The human ESCC cell line ECA109 was supplied by the Cell Resource Center of Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cell culture was performed in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal calf serum (FCS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), and 10 kU/ml penicillin and 10 mg/ml streptomycin (Beyotime Institute of Biotechnology, Haimen, China).

Firstly, recombinant lentiviral vectors were constructed to knockdown CtBP2 (LV-CtBP2-RNAi) or overexpress CtBP2 (LV-CtBP2). The sequences were designed using the software EPtiRNA (http://optirna.unl.edu/) and synthesized by GeneChem Co., Ltd. (Shanghai, China). The vector construction and virus packaging were conducted by GeneChem Co., Ltd. For the former, small hairpin RNA (shRNA) of CtBP2, whose target sequence was 5′-GCGCCTTGGTCAGTAATAG-3′, was cloned into a GV248 vector (GeneChem Co., Ltd.) via EcoRI and AgeI restriction endonuclease sites. For the latter, the coding sequence of CtBP2 was cloned into a GV492 vector (GeneChem Co., Ltd.) via BamHI and AgeI restriction endo-nuclease sites. The primers used for overexpression were as follows: 5′-AGG TCG ACT CTA GAG GAT CCC GCC ACC ATG GCC CTT GTG GAT AAG CAC-3′ (forward) and 5′-TCC TTG TAG TCC ATA CCT TGC TCG TTG GGG TGC TCT CGA TTG-3′ (reverse). Schematic diagrams of the recombinant lentiviral vector constructs are presented in Fig. 1A.

ECA109 cells were transfected with LV-CtBP2-RNAi or LV-CtBP2 to knockdown or overexpress CtBP2, respectively. ECA109 cells transfected with empty vector GV248 and empty vector GV492 served as the negative controls, LV-CtBP2-RNAi⁻ and LV-CtBP2⁻, respectively. The following formula was used to calculate to volume of virus to be added: Virus volume = multiplicity of infection x cell number/virus titer. The cells (1×10⁶ cells/well) were seeded into 6-well plates prior to transfection. In order to improve the transfection rate, 5 µg/ml polybrene and enhanced infection solution (without FCS) were incubated overnight with the cells at 37°C with 5% CO₂. Puromycin (2 µg/ml; Gibco; Thermo Fisher Scientific, Inc.) was added to the culture medium (DMEM containing 10% FCS) on the third day and refreshed every 2 days for 1 week to select the transfected cells. The knockdown or overexpression of CtBP2 in the ECA109 cells was confirmed using the observation of green fluorescence, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting.

Total RNA was extracted from the CtBP2 knockdown or overexpressing cells using TRIzol reagent (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. First-strand cDNA synthesis was conducted using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). The temperature protocol was 60 min at 42°C, with termination of the reaction by heating at 70°C for 5 min. qPCR was performed in triplicate using SYBR Green Master mix (Roche Diagnostics, Basel, Switzerland) in a 7500 Real-Time PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) to test the mRNA expression level of CtBP2. The thermocycling conditions were as follows: Firstly 95°C for 10 min, followed by 95°C for 10 sec, 60°C for 15 sec and 72°C for 20 sec, for 40 cycles. The relative expression of mRNA was calculated using the 2^−ΔΔCq method with GAPDH as an internal reference (30). The following primer sequences were used: GAPDH, 5′-GAC CTG ACC TGC CGT CTA-3′ (sense) and 5′-AGG AGT GGG TGT CGC TGT-3′ (antisense); CtBP2, 5′-CTG AGT TCC TGG CCT TTC TG-3′ (sense) and 5′-GAC TTG ATA TCC GCG TCC TC-3′ (anti-sense).

Briefly, cells were homogenized in lysis buffer containing 1 mM phenylmethane sulfonyl fluoride and complete protein inhibitor mixture (Beyotime Institute of Biotechnology) for 15 min on ice, and then centrifuged at 13,400 × g for 10 min to collect the supernatant. The supernatant was diluted in 5X sodium dodecyl sulfate (SDS) loading buffer (Beyotime Institute of Biotechnology), boiled for 5 min and then cooled on ice. The protein concentration was measured at 280 nm using a One Drop Spectrophotometer (Wuyi Technology Co., Ltd., Nanjing, China) prior to loading the protein onto a gel (50 µg protein/lane). The proteins were separated by 10% SDS-PAGE and then blotted to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membrane was blocked with Tris-buffered saline and 0.1% Tween-20 (TBST) supplemented with 5% non-fat milk for 2 h at room temperature, and then reacted with the following primary antibodies overnight at 4°C: Anti-CtBP2 (sc-17759; 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-activated caspase-3 (cleaved) (AB3623; 1:200; EMD Millipore), anti-B-cell lymphoma 2 (anti-Bcl-2; Ab-1; 1:1,000; EMD Millipore), anti-Bcl-2-associated X protein (anti-Bax; ab53154; 1:500; Abcam, Cambridge, MA, USA) and anti-β-actin (ab8227; 1:1,000; Abcam). After washing with TBST three times, the membrane was then reacted with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (A8919; 1:1,000) or HRP-conjugated rabbit anti-mouse IgG (A9044; 1,1000; both from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 2 h at 37°C. The protein bands were detected using ECL chemiluminescence reagent (Thermo Fisher Scientific, Inc.) and imaged using a chemiluminescence detection system (Tanon Science and Technology Co., Ltd., Shanghai, China). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the density of the bands and β-actin was used as a reference for normalization. All experiments were repeated three times.

ECA109 cells were seeded in triplicate in a 96-well plate and cultured in 100 µl DMEM medium containing 10% FCS. When the cells had become attached to the bottom of well, the cells were treated with DDP (Sigma-Aldrich; Merck KGaA) in serial dilutions (final concentration 1.5×10⁻³, 1.5×10⁻⁴, 1.5×10⁻⁵, 1.5×10⁻⁶, 1.5×10⁻⁷ or 1.5×10⁻⁸ M) in DMEM. Following incubation for 48 h, methylthiazolyl tetrazolium solution (MTT; Beyotime Institute of Biotechnology; 10 µl; 5 mg/ml) was added to each well and the plate was incubated for a further 4 h. The medium was eliminated and 100 µl dimethylsulfoxide (Merck KGaA) was added to each well. The plate was shaken to dissolve the MTT-formazan crystals and the absorbance at a wavelength of 570 nm was read using an ELX800 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

On the basis of the MTT results for the various concentrations of DDP, the half maximal inhibitory concentration (IC₅₀) of DPP was determined. This concentration of DDP was subjected to further testing of cell viability with different treatment times (0, 12, 24 and 48 h) in order to determine the appropriate treatment duration. For subsequent analysis of the role of CtBP2 in the cytotoxicity of DDP, transfected and untransfected ECA109 cells were exposed to the concentration of DPP closest to the IC₅₀ using the treatment duration identified to be appropriate in this assay.

ECA109 cells on glass coverslips in 24-well plates (5×10⁴ cells/well) were fixed with 4% paraformaldehyde for 30 min at room temperature and then washed with 0.01% PBS three times, for 10 min each time at room temperature. The cells were then stained with Hoechst 33342 (Shanghai Yeasen Biotechnology Co., Ltd., Shanghai, China) for 10 min at room temperature. The slides were mounted with anti-fade solution (Beyotime Institute of Biotechnology). The apoptotic cells were identified by detecting the condensation and fragmentation of the cell nuclei under a fluorescence microscope (Zeiss AG, Oberkochen, Germany). Apoptotic cell numbers were counted in three randomly selected fields to calculate the apoptosis rate in triplicate.

Following the aforementioned treatments, the cells were digested with trypsin and washed with 0.01% PBS twice. The cells were then stained for 10–20 min with Annexin V-fluorescein isothiocyanate and propidium iodide (Abcam) solution at room temperature for 1 h in the dark, and then subjected to FCM (BD Biosciences, San Jose, CA, USA). A total of 10,000 fluorescence signals of each group were collected. The data were analyzed using FACSuite software (BD Biosciences).

All data were analyzed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was used to analyze the data and Tukey's post hoc test was used to analyze the differences between specific groups. P<0.05 was considered to indicate a statistically significant difference.

In order to detect the impact of CtBP2 on DDP resistance in ESCC, the ECA109 cells were transfected with lentivirus to knockdown or overexpress CtBP2. As the recombinant lentiviral vector contained the enhanced green fluorescent protein (EGFP) gene, the rate of transfection could be determined by direct observation under a fluorescence microscope. The percentage of cells positive for EGFP was >90% in the ECA109 cells transfected with recombinant LV-CtBP2-RNAi or LV-CtBP2. Fluorescence images of the transfected cells are presented in Fig. 1B.

The expression levels of CtBP2 in the ECA109 cells transfected with recombinant LV-CtBP2-RNAi or LV-CtBP2 (the LV-CtBP2-RNAi⁺ and LV-CtBP2⁺ groups, respectively) were further confirmed by RT-qPCR and western blot analysis. The expression of CtBP2 mRNA was revealed to be significantly changed by RT-qPCR analysis. Compared with the blank and negative control (LV-CtBP2⁻) groups, the relative expression of CtBP2 mRNA in the LV-CtBP2⁺ transfection group was increased ~6-fold (P<0.01). By contrast, the relative expression of CtBP2 mRNA in the LV-CtBP2-RNAi⁺ transfection group was decreased by more than half compared with that in the blank and negative control (LV-CtBP2-RNAi⁻) groups (P<0.01). No significant difference in CtBP2 mRNA levels was detected between the blank group and the LV-CtBP2⁻ or LV-CtBP2-RNAi⁻ group (P>0.05; Fig. 2A and B).

The protein levels of CtBP2 detected by western blotting exhibited similar trends to the expression levels of CtBP2 mRNA in the ECA109 cells subjected to CtBP2 knockdown or overexpression. Compared with the blank and respective negative control groups, the expression of CtBP2 was increased ~2-fold in the LV-CtBP2⁺ transfection group (P<0.05) and decreased by two-thirds in the LV-CtBP2-RNAi⁺ transfection group (P<0.01; Fig. 2C and D). Furthermore, no significant difference was detected between the blank group and the LV-CtBP2⁻ or LV-CtBP2-RNAi⁻ group (P>0.05).

These results suggest that stable cell lines with the knockdown or overexpression of CtBP2 were successfully obtained following recombinant lentiviral transfection for use in the following experiments.

In order to determine the optimized concentration and treatment time for DDP, ECA109 cells were treated with DDP solution in serial dilutions for different time periods. Firstly, an MTT assay was conducted to test the changes in cell viability following treatment with serial dilutions of DDP. The cell viability was markedly decreased following treatment with increasing concentrations of DDP for 24 h. Compared with the blank group, the cell viability in the 1.5×10⁻³, 1.5×10⁻⁴ and 1.5×10⁻⁵ M DDP treated groups was significantly reduced (P<0.01). Notably, the cell viability of the 1.5×10⁻⁵ M DDP treatment group was reduced by 52.15% (Fig. 3A).

Secondly, the effects on cell viability of incubation with 1.5×10⁻⁵ M DDP for different treatment times were examined via MTT assay (Fig. 3B) and by observation under an inverted phase contrast microscope (Fig. 3C). When observed under the microscope, the number of dead cells appeared to increase gradually as the treatment time with DDP was prolonged. The MTT assay demonstrated that compared with the blank group, the viability of the cells treated with DDP for 24 and 48 h was significantly decreased (P<0.01), but the reduction in viability of the cells treated with DDP for 12 h was not significant (P>0.05; Fig. 3B).

These results indicate that the effect of DDP on cell viability was dependent on concentration and reaction time. The optimized concentration and treatment time for DDP in ECA109 cells were 1.5×10⁻⁵ M and 24 h, respectively, and were used in the following experiments.

The impact of CtBP2 on the viability of the DDP-treated ECA109 cells was examined by microscopy and MTT assay (Fig. 4). The microscopy images revealed that cell shrinkage occurred following treatment with DDP, and the number of dead cells was increased. The viability of the ECA109 cells treated with DDP for 24 h was significantly reduced compared with that of the control cells (P<0.01). The cell viability of the LV-CtBP2-RNAi⁺ + DDP group was significantly reduced compared with that of the LV-CtBP2-RNAi⁻ + DDP group (P<0.05). Furthermore, the cell viability of the LV-CtBP2⁺ + DDP group was significantly increased compared with that of the LV-CtBP2⁻ + DDP group (P<0.01). No statistically significant difference was detected between the LV-CtBP2⁻ + DDP and LV-CtBP2-RNAi⁻ + DDP groups and the blank + DDP group (P>0.05). These results indicate that the overexpression of CtBP2 attenuated the reduction of cell viability induced by DDP, and the knockdown of CtBP2 augmented the DDP-induced reduction of cell viability.

Hoechst 33342 staining and FCM were used to investigate the effect of CtBP2 on the cell apoptosis induced by DDP. The results of Hoechst 33342 staining demonstrated that the numbers of apoptotic bodies were significantly increased (P<0.01) in the DPP-treated cells compared with untreated control group. The number of apoptotic bodies was increased significantly in the LV-CtBP2-RNAi⁺ + DDP group compared with the LV-CtBP2-RNAi⁻ + DDP group, and decreased significantly in the LV-CtBP2⁺ + DDP group compared with the LV-CtBP2⁻ + DDP group (P<0.05; Fig. 5A). On the basis of these results, it appears that CtBP2 overexpression attenuates the increase of apoptotic bodies induced by DDP.

FCM was used to further verify the impact of CtBP2 on the DDP-induced apoptosis of ECA109 cells. The results are consistent with those of Hoechst 33342 staining. The percentages of apoptotic cells in the untreated control, blank + DDP, LV-CtBP2-RNAi⁻ + DDP and LV-CtBP2-RNAi⁺ + DDP groups were 5.34, 13.7, 13.98 and 21.59% respectively. Compared with the blank + DDP group and the LV-CtBP2-RNAi⁻ + DDP group, the percentage of apoptotic cells in the CtBP2 knockdown (LV-CtBP2-RNAi⁺ + DDP) group was significantly increased (P<0.05). The percentages of apoptotic cells in the blank + DDP, LV-CtBP2⁻ + DDP and LV-CtBP2⁺ + DDP groups were 13.7, 14.75 and 5.86% respectively. The percentage of apoptotic cells in the CtBP2 overexpression (LV-CtBP2⁺ + DDP) group was significantly lower than those of the LV-CtBP2⁻ + DDP and blank + DDP groups (P<0.05; Fig. 5B). The FCM results further indicate that CtBP2 overexpression inhibited the DDP-induced apoptosis of ECA109 cells.

Caspase serves essential roles in cell apoptosis, which is a cellular event considered as programmed cell death (31,32). Caspase-3 is one of the crucial downstream effectors of apoptosis (33). The effects of CtBP2 on the protein levels of p53, Bcl-2, Bax and activated caspase-3 were analyzed by western blotting (Fig. 6). The results shown in Fig. 6A and B demonstrate that the expression of p53 was significantly increased (P<0.01) in the ECA109 cells treated with 1.5×10⁻⁵ M DDP for 24 h compared with the untreated control cells. Notably, the expression of p53 in the CtBP2 overexpression (LV-CtBP2⁺ + DDP) group was significantly decreased compared with that of the LV-CtBP2⁻ + DDP group (P<0.05). The changes in cleaved caspase-3 levels (Fig. 6D) exhibited a similar pattern to those of p53. These results demonstrate that the overexpression of CtBP2 inhibited the DDP-induced increase in the protein levels of p53 and activated caspase-3 in ECA109 cells.

The protein levels of Bax and Bcl-2 were also detected using western blotting. The results clearly demonstrate that Bcl-2 expression was downregulated while Bax expression was upregulated in the CtBP2 knockdown (LV-CtBP2-RNAi⁺ + DDP) group. Therefore, the Bcl-2/Bax ratio in the LV-CtBP2-RNAi⁺ + DDP group was significantly decreased compared with those in the untreated control, blank + DDP and LV-CtBP2-RNAi⁻ + DDP groups (P<0.05). By contrast, the ratio of Bcl-2/Bax in the CtBP2 overexpression (LV-CtBP2⁺ + DDP) group was significantly increased compared with those in the blank + DDP and LV-CtBP2⁺ + DDP groups (P<0.05; Fig. 6C). These results indicate that CtBP2 overexpression alleviated the DDP-induced apoptosis of ECA109 cells by reducing the protein levels of p53, cleaved caspase-3 and Bax, and increasing Bcl-2 expression.