Gastric cancer samples and adjacent normal tissue samples used in the present study were obtained from 139 patients with gastric cancer that underwent resection at The Third Affiliated Hospital, Nanjing University of Traditional Chinese Medicine (Nanjing, China) and Wuxi XiShan People's Hospital (Wuxi, China) between January 2005 and August 2010. Normal gastric mucosa tissue (≤5 cm) adjacent to the tumor was excised and confirmed to be tumor-free following pathological analysis. Every resection specimen was examined by the Department of Pathology, The Third Affiliated Hospital, Nanjing University of Traditional Chinese Medicine (Nanjing, China), to confirm their histology features. The patients consisted of 81 males and 58 females, aged between 30 and 75 years (median, 50 years). The following inclusion criteria were sued for the present study: i) Complete surgical R0 resection of the primary tumor; ii) pathologically confirmed diagnosis of gastric adenocarcinoma; iii) no chemotherapy or radiotherapy administered; and iv) absence of secondary malignancies. All patients provided a signed agreement for participation in the study and the protocol was approved by the Ethics Committees of The Third Affiliated Hospital of Nanjing University of Traditional Chinese Medicine and Wuxi XiShan People's Hospital. Written informed consent was obtained from all patients involved in the present study. The epidemiological, clinical and pathological features of patients included in the present study are summarized in Table I. The clinical outcome of the patients was followed for 1–60 months, from the date of surgery to either the date of mortality or August 30, 2015.

The tissues were fixed for 24 h in 4% paraformaldehyde and embedded in paraffin. The paraffin-embedded tissues were cut into 4 µm sections. Then, tissue sections were deparaffinized in xylene and rehydrated through graded ethanol. Tissues were placed in 0.01 M citrate buffer and incubated at 100°C for 20 min for antigen retrieval. Tissues were blocked with a 3% hydrogen peroxide solution to inhibit endogenous peroxidase activity and washed with PBS, Subsequently, sections were incubated with 5% normal rabbit serum (Abcam, Cambridge, UK) for 30 min at room temperature to block non-specific binding sites. The slides were subsequently incubated with a TAK1 antibody (1:50; catalog no. sc-7162; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (dilution 1:1,000; catalog no. ab6721; Abcam) for 60 min at room temperature. The sections were then developed in 0.05% diaminobenzidine and counterstained with 0.1% hematoxylin and eosin (H&E) for 5 min at room temperature prior to dehydration and mounting. Evaluation of immunostaining in tumor cells was objectively performed by two pathologists under a light microscope at high magnification (×400). TAK1 staining was determined semi-quantitatively according to the intensity observed (0=no staining; 1=weak staining; 2=moderate staining; and 3=strong staining) and the percentage of positive cells (0, none or <5%; 1, 5–20%; 2, 21–40%; and 3, >40%). Scores of 0–2 were considered to be negative expression and scores of 3–6 were considered to be positive expression. Cells were counted in at least three randomly selected fields (at ×400 magnification) in the tumor areas.

The MGC803 human gastric cancer cell line was purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). MGC803 cells were cultured at 37°C and 5% CO₂ and saturation humidity in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.), with 10% fetal bovine serum, 100 U/ml penicillin (Thermo Fisher Scientific), and 100 mg/ml streptomycin (Thermo Fisher Scientific, Inc.). The cells adhered to the flask wall and grew into a single-cell monolayer and were passaged every 2–3 days. Cells in the exponential growth phase were harvested for subsequent experiments. There were four experimental groups: Control (without any intervention); vehicle treatment [1% dimethylsulfoxide (DMSO)]; low-dose OZ (3 µM); and high-dose OZ (6 µM). TAK1 kinase inhibitor, OZ was purchased from Tocris Bioscience, Bristol, UK (catalog no. 3604).

Cells were plated in 96-well plates at a density of 5×10³/well and treated with 6 µM OZ or vehicle for 24, 48 and 72 h at 37°C. At the aforementioned time points, 20 µl MTT substrate (5 mg/ml) was added to the cells and incubated at 37°C for 4 h. The resulting colored product was made soluble in 200 µl DMSO. Spectrometric absorbance at 490 nm was quantified using a microplate reader. Each cell line was established in quadruplicate wells and repeated three times.

Cell invasion activity was determined using a BD BioCoat Matrigel Invasion Chamber (8-µm; BD Biosciences, Franklin Lakes, NJ, USA). Briefly, gastric cancer cells were harvested and added to the upper chamber at a cell density of 2×10⁵ cells/ml in RPMI-1640 medium without FBS and treated with 6 µM OZ or DMSO. RPMI-1640 medium with 10% FBS was added to the lower chamber. The chambers were incubated for 48 h at 37°C and 5% CO₂. At the end of the incubation period, cells that had invaded through the membrane were subsequently fixed with 4% paraformaldehyde for 15 min and stained with 0.5% crystal violet for 30 min at room temperature. Cells were observed under ×40 magnification with a ZEISS light microscope and counted. Each experiment was performed in triplicate and repeated three times.

Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was used to analyze the apoptosis rate according to the manufacturer's protocol (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). MGC803 cells were seeded in six-well plates (1×10⁶ cells/well) at 37°C and treated with 6 µM OZ or vehicle for 24, 48 and 72 h. Cells were dissociated using trypsin, then centrifuged at 400 × g for 5 min. Next, cells were washed twice with PBS and centrifuged at 400 × g for 5 min. For apoptosis analysis, the cell pellet was resuspended in 500 µl binding buffer. Then, 5 µl Annexin V-FITC and 5 µl propidiumiodide (PI) was added to the cell suspension, which was gently mixed and incubated at room temperature, and was protected from light, for 15 min. Within 1 h, the cells were analyzed via flow cytometry using a BD FACSCanto II instrument (BD Biosciences, San Jose, CA, USA), and FlowJo software version 9.5.3 (Tree Star, Inc., Ashland, OR, USA).

MGC803 cells were treated with 6 µM OZ for 48 h at 37°C. Then, washed twice with PBS and centrifuged at 12,000 × g for 15 min at 4°C. A total cellular protein extraction kit (Beyotime Institute of Biotechnology, Haimen, China) was used to extract the total protein, and the nucleoprotein extraction kit (Beyotime Institute of Biotechnology) was used to extract nucleoprotein, according to the manufacturer's protocol. Isolation of mitochondrial and cytosolic proteins was performed using the Mitochondria/Cytosol Fractionation kit (Bi Yuntian Biological Technology Institution). Protein concentrations were determined using a BCA protein assay (Beyotime Institute of Biotechnology). Cell lysate was boiled for 12 min, and samples (40 µg protein per lane) were separated on 5–20% gradient SDS-PAGE gels. Proteins were subsequently transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), which were blocked overnight in 5% non-fat milk at 4°C. The membranes were incubated with the following primary antibodies at a dilution of 1:1,000: (p)-TAK1 (Thr187) (catalog no. 4536), pro-caspase 3 (catalog no. 9665), cyt c (catalog no. 11940), cyclin D1 (catalog no. 2978), Bcl-2 apoptosis regulator (Bcl-2; catalog no. 2827), voltage-dependent anion channel (catalog no. 4661), cleaved caspase 3 (catalog no. 9654), matrix metallopeptidase (MMP) 9 (catalog no. 13667), p65 (catalog no. 4764), histone 3 (catalog no. 4499) and β-actin (catalog no. 8457) at 4°C overnight. All primary antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The membranes were then washed with 1x TBS containing 0.1% Tween-20, incubated with anti-rabbit IgG conjugated to HRP (dilution, 1:1,000; Cell Signaling Technology, Inc. catalog no. 7074) for 1 h at room temperature, and washed with 1xTBS containing 0.1% Tween-20 three times for 10 min each. Proteins were visualized using an Enhanced Chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc., catalog no. 32106). The experiments were repeated at least 3 times. Densitometry analysis was performed using ImageJ software version 1.48 (National Institutes of Health).

Data are expressed as mean ± standard deviation. SPSS version 19.0 (IBM, Armonk, NY, USA) was used for statistical analysis. Count data were analyzed with a χ² test. Survival curves of the patients were compared using the Kaplan-Meier method and analyzed by the log-rank test. One-way analysis of variance followed by the Tukey post test was used to analyze differences between groups. P<0.05 was considered to indicate a statistically significant difference.

All gastric cancer tissue specimens and the adjacent normal tissue specimens used in the current study were verified using hematoxylin and eosin staining (Fig. 1). Immunohistochemistry revealed that the cytoplasm of gastric cancer cells appeared yellow or brown in a diffuse pattern, indicating high TAK1 expression; however, TAK1 expression was reduced in the adjacent normal tissues (Fig. 1).

To investigate the biological significance of TAK1 expression in gastric cancer, the patients were divided into two groups according to TAK1 immunostaining: the TAK1 negative group and the TRAF6 positive group. TAK1 positive expression was quantified as 70.5% in gastric cancer tissue samples and 25.9% in the adjacent normal tissues (P<0.001; Table II). Furthermore, TAK1 expression was positively associated with advanced N stage and pathological stage, indicating that TAK1 protein expression level may be elevated during gastric cancer progression. No significant association was identified between TAK1 protein expression level and gender, age, tumor size, tumor grade, neural or vascular invasion, T stage or M stage (P>0.05; Table I). Kaplan-Meier survival analysis (Fig. 2) demonstrated that the median 5-year survival was 21 months in patients with positive TAK1 expression, which was significantly lower compared with patients with negative TAK1 expression (41 months; P=0.009).

To evaluate the effects of OZ, the TAK1 inhibitor, on MGC803 cells, the present study examined the apoptotic properties of MGC803 cells incubated with OZ. The Annexin V and propidium iodide dual staining revealed that MGC803 cells from the TAK1 inhibitor treatment groups (3 and 6 µM) had a significantly greater percentage of apoptotic cells compared with the vehicle-treated group (P<0.05; Fig. 3A). To further investigate the cellular basis of the apoptotic response observed in the MGC803 cell line, the expression of apoptosis-associated proteins was investigated using western blotting. The aforementioned experiments confirmed that the high dose OZ (6 µM) effectively promoted apoptosis in MGC803 cells, 6 µM OZ was used for the subsequent experiments investigating the apoptotic mechanism. As demonstrated in Fig. 3B-D, OZ treatment significantly reduced the expression of mitochondrial cyt c (P<0.05; Fig. 3B), Bcl-2 (P<0.05; Fig. 3C) and procaspase 3 (P<0.05; Fig. 3C) compared with the vehicle treatment group. Conversely, cleaved caspase 3 (P<0.05; Fig. 3C) and cytosolic cyt c (P<0.05; Fig. 3D) expression levels were significantly greater in the OZ-treated group compared with the vehicle group.

To verify the effect of OZ treatment on tumor growth in MGC803 cells, cell proliferation was examined using an MTT assay. It was revealed that OZ treatment significantly inhibited the growth of MGC803 cells in a time-dependent manner (P<0.05; Fig. 4A) compared with vehicle-treated cells. As cyclin D1 has previously been reported to have an important role in gastric cancer proliferation (15,16), the cyclin D1 protein expression level was examined by western blot analysis. The findings indicated that cyclin D1 expression was significantly downregulated in the OZ treatment group compared with the vehicle-treated group (P<0.05; Fig. 4B).

The present study further investigated the effect of OZ treatment on the invasive behavior of MGC803 cells. As presented in Fig. 5A and B, OZ treatment significantly reduced the invasive ability of MGC803 cells compared with the vehicle-treated group (P<0.05). As MMP9 has been previously reported to have an important role in gastric cancer invasion (17), the present study also investigated the expression level of MMP9 protein by western blot analysis and demonstrated that MMP9 expression was significantly downregulated in the OZ treatment group compared with the vehicle treatment group (P<0.05; Fig. 5C).

Following treatment with OZ for 48 h, the expression levels of p-TAK1 (Thr187) and nuclear p65 protein were detected by western blot analysis. As presented in Fig. 6, OZ treatment significantly reduced p-TAK1 (Thr187) and nuclear p65 expression levels compared with the vehicle treatment group (both P<0.05).