EC109 and EC9706 cells (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China), which are well and poorly differentiated human ESCC cell lines, respectively, were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 10 U/ml penicillin and 10 µg/ml streptomycin at 37°C and 5% CO₂. Sporamin, a primary soluble protein in sweet potatoes with trypsin inhibitory activity, was purified from fresh sweet potato tuberous roots (Ipomoea batatas cv. Tainong 57; Hangzhou Lianhua Huashang Group Co., Zhejiang, China), as described previously (6). The primary antibodies against Bcl-2 (catalog no. sc-509), Bcl-2 like 1 (Bcl-XL; catalog no. sc-7122), Bcl-2-associated X (Bax; catalog no. sc-23959) and NF-κB p65 (catalog no. sc-8008) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). AKT (catalog no. 9272), phosphorylated (p)-AKT (Thr308; catalog no. 4056), p70 S6 kinase (catalog no. 9202), p-p70 S6 kinase (Thr421/Ser424; catalog no. 9204), NF-κB inhibitor α (IκBα; catalog no. 9242) and p-IκBα (Ser32; catalog no. 2859) primary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), and tubulin (catalog no. ab59680), histone (H)2A (catalog no. ab13923) and β-actin (catalog no. ab8227) primary antibodies were purchased from Abcam (Cambridge, UK).

The in vitro cell viability of EC109 and EC9706 cells treated with sporamin was measured using the MTT assay. A total of 1×10³ cells/well were cultured in 96-well plates and incubated with 0, 12.5, 25, 50 or 100 µM sporamin for 24, 48 or 72 h at 37°C. Dimethyl sulfoxide (DMSO) was cells treated with as a control instead of sporamin. Medium was discarded and wells were replaced with 100 µl fresh medium containing 10% MTT solution (5 mg/ml stock) at 37°C for 4 h, 100 µl DMSO was subsequently added to each well before agitating the plates at room temperature for 10 min. Colorimetric determination of MTT reduction was determined at a wavelength of 570 nm using a microplate ELISA reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The in vitro cell proliferation activity of EC109 and EC9706 cells treated with sporamin was measured by a [³H] thymidine incorporation assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). A total of 2.5×10⁵ cells/well were cultured in 24-well plates in DMEM with 10% dialyzed and charcoal-stripped FBS. When cells reached 80% confluence, the cultures were rinsed in phenol-free DMEM medium and incubated with 0, 12.5, 25, 50 or 100 µM sporamin in phenol-free and serum-free DMEM for 24, 48 and 72 h. [³H] thymidine (1.35×10⁴ Bq/l) was added and cultured with cells for a further 12 h at 37°C. Subsequently, the supernatant was aspirated and excess [³H] thymidine was removed by washing with PBS. Finally, 0.2 mol/l NaOH was added at 37°C and radioactivity ([³H] thymidine incorporation activity) was determined by scintillation counting (LS6500; Beckman Instruments, Inc., Fullerton, CA, USA).

Cells were cultured in 12-well plates at a density of 2×10⁴ cells per well and incubated with 25 µM sporamin or DMSO for 48 h at 37°C. Cells were then fixed with 4% cold paraformaldehyde at 4°C for 15 min (pH 7.4) and stained with 0.5 µg/ml DAPI (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for 5 min at room temperature. The cells were subsequently visualized under a fluorescence microscope (model DMI4000B; Olympus Corporation, Tokyo, Japan) using a 360–370 nm excitation light and a 420–460 nm emission filter. Cells undergoing apoptosis exhibit profound structural alterations, including apoptotic chromatin condensation, nuclear envelope shrinkage and fragmentation. Apoptotic morphological changes in the nucleus were distinguished from intact nuclei and counted, and the percentages were subsequently calculated. A total of six randomly chosen fields of view were examined with a minimum number of 500 cells scored in each condition.

Apoptosis was measured using an Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. Briefly, 1.5×10⁶ cells/well were incubated with 25 µM sporamin or DMSO for 48 h at 37°C. Both attached and floating cells were pooled, pelleted by centrifugation at 100 × g for 5 min, 4°C, washed twice with cold PBS and 1×10⁶ cells/ml were resuspended in 1X binding buffer [10 mM hydroxyethyl piperazine ethanesulfonic acid, (pH 7.4), 2.5 mM CaCl₂, and 140 mM NaOH]. A total of 1×10⁵ cells were transferred to a 5-ml tube and stained with 5 µl annexin V-FITC and 5 µl PI for 15 min at room temperature in the dark. Finally, 400 µl 1X binding buffer was added to each tube and apoptosis was determined using a BD FACSCanto II flow cytometer with BD Accuri™ C6 software (BD Biosciences, San Jose, CA, USA).

Total protein was extracted from treated cells using M-Per Mammalian Protein Extraction reagent (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Nuclear fractions were prepared using a Nuclear Protein Extraction kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol. The protein concentrations were determined using a Bradford assay. Cell protein extracts were either used immediately or stored at −70°C.

For SDS-PAGE, a total of 20 µg protein was loaded into each well of 10–12% gel and electrophoresed at 98 V. Proteins on the gel were transferred onto a polyvinylidene fluoride membrane by wet electrotransfer for 2 h. The membrane was blocked in 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) in TBS containing 0.1% Tween-20 at room temperature for 90 min. The primary antibodies (all used at a 1:200 dilution) against Bcl-2, Bcl-XL, Bax, NF-κB p65, IκBα, AKT, p70 S6 kinase, p-IκBα (Ser32), p-AKT (Thr308), p-p70 S6 kinase (Thr421/Ser424), tubulin, H2A and β-actin were added separately and incubated with the membrane overnight at 4°C. The membranes were subsequently washed three times with TBS/0.1% Tween-20. The following day, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.), anti-mouse (1:2,000; cat. no. 7076; Cell Signaling Technology, Inc.), and anti-goat (1:2,000; cat. no. sc-2350; Santa Cruz Biotechnology) immunoglobulin G secondary antibodies. The membrane was subsequently washed four times with TBS/0.1% Tween-20, 5 min per wash. All bands were visualized using Pierce™ enhanced chemiluminescence Western Blotting Substrate (Merck KGaA, Darmstadt, Germany) and scanned using an LAS-4000 imaging system (Fujifilm, Tokyo, Japan).

For EMSA, cells were treated with 0, 12.5, 25 or 50 µM sporamin for 48 h at 37°C. EMSA was performed using a commercial kit (Gel Shift Assay System; Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Nuclear Protein Extraction kit (Beyotime Institute of Biotechnology, Haimen, China) was adopted to prepare nuclear fractions, according to the manufacturer's protocol. A bicinchoninic assay was used to determine the protein concentration. Nuclear protein (2 µg) was preincubated in binding buffer [10 mM Tris-Cl, (pH 7.5), 50 mM NaCl, 1 mM MgCl₂, 0.5 mM EDTA, 4% glycerol, 0.5 mM DTT and 0.05 g/l poly (deoxyinosinic deoxycytidylic acid)] for 15 min at room temperature. After addition of 1 µl ³²P-labeled oligonucleotide probe (1.75 pmol/µl), the incubation was continued for 30 min at room temperature. The reaction was stopped by adding 1 µl gel loading buffer and the mixture was subjected to 4% nondenaturing polyacrylamide gel electrophoresis in 0.5X Tris/Borate/EDTA buffer. The gel was vacuum-dried and exposed to X-ray film at −70°C. The dried gels were visualized by autoradiography using x-ray film.

Data are presented as the mean ± standard deviation. A one-way analysis of variance followed by Tukey's post hoc test, or student's t-test were used to compare differences between groups using SPSS version 18.0 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of sporamin on cell viability and the proliferation of ESCC cells, EC9706 and EC109 cells were treated with sporamin at increasing concentrations (12.5, 25, 50 and 100 µM) and for varying durations (24, 48 and 72 h), and cell viability and proliferation were determined using an MTT assay (Fig. 1A) and a [³H] thymidine incorporation assay (Fig. 1B), respectively. The results demonstrated that sporamin significantly inhibited cell viability and proliferation activity in a concentration- and time-dependent manner.

When EC9706 and EC109 cells were treated with 25 µM sporamin for 48 h, the inhibition of cell viability and proliferation activity were significant. Therefore, cells were treated with 25 µM sporamin for 48 h for further experiments. Apoptotic cells were examined under fluorescence microscopy following DAPI staining. Following exposure to sporamin, the cell volume reduced, the chromatin became condensed and the nuclei became fragmented, compared with DMSO-treated cells (Fig. 2A). Compared with DMSO-treated control cells, the percentage of apoptotic cells was increased significantly following sporamin treatment in EC9706 and EC109 cells (Fig. 2B).

As sporamin induced morphological changes associated with apoptosis at a concentration of 25 µM for 48 h, annexin V and PI staining was additionally performed to determine cell apoptosis. The results demonstrated that the percentages of apoptotic cells (annexin V positive cells) were increased significantly following culture with sporamin, compared with the DMSO control cells (Fig. 2C and D). These results indicate that sporamin may induce cell apoptosis in EC9706 and EC109 cells.

As Bcl-2, Bcl-XL and Bax are involved in the promotion of apoptosis, the expression levels of Bcl-2, Bcl-XL and Bax in EC9706 and EC109 cells were measured by western blotting, to identify the potential underlying molecular basis for the effects of sporamin on ESCC cells. The results indicated that sporamin inhibited the expression of the anti-apoptotic proteins, Bcl-2 and Bcl-XL, and induced the expression of the proapoptotic protein Bax, in a concentration-dependent manner in EC9706 and EC109 cells (Fig. 3). The results indicate that sporamin may induce apoptosis via the regulation of Bcl-2, Bcl-XL and Bax expression.

As Bcl-2 and Bcl-XL may be regulated by NF-κB (10), it was hypothesized that sporamin may mediate its effects by regulating NF-κB. Therefore, nuclear extracts of EC9706 and EC109 cells treated with various concentrations of sporamin (12.5, 25 and 50 µM) or control diluent were prepared and NF-κB DNA-binding activity was examined by EMSA. As demonstrated in Fig. 4, a marked reduction of NF-κB DNA-binding activity was observed in sporamin-treated cells in a concentration-dependent manner. These results indicate that sporamin may inhibit NF-κB DNA-binding activity.

Treatment with sporamin resulted in reduced nuclear expression of NF-κB p65 in a concentration-dependent manner in EC9706 and EC109 cells (Fig. 5A and B). Consistently, there was a marked reduction in the expression levels of p-IκBα (Fig. 5C). However, the expression and phosphorylation levels of AKT, or its downstream target, p-p70 S6 kinase, were not altered following sporamin treatment (Fig. 5C). These results indicate that NF-κB inhibition by sporamin may be partially regulated via the dephosphorylation of IκBα, without inhibition of AKT.