BER (purity, 98%), evodiamine (EVO; purity, 98%) and 5-fluorouracil (5-Fu; purity, 98%) were obtained from Melonepharma Co., Ltd. (Dalian, China). Trypsin and fetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific, Inc. Waltham, MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). IL-8 (cat. no. 88-8086-88) and TNF-α (cat. no. 88-7346-88) enzyme-linked immunosorbent assay (ELISA) kits were obtained from eBioscience, Inc., (San Diego, CA, USA). Anti-GAPDH (cat. no. 5174), anti-phospho (p) p38 MAPK (cat. no. 4511), anti-pERK1/2 (cat. no. 9154), anti-pJNK (cat. no. 4668) and anti-β-actin (cat. no. 12413) antibodies were supplied by Cell Signaling Technology, Inc. (Danvers, MA, USA). Enhanced chemiluminescence (ECL) Prime kit was purchased from GE Healthcare Life Sciences (Chalfont, UK). SB202190, SP600125 and PD98059 were purchased from Selleck Chemicals (Houston, TX, USA). Anisomycin was obtained from EMD Millipore (Billerica, MA, USA).

MGC 803 cells obtained from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) were cultured in RMPI 1640 medium (Thermo Fisher Scientific, Inc.), supplemented with 10% FBS. The cells were cultured at 37°C in a humidified incubator with 5% CO₂.

Cells were seeded in 100 µl medium at 1.0×10⁴ cells/ml in 96-well culture plates and cultured overnight. Following pre-incubation with or without inhibitors of p38 MAPK (25 µM SB202190), ERK1/2 (20 µM PD98059), JNK (20 µMSP600125) and the activator of MAPKs (0.05 µg/ml anisomycin) for 1 h, cells were treated with BER (0, 7.5, 15, 30 and 60 µM) for 24 or 48 h. The medium was then removed and replaced with equal volume of fresh medium with additional 10 µl CCK-8 solution and incubated at 37°C for 20 min. Absorbance of the dissolved solutions was detected at 450 nm using a Varioskan Flash microplate reader (Thermo Fisher Scientific, Inc.). Cell viability rate (%) was calculated as follows: (Absorbance of drug-treated sample/absorbance of control sample) ×100.

For in vitro experiments, MGC 803 cells were seeded in 96-well culture plates and cultured overnight. Following treatment with BER (0, 15, 30 and 60 µM) for 48 h, culture medium was collected and subjected to IL-8 and TNF-α ELISA assay using the respective kits. To identify the involvement of MAPKs in modulation of IL-8 expression, the cells were pre-treated with SB202190 (25 µM), SP600125 (20 µM), PD98059 (20 µM) and anisomycin (0.25 µg/ml) for 1 h, then treated with or without BER (60 µM) for 24 or 48 h. The culture medium was collected for IL-8 ELISA.

For in vivo experiments, serum and the supernatant of tumor homogenates from nude mice xenografts were used for IL-8 ELISA.

Cells or tumor tissues were lysed with CelLytic MT Cell Lysis Reagent (Sigma-Aldrich, St. Louis, MO, USA) and sonicated three times, each for 15 sec. The lysate was centrifuged at 14,000 × g for 15 min at 4°C and the supernatant was collected. Protein concentration was determined by the bicinchoninic acid method. Protein samples (30 µg) were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane using the wet transfer method. Then, PVDF membranes were blocked with 5% non-fat milk solution at room temperature for 1 h and incubated with the different primary antibodies (anti-GAPDH, 1:5,000; anti-p-p 38, 1:1,000; anti-p-ERK, 1:1,000; anti-p-JNK, 1:1,000; and anti-β-actin, 1:2,000) overnight at 4°C. After washing with 1X phosphate-buffered saline Tween 20 (PBST), PVDF membranes were incubated with the respective secondary antibodies (1:5,000; cat. no. 111-035-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 h at room temperature. The protein bands were visualized with the ECL Prime kit and X-ray films.

Total RNA was extracted from the MGC 803 cells by using TRIzol reagent. RT was performed using a Prime-Script RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). Forward and reverse primers used for qPCR are presented in Table I. qPCR reactions were performed using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.) under the following cycling conditions: Initial step of 95°C for 30 sec; followed by 95°C for 5 sec and 60°C for 34 sec for 40 cycles; with final steps of 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. The relative expression level of IL-8 was normalized to that of GAPDH in the same sample. Relative expression of target genes was normalized to GAPDH, analyzed by 2^−ΔΔCq method (21) and presented as a ratio compared with the control.

Thirty 4-week-old male BALB/C nude mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China; license no. SCXK 2014-0008). The tumor xenograft model was established by subcutaneous injection of MGC 803 cells (5×10⁶ cells in 200 µl PBS) into the right flank of the mouse. Animals bearing tumors were randomly divided into five groups (n=6) as follows: i) Control group; ii) EVO group (45 mg/kg); iii) BER group (15 mg/kg); iv) BER + EVO group (EVO 45 mg/kg, BER 15 mg/kg) and v) 5-Fu group (25 mg/kg). Prior to injection of the MGC 803 cells, mice were orally administrated with BER, EVO or 5-Fu, and administration was continued for 23 days. Body weight and two perpendicular tumor diameters (width, a; length, b) were recorded every 4 days. The tumor volume was calculated as ab²/2. Following completion of treatment, the mice were sacrificed using 1% pentobarbital sodium (DingGuo Biotech Co., Ltd., Shanghai, China). The tumors were dissected, weighed, and stored at −80°C for use in ELISA and western blotting.

All animal experiments were performed according to the protocols approved by Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine, (Shanghai, China) which complies with international rules and policies. All efforts were made to minimize suffering and reduce the number of animals used.

Values are presented as the mean ± standard error. Differences among groups were analyzed by one-way analysis of variance with Newman-Keuls test using Prism software (version 5; GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

BER demonstrated an inhibitory effect on the viability of MGC 803 cells in a dose- and time-dependent manner. As demonstrated in Fig. 1, BER treatment (7.5–60 µM) significantly decreased the cell viability compared with the control (P<0.001) at 24 and 48 h. Furthermore, prolonged BER treatment (48 h) led to increased injury to the MGC 803 cells as demonstrated by the reduced cell viability.

To determine the association between MAPK signaling pathways, including p38 MAPK, ERK1/2 and JNK pathway, and cell survival of MGC 803 cells, western blot analysis was performed. As demonstrated in Fig. 2, BER treatment at 15, 30 and 60 µM for 24 and 48 h reduced the phosphorylation of p38 MAPK, ERK1/2 and JNK in a dose- and time-dependent manner.

In order to further clarify the importance of MAPK signaling in the cell proliferation, and whether BER inhibited cell proliferation of gastric cancer cells via deactivating MAPKs, the inhibitors of p38 MAPK (SB202190), JNK (SP600125) and ERK1/2 (PD98059) were used in a CCK-8 assay. As demonstrated in Fig. 3A, in MGC 803 cells, these inhibitors enhanced the inhibitory effect of BER on cell proliferation compared with BER treatment (P<0.001, P<0.001 and P<0.05). Notably, anisomycin, an activator of p38 MAPK and JNK, also significantly reduced cell viability of MGC 803 cells compared with controls (Fig. 3C). As demonstrated in Fig. 3B and D, as BER treatment for 48 h killed the majority of the cancer cells, combination of BER with the inhibitors or activator of MAPKs did not demonstrate a synergistic effect at this time point.

In previous research, BER was demonstrated to inhibit IL-8 expression in a dose- and time-dependent manner in AGS and MDA-MB-231 cells (10,12). However, whether the inhibitory effect of BER on IL-8 expression is cell-type specific remains unclear. The present study demonstrated that BER also reduces the secretion (P<0.001; Fig. 4A) and gene expression levels (P<0.001; Fig. 4B) of IL-8 in MGC 803 cells compared with controls. Further investigation demonstrated that anisomycin significantly increased the secretion of IL-8 and reduced cell viability of MGC 803 cells compared with controls (P<0.001), which was completely abolished by combination with BER treatment (P<0.001; Fig. 4C and D). Furthermore, as demonstrated in Fig. 4E, treatment with SB202190, SP600125 or PD98059 significantly decreased IL-8 secretion in MGC 803 cells compared with controls (P<0.001). However, BER did not alter TNF-α production of the cells (Fig. 4F). Thus, the results of the present study indicated that BER specifically affects IL-8 production in MGC 803 cells.

To examine the anti-tumor effect of BER in vivo, a human gastric cancer xenograft model was used in BALB/C nude mice. In nude mice transplanted with MGC 803 cells were treated with the drugs for 23 days. 5-Fu (25 mg/kg), was administered as a positive control and significantly prevented the growth of tumor (Fig. 5), with the tumor weight and volume of mice in this group significantly reduced compared with the control group (P<0.001). However, long-term treatment with 5-Fu led to a significant reduction in the body weight compared with control mice (P<0.001). By contrast, BER (15 mg/kg daily) significantly reduced the tumor weight and tumor volume compared with controls (P<0.05 and P<0.001, respectively), and also did not lead to body weight loss. EVO treatment also significantly inhibited tumor weight and size compared with controls (P<0.05 and P<0.01, respectively). Co-administration of EVO and BER demonstrated a trend to inhibit tumor weight and volume compared with BER or EVO alone, but without a statistically significant difference. Both EVO and combination of BER and EVO demonstrated no significant effect on body weight.

In a previous study, EVO was demonstrated to upregulate IL-8 expression in AGS cells in vitro (10), whether it induces IL-8 expression in vivo remains to be determined. In the present study, EVO, the alkaloid from Evodia fructus, significantly suppressed MGC 803 tumor development in nude mice compared with controls (Fig. 5), but increased IL-8 production in tumor tissue (P<0.01; Fig. 6A) and serum (P<0.05; Fig. 6B). BER treatment alone, or in combination with EVO significantly attenuated the increase of IL-8 in tumor tissue and serum compared with EVO treatment (P<0.01 and P<0.05, respectively).

Following treatment with BER at a dose of 15 mg/kg for 23 days, the phosphorylation levels of p38 MAPK, ERK1/2 and JNK in tumor tissue was significantly reduced compared with controls (Fig. 7). By contrast, EVO alone did not alleviate the phosphorylation of p38 MAPK, ERK1/2 and JNK compared with control levels. However, co-treatment with BER and EVO markedly reduced the phosphorylation of the MAPKs in tumor tissues compared with control and EVO-treated nude mice.