RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). 3-BrPA, SCT and the chemotherapeutic agent 5-fluorouracil (5-FU) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The human gastric cancer cell line SGC-7901 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100,000 U/l penicillin, and 100 mg/l streptomycin at 37°C in an incubator with 5% CO₂. The cells were harvested after trypsinization by 0.025% trypsin with 0.02% EDTA and washed twice with phosphate-buffered saline (PBS). The cells were split for further culture once they reached ~80% confluency. Experiments were not conducted until the cells were in logarithmic growth phase. The 5-to-6-week old female BALB/c nude mice (weighing between 18 and 20 g) were purchased from the Animal Experimental Center of Guangxi Medical University (Guangxi, China) and fed under specific pathogen-free conditions. The experimental protocol was carried out under the supervision of the Ethics Committee of Guangxi Medical University, and in accordance with internationally recognized guidelines on Animal Welfare.

Cell viability was assessed with the modified tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) method. Briefly, SGC-7901 cells were exposed to different concentrations of 3-BrPA or SCT (or 5-FU or the PBS control) for 24 or 48 h after being seeded onto 96-well plates (~2,000 cells/well) for 24 h. The 3-BrPA and SCT solutions were prepared in RPMI-1640 medium, adjusted to pH 7.4 with NaOH, and then sterilized using a 0.22-µm filter unit (Millipore, Billerica, MA, USA). To each well, 0.01 ml MTT solution (5 mg/ml) was added and incubated at 37°C for 4 h. Formazan crystals were then dissolved with DMSO, and the optical densities at 490 nm were measured using a microplate reader (Bio-Rad, Hercules, CA, USA). The IC₅₀ value (concentration of 50% inhibition) was obtained from three independent repetitive trials.

SGC-7901 cells were cultured with 5-FU (0.5 mmol/l) and different concentrations of 3-BrPA and SCT for 24 h. The morphological changes in cells were photographed using inverted microscopy (Nikon, Tokyo, Japan).

The apoptosis of SGC-7901 cells was analyzed by flow cytometry (FCM) with the Annexin V:PE Apoptosis Detection kit I (BD Biosciences, USA). Briefly, the cells were collected after being treated for 24 or 48 h with 3-BrPA, SCT, 5-FU or the PBS control, resuspended in 100 µl of binding buffer with 5 µl Annexin V-PE and 5 µl 7-amino-actinomycin D (7-AAD), washed twice with cold PBS, and then incubated in the dark at room temperature for 15 min. The samples were then analyzed by FCM (Beckman Coulter, Miami, FL, USA).

SGC-7901 cells were collected after being treated for 24 h with 3-BrPA, SCT, 5-FU or the PBS control, washed twice with PBS and fixed in 70% cold ethanol. The cells were then incubated with RNase A (0.1 mg/ml) at 37°C for 30 min, and then with propidium iodine (PI, 0.2 mg/ml) at 4°C for 60 min before analysis. Cell cycle progression was measured using the Cell Cycle Detection kit (Keygen, Nanjing, China) and analyzed by FCM.

According to the manufacturer's instructions in the HK, PFK-1, PK, ATP and lactate assay kits (Jiancheng, Nanjing, China), we measured the activity or concentration of HK, PFK-1, PK, ATP and lactate spectrophotometrically (UV-2450; Shimadzu, Japan) at an absorbance of 340 nm. Samples were obtained from SGC-7901 cells after being treated for 24 or 48 h with 3-BrPA, SCT, 5-FU or the PBS control. The activities of the enzymes were calibrated with cellular protein concentration.

Total RNA was prepared by using the total RNA Extraction kit (Axygen, Union City, CA, USA) and reverse-transcribed with the PrimeScript^® RT reagent kit (Takara, Tokyo, Japan). The primers for Bax, Bcl-2, Cyt-C, survivin, and GAPDH synthesized by Sangong Biotech (Shanghai, China) are shown in Table I. The reaction was carried out using the SYBR Green PCR Master Mix (Roche, USA). Real-time PCR assays were performed using Applied Biosystems^® 7500 Real-Time PCR systems (Life Technologies, MA, USA) according to the manufacturer's instructions. PCR was carried out for 40 cycles of 95°C for 15 sec and 60°C for 30 sec. The mRNA expression levels were calculated relative to GAPDH by using the 2^−ΔΔCT method.

The total protein was extracted from the SGC-7901 cells after being treated for 24 h with 3-BrPA, SCT, 5-FU or the PBS control, and protein concentration was determined using the BCA method. A 15% separation gel was used to separate the proteins using SDS-PAGE at a constant voltage (80 mV) for 90 min. A 5% stacking gel and water bath method were used to transfer the membranes at a constant current (180 mA) for 30–75 min. The NC membrane (Millipore) was blocked with 5% skimmed milk for 2 h at room temperature. The rabbit antibodies against Bcl-2, Bax, Cyt-C, survivin, GAPDH and the anti-rabbit IgG DyLight 800 conjugated antibody were purchased from Cell Signaling Technology (Boston, MA, USA). The rabbit antibody against cleaved caspase-3 (P17KD fragment) was purchased from Neomarker (USA). The primary antibodies were diluted with primary antibody dilution buffer (Beyotime, Shanghai, China) and incubated at 4°C in the refrigerator overnight. The secondary antibodies were added to the slides, and then incubated for 90 min at room temperature. The gray scale values of the protein bands were determined by Odyssey software (LI-COR, Lincoln, NE, USA).

The back of the axillary region of each nude mouse was subcutaneously injected with 0.2 ml (2×10⁶/ml) of the SGC-7901 cell suspension. When the subcutaneous tumor became palpable (i.e., ~5 mm after ~2 weeks), the tumor tissue was excised from the nude mouse, and then minced. The minced tumor tissue became the subcutaneous transplant for the next group of nude mice, and this subcutaneous transplantation was repeated for six generations. The tumor tissue from the sixth generation nude mice was minced to 1–2 mm³ in size before being orthotopic transplanted inside the seromuscular layer of greater curvature. We chose the seromuscular layer near the antrum of the greater curvature as the preferable operation site for its rich blood flow. On account of its secure wound adhesion, we used 1–2 drops of medical OB glue (32) to bind the serosal layer incision, before covering it with the greater omentum. We waited for ~10 sec for the glue to fully congeal to avoid the tumor tissue masses falling off. The tumor was allowed to grow for 2 weeks before treatment was initiated.

Mice were then randomized into 8 groups (8 mice/group) as follows: 3-BrPA low-dose group (3-BrPA-L, 1.85 mg/kg/day), 3-BrPA medium-dose group (3-BrPA-M, 2.23 mg/kg/day), 3-BrPA high-dose group (3-BrPA-H, 2.67 mg/kg/day), SCT low-dose group (SCT-L, 7.5 mg/kg/day), SCT medium-dose group (SCT-M, 15 mg/kg/day), SCT high-dose group (SCT-H, 30 mg/kg/day), 5-FU group (5-FU, 10 mg/kg/day), and control group (PBS, 10 ml/kg/day). 3-BrPA, SCT and 5-FU were dissolved in PBS, adjusted to pH 7.4 with NaOH, and then sterilized with a 0.22-µm filter unit. All mice were intraperitoneally injected with 0.2 ml of the drug or PBS once per day for 4 weeks. Any changes observed in the nude mice, such as behavior, eating and excretion were monitored. The tumors were harvested after 4 weeks of treatment, and tumor volume was calculated as 0.5 × length × width × thickness.

Apoptosis in the tumor tissue was investigated by TUNEL staining using the In Situ Cell Death Detection kit (Roche, USA). The percentages of positive cells from total cells were considered as apoptotic indices (AI) after being counted in six different high power fields. The tumor tissue sections were observed under an IX73 inverted microscope (Olympus, Tokyo, Japan).

The ultra-structure of the tumors was observed by TEM. The tumor tissues were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide, dehydrated by graded ethanol, and embedded in Epon. Ultrathin sections were stained with 2% uranyl acetate and lead citrate, and then examined under a JEM-2000EX transmission electron microscope (Jeol, Tokyo, Japan).

All statistical analyses were performed using SPSS 17.0 software (SPSS, Chicago, IL, USA). The data are presented as the mean ± standard deviation (SD). Differences between multiple groups were analyzed with one-way analysis of variance. Pairwise comparison was performed using the t-test, with significant differences determined as a value of P<0.05 (P<0.05 vs. the control group and P<0.05 vs. the 5-FU group).

As shown in Fig. 1A, untreated cells attached closely to one another, and were polygonal in shape. However, the cells in the 5-FU-, 3-BrPA-, or SCT-treated groups became round or inflated, with fewer cellular contacts.

We measured the proliferative effect of 3-BrPA and SCT on SGC-7901 cells using the MTT assay. As shown in Fig. 1B, we found a time- and dose-dependent decrease in cell viability after exposure to 3-BrPA or SCT. The IC₅₀ values of 3-BrPA against SGC-7901 cells after being treated for 24 or 48 h were 9.88±1.07 and 5.97±0.95 µg/ml, respectively; however, the IC₅₀ values of SCT were 6.47±0.87 and 3.84±0.59 mg/ml. Therefore, for our in vitro study we created the following eight treatment groups: 3-BrPA low-dose group (3-BrPA-L, 4 µg/ml), 3-BrPA medium-dose group (3-BrPA-M, 6 µg/ml), 3-BrPA high-dose group (3-BrPA-H, 8 µg/ml), SCT low-dose group (SCT-L, 1.5 mg/ml), SCT medium-dose group (SCT-M, 3 mg/ml), SCT high-dose group (SCT-H, 6 mg/ml), 5-FU group (5-FU, 0.5 mmol/l), and control group (untreated).

The effect of 3-BrPA and SCT on the cell cycle was detected by FCM. As shown in Fig. 1C and D, after exposure to 3-BrPA, SCT or 5-FU for 24 h, the cell population in the G2/M phases was significantly increased (P<0.05). The cell population in the S phase of the 5-FU group was also obviously increased (P<0.05). The results suggest that one of the inhibitory mechanisms of 3-BrPA or SCT is to induce cell cycle arrest to increase the rate of apoptosis.

To determine whether 3-BrPA- and SCT-induced cell death is related to apoptosis, an Annexin V:PE staining assay was conducted. As shown in Figs. 2 and 3, the percentage of apoptosis was increased from 15.60±1.83% after 24 h to 84.45±3.97% after 48 h in the 3-BrPA-treated groups, and increased from 19.31±1.89 to 88.34±5.22% in the SCT groups. As a positive control, the percentage was increased from 61.57±4.30 to 92.64±4.15% after treatment with 5-FU. These increases in apoptosis percentages were significant compared with the control group (3.24±0.72% after 24 h and 9.70±1.52% after 48 h) (P<0.05). The result demonstrated that dose- and time-dependent apoptosis occurs after exposure to 3-BrPA or SCT. Moreover, the reductions in cell viability using the MTT assay correlated well with the induction of apoptosis by 3-BrPA or SCT.

We evaluated the activities of HK, PFK-1 and PK to investigate whether 3-BrPA and SCT kill cancer cells through regulation of glycolytic enzymes. As shown in Fig. 4A, 3-BrPA significantly reduced HK activity in a time- and dose-dependent manner (P<0.05). The HK activity observed in the 5-FU group was less than that in the 3-BrPA-L group but more than that in the 3-BrPA-M group (P<0.05). HK activity in the SCT groups had no difference when compared with the activity in the control group (P>0.05). As shown in Fig. 4B, PFK-1 activity decreased in a time- and dose-dependent manner in the SCT groups (P<0.05). However, PFK-1 activity was not inhibited by 3-BrPA or 5-FU (P>0.05). As shown in Fig. 4C, there were no significant difference noted in PK activity in the 5-FU, 3-BrPA, and SCT groups compared with the control group (P>0.05).

ATP and lactate are the main end products of glycolysis, and are regarded as efficient indicators of the glycolysis rate. As shown in Fig. 5, both the 3-BrPA and SCT groups exhibited a significant time- and dose-dependent decrease in cellular ATP levels and lactate production (P<0.05). Moreover, the cellular ATP and lactate levels in the 5-FU group after treatment for 8 h were obviously reduced compared with the control group (P<0.05). These results indicate that 3-BrPA and SCT may cause apoptosis by blocking glycolysis, which is required for energy metabolism.

To determine the effect of apoptosis-related genes induced by 3-BrPA and SCT, the mRNA levels of Bax, Bcl-2, Cyt-C and survivin were detected by RT-qPCR analysis. As shown in Fig. 6, compared with the control group, the mRNA levels of Bax and Cyt-C were increased, while the mRNA levels of Bcl-2 and survivin were decreased after exposure to 3-BrPA, SCT, or 5-FU (P<0.05). Furthermore, we found that 3-BrPA or SCT produced these effects in a dose-dependent manner. The mRNA levels of Bax and Cyt-C in the SCT-H group were higher than these levels in the 3-BrPA-H group (P<0.05). While 5-FU also increased the mRNA expression of Bax and Cyt-C, and decreased the mRNA expression of Bcl-2, it was less effective than 3-BrPA-H and SCT-H (P<0.05). However, the inhibitory effect of 5-FU on survivin mRNA expression was the highest among all the groups (P<0.05).

To further confirm that 3-BrPA and SCT regulate the expression of apoptosis-related genes, we determined the protein expression of Bax, Bcl-2, Cyt-C, cleaved caspase-3, and survivin by western blot analysis. As shown in Fig. 7, 3-BrPA and SCT both upregulated Bax, Cyt-C, and cleaved caspase-3 protein expression, and downregulated Bcl-2 and survivin protein expression in a dose-dependent manner (P<0.05). 5-FU also increased the protein expression of Bax, Cyt-C, cleaved caspase-3 and decreased the expression of Bcl-2 and survivin, but its ability was weaker than 3-BrPA-M and SCT-M (P<0.05). The increased expression of Bax, and downregulation of Bcl-2 and survivin, in the 3-BrPA-H group was less than that in the SCT-H group (P<0.05). Expression levels of Cyt-C and cleaved caspase-3 in the 3-BrPA-H group were not significantly different when compared with the SCT-H group (P>0.05).

To determine whether 3-BrPA and SCT are effective against gastric tumors in vivo, we produced a gastric orthotopic transplantation tumor model and administered an intraperitoneal injection of 3-BrPA, SCT, 5-FU or PBS for 4 weeks. As shown in Fig. 8A, models of human gastric orthotopic transplantation tumors in nude mice were successfully made, and the tumor formation rate was 85%. As shown in Fig. 8A and B, tumor volumes in the 3-BrPA-, SCT-, or 5-FU-treated mice were significantly reduced compared with the PBS-treated mice (P<0.05). Furthermore, 3-BrPA and SCT inhibited the orthotopic transplantation tumor growth in a dose-dependent manner (P<0.05).

We next evaluated whether 3-BrPA and SCT induce apoptosis in orthotopic transplantation tumors by TUNEL staining. As shown in Fig. 8C, a greater proportion of apoptotic cells, appearing with brownish granules in the cytoplasm and nucleus, were observed in the 3-BrPA, SCT, and 5-FU groups. As shown in Fig. 8D, the apoptosis index (AI) in each group was as follows: 3-BrPA-L group 12.50±2.43%; 3-BrPA-M group 26.29±5.76%; 3-BrPA-H group 52.21±6.92%; SCT-L group 13.98±2.28%; SCT-M group 30.93±3.79%; SCT-H group 55.07±7.38%; 5-FU group 52.71±6.73%; and control group 3.29±0.76%. A significant difference in AI values was identified between the 3-BrPA-, SCT-, or 5-FU-treated group and the control group (P<0.05). The AI of the 5-FU group was similar to the 3-BrPA-H and SCT-H groups (P>0.05). Furthermore, 3-BrPA-H and SCT-H both induced apoptosis of the orthotopic transplantation tumors in a dose-dependent manner.

To further confirm the apoptotic effects, ultrastructural changes in the tumors treated by 3-BrPA, SCT, or 5-FU were observed by TEM. As shown in Fig. 9, typical features of apoptosis, such as the formation of apoptotic bodies, mitochondrial crest fracture, or disappearance and chromatin concentration or fragmentation, were observed in the 3-BrPA, SCT and 5-FU groups. However, the control group had large nuclei, with prominent nucleoli, and no apoptotic bodies were observed.