ESCC TE-1 cells were purchased from the Health Science Research Resources Bank (Osaka, Japan). The cells were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in an atmosphere containing 5% CO₂.

GA was purchased from Sigma-Aldrich. A total of 1×10⁴ TE-1 cells per well were plated in 96-well plates (Corning Incorporated, Corning, NY, USA) and cultured for 24 h at 37°C with 5% CO₂ in a humidified atmosphere. At the indicated time points (12, 24 and 36 h) following various concentrations of GA treatment (2–10 µg/ml), 10 µl of Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) reagent was added to each well and incubated at 37°C for 3 h. Absorbance was measured at 450 nm in a spectrophotometer (UV-2802PCU; CRAIC Technologies, Inc., San Dimas, CA, USA). Each experiment was performed in triplicate and repeated at least two times.

TE-1 cells were harvested and then disrupted in lysis buffer A [0.33 M sucrose, 10 mM Hepes (pH 7.4; Sigma-Aldrich), 1 mM MgCl₂, 0.1% Triton X-100 (Sigma-Aldrich), protease inhibitor cocktail (PIC; Sigma-Aldrich) and phenylmethanesulfonyl fluoride (PMSF)]. The cell lysates were centrifuged for 5 min at 800 × g, and the supernatants were collected to use as the cytosolic fractions. The resulting pellets were resuspended in lysis buffer B [0.45 M NaCl, 10 mM Hepes (pH 7.4), PIC and PMSF] and centrifuged for 5 min at 18,000 × g. The supernatants were collected to use as the cytosolic fractions. Samples were frozen in aliquots in liquid nitrogen and stored at −80°C. PMSF, sodium orthovanadate, sodium fluoride and PIC were purchased from Roche Diagnostics (Indianapolis, IN, USA).

Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, using an In Situ Cell-Death Detection kit (Boehringer Mannheim; Roche Diagnostics), according to the manufacturer's protocol. Briefly, 5,000 TE-1 cells/well were treated with various concentrations (0, 4, 6, 8 and 10 µg/ml) of GA for 24 h in 96-well plates (Corning Incorporated). Following treatment, cells were trypsinized (Thermo Fisher Scientific, Inc.) and cytospin (9,000 × g at 4°C; Eppendorf 5810R; Eppendorf, Hamburg, Germany) preparations were obtained. Cells were fixed with freshly prepared paraformaldehyde (Thermo Fisher Scientific, Inc.) [4% in phosphate-buffered saline (PBS; Thermo Fisher Scientific, Inc.); pH 7.4], rinsed with PBS three times (5 min each time), and incubated in permeabilization solution (Sigma-Aldrich). Subsequent to cross-reaction with the TUNEL reaction mixture for 60 min at 37°C and cross-reaction with converter-alkaline phosphatase solution (Sigma-Aldrich) for 30 min at 37°C in a humidified chamber, the slides were reacted with alkaline phosphatase substrate solution for 5–10 min (Vector Laboratories, Inc., Burlingame, CA, USA), rinsed with PBS three times (5 min each time) and mounted under a coverslip for analysis with a light microscope (CX23; Olympus Corporation, Tokyo, Japan). The number of TUNEL-positive cells was assessed at ×40 magnification, and representative fields were photographed (using a Nikon L200 camera; Nikon Corporation, Tokyo, Japan). The percentages of apoptotic cells were calculated from the ratio of apoptotic cells to total cells counted. A minimum of 500 cells was counted in five isolated fields, and assays were performed in duplicate, three times (six times in total). An AKT selective inhibitor, GSK690693, was purchased from Selleck Chemicals (Houston, TX, USA).

The activity of caspase-3 was measured using a caspase-3 assay kit (#KA0740; Abnova, Walnut, CA, USA), according to the manufacturer's protocol. Briefly, TE-1 cells were treated with various concentrations (0, 4, 6, 8 and 10 µg/ml) of GA with or without z-VAD for 24 h, and then the cell lysis buffer containing 50 µg of protein was incubated with 5 µl of 4 mM peptide nucleic acid (pNA)-conjugated substrate at 37°C for 2 h. The amount of pNA released was measured at 405 nm using a spectrophotometer (UV-2802PCU; CRAIC Technologies). Samples without cell lysates or substrates acted as controls. All measurements of caspase activity were performed in duplicate and only samples in which the differences between the values obtained across replicates did not exceed 10% were used to calculate the mean value.

Culture cells were lysed in cell lysis buffer containing phosphatase inhibitor cocktail and proteinase inhibitor cocktail (Sigma-Aldrich), and the protein concentrations were determined using the BCA Protein Assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Total protein (20–40 mg) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Inc., Hercules, CA, USA) under reducing conditions and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked with Tris-buffered saline (Sigma-Aldrich) containing 0.05% Tween-20 (Sigma-Aldrich) and either 5% skim milk or 5% bovine serum albumin (Sigma-Aldrich), and incubated with monoclonal rabbit antibodies against NF-κB (#4764; dilution, 1:1,000), AKT (#8596; dilution, 1:1,000) and phosphorylated-AKT (p-AKT; clone Ser473; #4060; dilution, 1:1,000), which were all purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Subsequent to washing three times with PBS, the membranes were incubated for 1 h at room temperature with species-specific horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (#7074; dilution, 1:2,000; Cell Signaling Technology, Inc.). Immunoreactive bands were visualized using SuperSignal West Dura Extended Duration Substrate Enhanced Chemiluminescent Substrate (Pierce Biotechnology, Inc.). Each experiment was performed at least 3 times independently.

Monodansylcadaverine (MDC) is also a specific marker for autophagic vacuoles. To confirm that GA treatment induces autophagy, TE-1 cells were treated with various concentrations (0, 4, 6, 8 and 10 µg/ml) of GA for 24 h. The autophagic vacuoles were labeled with MDC by incubating with 0.05 mM/l MDC (Sigma-Aldrich) in PBS at 37°C for 2 h. Cells were then washed three times (5 min each time) with cold PBS buffer and immediately measured under a flow cytometer (FACScan; BD Biosciences, San Jose, CA, USA) using the Cell Quest software version 4.0 (BD Biosciences) to determine the percentage of cells undergoing autophagy that recruited MDC-positive particles.

All data and results presented were confirmed in at least three independent experiments, unless otherwise indicated. Data were expressed as the mean ± standard error unless otherwise noted. The differences between groups were analyzed using a two-tailed Student's t-test and the null hypothesis was rejected at the 0.05 level. Significant differences were considered to be indicated by P<0.05.

To analyze the effect of GA on cell growth, the CCK-8 assay was used to measure TE-1 cell viability following exposure to various concentrations (0, 4, 6, 8 and 10 µg/ml) of GA for 12, 24 and 36 h. The result showed that GA significantly inhibited the growth of TE-1 cells in a dose- and time-dependent manner (Fig. 1). The maximum cell growth inhibition was evident at 36 h exposure to 10 µg/ml GA. The half maximal inhibitory concentrations (IC₅₀) of 12, 24 and 36 h of GA treatment for TE-1 cells were 6.5, 5.8 and 5.3 µg/ml respectively.

The inhibition of cell proliferation may result from the induction of apoptosis. Apoptosis rate was first observed using TUNEL analysis in TE-1 cells following treatment with various concentrations (0, 4, 6, 8 and 10 µg/ml) of GA. The result indicated that GA may significantly increase the percentage of TE-1 cell apoptosis in a dose-dependent manner (between 11.2 and 42.6%), which may be augmented by the AKT inhibitor, GSK690693 (Fig. 2A and B).

Since caspase-3, a member of the family of aspartate-specific cysteinyl proteases, has been identified as a key mediator of apoptosis in mammalian cells by cleaving a variety of key cellular proteins (11), the activation of caspase-3 was investigated following treatment with various concentrations (0, 4, 6, 8 and 10 µg/ml) of GA in TE-1 cells. The result showed that caspase-3 activity may be promoted by GA in a dose-dependent manner, which may be reduced by z-VAD (Fig. 3A). Another CCK-8 assay was performed to ascertain whether z-VAD reverses GA-induced growth inhibition in TE-1 cells. The results indicated that z-VAD attenuated GA-induced growth inhibition in the cells (Fig. 3B).

Since p-AKT and transcription factor NF-κB mainly mediate cell apoptosis, western blot analysis was conducted to ascertain the expression status of p-AKT and NF-κB following treatment with GA in TE-1 cells. In the current study, GA was indicated to decrease the expression of p-AKT and NF-κB in TE-1 cells in a dose-dependent manner (Fig. 4).

The involvement of autophagy in GA-treated TE-1 cells was then examined. Compared with the control group, GA only caused a slight increase in the MDC positive ratio (Fig. 5). In addition, there was no significant difference between the treatment and the control group, regarding cell morphology. The results indicated that GA may not induce autophagy in TE-1 cells.