The human gastric cancer cell lines BGC-823 and SGC-7901 were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal bovine serum (Tianhang Biological Technology Co., Ltd., Hangzhou, China). All cells were maintained in a 5% CO₂ atmosphere at a temperature of 37°C.

To determine cell growth, a colorimetric water-soluble tetrazolium salt assay (Cell Counting Kit 8; Dojindo Laboratories, Kumamoto, Japan) was performed. This allowed the number of viable cells to be evaluated following treatment with various agent combinations.

The detection of apoptotic cells was performed using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Beyotime Institute of Biotechnology, Nantong, China), according to the manufacturer's instructions. Cells were plated in 6-well plate (5×10⁵ cells/well) and allowed to grow to 75–80% confluence. Following treatment with DIM (10 µmol/l), TRAIL (25 or 50 ng/ml) or DIM (10 µmol/l) + TRAIL (25 ng/ml) for 24 h, the cells were collected, resuspended in 500 µl binding buffer, and 5 µl Annexin V-FITC and 5 µl propidium iodide were added. Untreated cells were washed twice with phosphate-buffered saline (Beyotime Institute of Biotechnology), collected, resuspended in 500 µl binding buffer, and 5 µl Annexin V-FITC and 5 µl propidium iodide were added, which served as the control. The data was quantified and analyzed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Western blot analysis was conducted as previously described (11). Briefly, cells were lysed by incubating in lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS; pH 7.4; Beyotime Institute of Biotechnology) for 20 min at 4°C and the protein concentration was determined using the Beyotime assay system (Beyotime Institute of Biotechnology). Protein (60 µg) was run on 12.5% polyacrylamide gel and transferred to a nitrocellulose membrane (Hybond-ECL; Amersham Pharmacia Biotech, Buckinghamshire, UK). Next, the membrane was blocked with Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% nonfat milk for 2 h at room temperature and incubated overnight at 4°C with primary monoclonal antibodies against DR5 (cat. no. sc-166624), DR4 (cat. no. sc-8411), CCAAT/CHOP (cat. no. sc-7351), GRP78 (cat. no. sc-376768) and GAPDH (cat. no. sc-365062) purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The membrane was washed three times with TBST and incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (cat. no. A0216, Beyotime Institute of Biotechnology) for 1 h at room temperature. Immunoreactive bands were subsequently identified using LumiGLO® chemiluminescent substrate (Cell Signaling Technology, Inc.). The light emitted by destabilized LumiGLO® reagent was subsequently captured on X-ray film, (Cell Signaling Technology, Inc.), normalized to the corresponding GAPDH level and analyzed with ImageJ software (version 1.44; National Institutes of Health, Bethesda, MD, USA).

The SPSS software package (version 15.0; SPSS, Inc, Chicago, IL, USA) was used for data analysis, with data presented as the mean ± standard deviation (SD) and n indicating the number of independent experiments performed. After determining equal variance, comparisons among the means of multiple groups were performed using one-way analysis of variance and Fisher's least significant difference test. Additionally, two-tailed independent samples t-tests were used when appropriate. P<0.05 was considered to indicate a statistically significant difference.

The present study examined the role of TRAIL and DIM treatment, alone or in combination, on the proliferation of human gastric cancer cell lines BGC-823 and SGC-7901. The treatment of gastric cancer cells with 10 µmol/l DIM for 24 h induced marginal cytotoxicity (<20%). Similarly, limited cytotoxicity (20%) was observed following treatment with TRAIL at concentrations of 25–50 ng/ml. However, compared to treatment with TRAIL or DIM alone, significant cytotoxic effects were induced following co-treatment of the two gastric cancer cell lines with TRAIL (25 ng/ml) and DIM (10 µmol/l), compared with control (P=0.0001 in BGC-823, P=0.0001 in SGC-7901), TRAIL (P=0.0002 in BGC-823, P=0.0006 in SGC-7901) or DIM alone (P=0.0002 in BGC-823, P=0.0006 in SGC-7901 (Fig. 1).

To investigate whether apoptosis is correlated with the inhibition of cell proliferation following DIM and TRAIL co-treatment, flow cytometric analysis was used to detect the increase in hypodiploid cell populations. The mean (n=3) proportions of late apoptotic BGC-823 cells were 15.1, 16.9, 19.4 and 39.7% for DIM (10 µmol/l), TRAIL (25 ng/ml), TRAIL (50 ng/ml), and TRAIL (25 ng/ml) plus DIM (10 µmol/l), respectively (Fig. 2A). Similarly, in SGC-7901 cells, the mean (n=3) proportions were 10.9, 13.9, 16.1 and 37.3% for DIM (10 µmol/l), TRAIL (25 ng/ml), TRAIL (50 ng/ml), and TRAIL (25 ng/ml) plus DIM (10 µmol/l) (Fig. 2B). These results indicate that DIM sensitizes gastric cancer cells to TRAIL-induced apoptosis.

BGC-823 cells were treated with DIM for 24 h and subsequently analyzed by western blot to investigate the expression levels of various TRAIL receptor-associated proteins. The group of cells treated with TRAIL plus DIM were demonstrated to significantly induce the protein expression levels of DR5 (P<0.001) but exhibited no effect on DR4 expression. This data indicates that DR5 may be involved in DIM enhancement of the apoptotic effects of TRAIL in BGC-823 cells. Significant upregulation of DR5 by DIM plus TRAIL was also identified in SGC-7901 cells (P<0.05). Furthermore, DIM and TRAIL co-treatment was observed to induce a significant increase in the protein expression levels of CHOP and GRP78 (P>0.05; Fig. 3), two proteins that accumulate during endoplasmic reticulum (ER) stress (12).