1 cells were examined. A CCK-8 assay was used to measure the EC1 cell survival rate at 12, 24, 48, 72 and 96 h following treatment. As compared with the H101 monotherapy group, the cell survival rate in the TSA and H101 combination group exhibited a significant decrease at 24, 48 and 72 h (P<0.001; Fig. 2B).

TSA and H101 in combination enhanced tumor cell cytotoxicity in vitro. Therefore, in order to examine whether this treatment is able to inhibit EC1 tumor growth in vivo, tumor bearing mice were divided into various treatment groups as described in material and methods. As compared with the PBS control group, mice treated with TSA alone did not exhibit tumor regression and there was no significant difference in the tumor volume at the end of treatment (P=0.148). In the TSA and H101 combination treatment group, a significant decrease in tumor volume (286.53±28.99 mm³) was observed, as compared with the untreated controls (1459.79±76.81 mm³) (P<0.001). Furthermore, a significant decrease in the tumor volume was detected in the TSA and H101 combination group, as compared with the two treatments administered individually (both P<0.001). The results indicate that TSA and H101 in combination produce an enhanced antitumor effect, compared with the two treatments administered individually (Fig. 3A). In addition, marked variations were identified in the degree of inflammation and necrosis observed in the tumor specimens in the TSA and H101 combination group, compared with the groups in which TSA and H101 were administered individually (both P<0.001; Fig. 3A and B).

To investigate whether the enhanced antitumor effects of TSA and H101 combined in vivo were achieved via the upregulation of CAR, the expression levels of CAR in the xenograft tumor tissues were detected using immunohistochemistry. An increase in the expression levels of CAR in xenograft tumors was observed in the TSA group and in the TSA and H101 combination group, as compared with the control group and the H101 group (Fig. 4A). Western blot analysis also demonstrated that the CAR protein levels were increased in the TSA group and the TSA in combination with H101 group (both P<0.001; Fig. 4B and C). These results indicated that TSA intratumoral injections may result in increased levels of CAR expression in xenograft tumors in mice.