A preparation of dry TEB extract powder (Tongrentang, Fuzhou, China) was weighed (58.7 g) and added to a 1,000-ml flask with 500 ml 95% (v/v) ethanol. The mixture was refluxed at 95°C for 3 h. A second sample was mixed with an additional 500 ml 95% (v/v) ethanol and a second reflux was performed. The two samples were mixed and concentrated on a rotary evaporator at a temperature of 50°C for 48 h. Subsequently, the sample was successively washed two times with distilled water and three times with petroleum ether to obtain the final extract (EETEB), weighing 1.71 g.

SGC-7901 human GC cells, from the Tumor Hospital of Fujian (Fujian, China) were grown and maintained in RPMI 1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum, 80 U/ml penicillin and 80 U/ml streptomycin (all from Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere of 5% CO₂. The SGC-7901 cells were inoculated into 96-well plates at a concentration of 100 µl/well and cultured for 24 h. Subsequently, the cells were treated with various concentrations of EETEB (0.75, 1.0 and 1.5 mg crude EETEB/ml); the control cells were treated with PBS. Following incubation for 24 h, 50 µl MTT solution (1.1 mg/ml) was added to each well and incubated for an additional 4 h. Finally, the medium was replaced with 1.5 mg/ml MTT and incubated at 37°C for ~4 h. The crystallization was resolved using dimethyl sulfoxide and the absorbance was measured using a microplate spectrophotometer (SpectraMax 190; Molecular Devices, LLC, Sunnyvale, CA, USA) at a wavelength of 580 nm.

SGC-7901 cells were seeded into 6-well plates in 2 ml medium and treated with various concentrations of EETEB (0.75, 1.0 and 1.5 mg/ml) for 24 h. Flow cytometric analysis with an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect the apoptosis of SGC-7901 cells. The staining procedure was performed according to the manufacturer's instructions. In the apoptosis assay, an Annexin V/PI double-negative population [labeled as LL in the fluorescence-activated cell sorting (FACS) diagram] indicated the viable cells; by contrast, an Annexin V-positive/PI-negative or Annexin V/PI double-positive population represented cells undergoing early or late apoptosis, respectively.

DAPI staining followed by laser scanning confocal microscopy (LSCM; LSM 710; Carl Zeiss AG, Oberkochen, Germany) was used to detect the apoptosis of the SGC-7901 cell nuclei. A minimum of 5×10⁴ cells treated with various concentrations of EETEB (0.75, 1.0 and 1.5 mg/ml) were evaluated per chamber. A 488-nm, 10-MW argon laser beam was used for excitation of blue DAPI fluorescence. Subsequently, LSCM was performed to observe the nuclei of the SGC-7901 cells and detect apoptosis. The collected data were analyzed using Microsoft Excel 2000 software (Microsoft Research, Redmond, WA, USA).

The cell lysates were collected by centrifugation at 15,000 × g for 10 min at 25°C, the cells treated with various concentrations of EETEB (0.75, 1.0 and 1.5 mg/ml) were fixed in 1.5% paraformaldehyde and 4% glutaraldehyde in 0.1 M phosphate-buffered saline (PBS) buffer (pH 7.2–7.4) for 12 h at 4°C. Next, the cells were washed with PBS buffer and post-fixed with 1% OsO₄ in 0.1 M PBS buffer for 2 h. The cells were then dehydrated in a graded ethanol alcohol series and embedded in Epoxy resin 618 (E-51, Ganxi Chemical Co. Ltd., Jiangxi, China). Ultrathin sections (100 nm) were cut using an ultramicrotome (EM UC6; Leica Microsystems GmbH, Wetzlar, Germany). The sections were stained in 2.0% uranyl acetate for 20 min and lead citrate for 15 min. Finally, transmission electron microscopy (TEM; H-7650; Hitachi, Ltd., Tokyo, Japan) was used to examine and capture images of the cell sections.

The mitochondrial membrane potential was detected using a JC-1 fluorescent probe (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), which is a cationic dye that exhibits potential-dependent accumulation in mitochondria. A change in JC-1 fluorescence emission from red to green indicates depolarization of the mitochondrial membrane; thus, this dye can be used as an indicator of changes in mitochondrial membrane potential. In the current experiment, following trypsin digestion (Thermo Fisher Scientific Inc.), SGC-7901 cells (1×10⁶) treated with various concentrations of EETEB (0.75, 1.0 and 1.5 mg/ml) were resuspended in 1 ml medium and incubated with 10 µg/ml JC-1 at 37°C in an atmosphere of 5% CO₂ for 30 min. JC-1 fluorescence was recorded using a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) at emission wavelengths of 525 and 590 nm for green and red fluorescence, respectively.

SGC-7901 cells (1×10⁶ cells/well) were seeded into 6-well plates with 2 ml medium and treated with various concentrations of EETEB (0.75, 1.0 and 1.5 mg/ml) for 24 h. Total RNA was extracted from the SGC-7901 cells using a Beyzol reagent kit (Shanghai Biyuntian Bio-Technology Co., Ltd., Shanghai, China). Subsequently, oligo(dT)-primed RNA (1 µg) was reverse-transcribed using SuperScript II® Reverse Transcriptase (Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions. PCR was performed to determine the quantity of Bcl-2 and Bax mRNA in the obtained cDNA samples. GAPDH was used as the internal control and the DNA bands were examined using a Gel Documentation system (Gel Doc 2000; Bio-Rad Laboratories, Hercules, CA, USA).

SGC-7901 cells (1×10⁶) were seeded in 6-well plates in 2 ml medium and treated with various concentrations of EETEB (0.75, 1.0 and 1.5 mg/ml) for 24 h. The cells were washed twice with cold PBS and then lysed in radioimmunoprecipitation assay buffer containing 1 mM phenylmethyl sulfonyl fluoride (Roche Diagnostics GmbH, Mannheim, Germany). Next, the lysates were centrifuged at 12,000 × g for 10 min at 4°C to acquire the total Bax and Bcl-2 proteins from the mitochondria. The cytosolic and mitochondrial fraction proteins were collected. A single aliquot of the supernatant (50 mg protein) was subjected to electrophoretic separation using SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). The membranes were incubated in blocking buffer (non-fat milk) and then incubated overnight at 4°C with rabbit polyclonal antibodies against Bax (1:1,000, 20 kDa, Cell Signaling Technology, Inc., Danvers, MA, USA), Bcl-2 (1:1,000, 26 kDa, Cell Signaling Technology, Inc.) or β-actin (1:4,000, 43 kDa, Beyotime Institute of Biotechnology, Haimen, China). The membranes were stringently washed and incubated with HRP-conjugated secondary antibodies (Proteintech Group, Chicago, IL, USA), for 1 h at room temperature. Images were captured using a Kodak Image Station 400R (Eastman Kodak Co., Rochester, NY, USA).

All the data are presented as the mean of three experiments and were analyzed using SPSS software (version 16.0; SPSS, Inc., Chicago, IL, USA). Statistically significant differences between the control and treatment groups were obtained by performing one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

The effect of EETEB on the proliferation of SGC-7901 cells was determined by performing an MTT assay. As demonstrated in Fig. 1, treatment with 0.75, 1.0 and 1.5 mg/ml EETEB resulted in cell viability of 71.99±7.26, 54.28±5.28 and 34.48±5.84%, respectively. These values were significantly higher compared with the untreated control cells (P<0.05), indicating that EETEB inhibited SGC-7901 cell proliferation in a dose-dependent manner.

To determine whether the cell-growth suppressive effect of EETEB was due to apoptosis, the pro-apoptotic activity of EETEB on SGC-7901 cells was determined using Annexin-V/PI staining followed by FACS analysis. As indicated in Fig. 2A and B, following treatment with 0, 0.75, 1.0 and 1.5 mg/ml EETEB, the percentage of cells undergoing early or late apoptosis was 8.90±0.81, 14.31±1.13, 18.28±2.32 and 50.68±2.05%, respectively. EETEB concentrations of 1.0 and 1.5 mg/ml resulted in a significant increase in apoptosis compared with the control group (P<0.05), indicating that EETEB promoted SGC-7901 cell apoptosis in a dose-dependent manner.

The promotion of cellular apoptosis by EETEB treatment was verified by examining nuclear morphological changes following staining of cell nuclei with the DNA-binding dye, DAPI. As indicated in Fig. 3, EETEB-treated cells (Fig. 3B-D) exhibited typical apoptotic morphological features, such as condensed chromatin and fragmented nuclei. By contrast, the untreated cell nuclei (Fig. 3A) exhibited homogenous and less intense staining.

The morphological effect of EETEB treatment on SGC-7901 cells was also evaluated using TEM. As demonstrated in Fig. 4A, untreated control cells did not display any morphological changes. However, EETEB treatment resulted in a decrease in the number of microvilli, shrinkage of the cytoplasmic organoid volume (as evidenced by the promotion of electron density), shrinkage of the nucleolus and margination of the heterochromatin (Fig. 4B-D).

Changes in the mitochondrial membrane potential were examined using FACS analysis with JC-1 staining. The membrane-permeable JC-1 dye exhibits potential-dependent accumulation in the mitochondria, indicated by a fluorescence emission shift from green (wavelength, ~525 nm) to red (wavelength, ~590 nm) (20). Therefore, a collapse in the mitochondrial membrane potential during apoptosis can be indicated by a decrease in the ratio of red/green fluorescence intensity. As demonstrated in Fig. 5, following treatment with 0, 0.75, 1.0 and 1.5 mg/ml EETEB, the JC-1 red/green fluorescence ratio in SGC-7901 cells was 22.67±0.56, 51.10±1.25, 71.48±1.18 and 97.13±3.21%, respectively. This indicated that EETEB treatment induced the loss of mitochondrial membrane potential in SGC-7901 cells in a dose-dependent manner.

The underlying mechanism mediating the pro-apoptotic activity of EETEB was investigated by performing RT-PCR to detect the mRNA expression levels and western blotting to determine the protein expression levels of Bcl-2 and Bax in SGC-7901 cells. As shown in Fig. 6, EETEB treatment markedly reduced the mRNA (Fig. 6A) and protein (Fig. 6B) expression levels of anti-apoptotic Bcl-2. By contrast, the expression of pro-apoptotic Bax appeared to be markedly increased following EETEB treatment, indicating that EETEB promoted SGC-7901 cell apoptosis by increasing the pro-apoptotic Bax/Bcl-2 ratio.