A total of 60 specimens of gastric carcinoma tumors and para-carcinoma tissues were sampled from patients during resection in Heilongjiang Province Land Reclamation Headquarter General Hospital (Harbin, China) between January 2014 and February 2016. The clinicopathological data of all cases were reviewed, including patient age, sex, tumor size, differentiation status, tumor node metastasis (TNM) stage, lymph node metastasis and invasion status (Table I) (15). No patients had received preoperative chemotherapy and radiotherapy, and those who succumbed to other diseases or accidents were excluded from the study. All specimens were fixed in 10% neutral formaldehyde at room temperature for 24–48 h, and embedded in paraffin at 65°C and cut into 5-µm sections. All specimens were subjected to hematoxylin staining for 5 min and eosin staining for 1 min at room temperature and diagnosed as gastric carcinoma by two pathologists. Informed consent was obtained from all patients or their relatives. The experimental protocol was approved by the Ethics Committee of the King Medical Diagnostics Center (Shanghai, China).

MKN-45, MKN-28, and SGC-7901 gastric cancer cell lines (Cancer Cell Bank, Chinese Academy of Medical Sciences, Beijing, China) were cultured in Dulbecco's modified Eagle's medium (DMEM; PAN Biotech GmbH, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C with 5% CO₂. Western blot analysis determined that Beclin-1 expression was relatively low in the MKN-45 cell line (data not shown), thus the MKN-45 cell line was selected for all follow-up overexpression experiments.

The Beclin-1 sequence was cloned into a PCDNA3.0 plasmid (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). For transfection, the cells were seeded at a density of 1×10⁵/well in a 24-well plate and grown to >50-70% confluency. A total of 1 µl Lipofectamine 2000^® (Invitrogen; Thermo Fisher Scientific, Inc.) was added to 50 µl serum-free Opti-MEMI medium (Invitrogen; Thermo Fisher Scientific, Inc.) and mixed gently at room temperature for 5 min; 2 µg Beclin-1 plasmid was added to 50 µl serum-free Opti-MEMI medium and mixed gently. After 5 min, the diluted Lipofectamine 2000^® was gently mixed with the diluted Beclin-1 plasmid at room temperature for 20 min. Subsequent to transfection with Beclin-1 or control plasmids (empty PCDNA3.0 vector), the 24-well plate was incubated at 37°C with 5% CO₂ for 6 h, following which the culture medium was replaced with complete medium.

The paraffin sections were incubated at 65°C for 2 h and dewaxed in xylene for 20 min at room temperature. The specimens were washed with distilled water three times, incubated in 3% H₂O₂ at room temperature for 10 min and washed with distilled water again. Antigen retrieval was performed using Tris-EDTA buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in a pressure cooker at medium to high pressure for 3 min. Subsequent to washing with PBS, the specimens were incubated with monoclonal primary antibody against Beclin-1 (1:500; cat. no. ab210498; Abcam, Cambridge, UK) at 4°C overnight. The specimens were then rinsed with PBS buffer, and incubated in a 1:1,000 solution of biotin-labeled anti-Human Immunoglobulin G (IgG) secondary antibody (cat. no. SAB3701279; Sigma-Aldrich; Merck KGaA) at room temperature for 60 min. The specimens were washed with PBS buffer and treated with 3,3-Diaminobenzidine (Sigma-Aldrich; Merck, KGaA) for 2 min at room temperature. Slides were counterstained with hematoxylin for 1 min at room temperature, washed with distilled water, dehydrated sequentially with a graded ethanol series (80, 95 and 100% ethanol), transparentized with xylene and sealed with neutral gum at room temperature.

For each section, 10 high-power fields at magnification, ×400 were randomly selected, and ~500 cells were counted in each field. Scores between 0–3 were assigned according to staining intensity and the number of positive cells. The cells were scored as follows: No staining, 0; light yellow, 1; pale brown, 2; dark brown, 3. The number of positive cells was scored as follows: <10%, 0; 10–45%, 1; 45–70%, 2; and >70%, 3. The two scores were totaled and the sum was divided into 4 levels: A total score of 1 was considered negative (−), a score of 2 as weakly positive (+), a score of 3–4 as positive (++), and a score of 5–6 as strongly positive (+++). Negative and weakly positive were regarded as low expression, while positive and strongly positive as high expression.

Tissues were lysed in buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 1% protease cocktail inhibitor, followed by centrifugation at 12,000 × g for 10 min at 4°C. The tissue lysis supernatants were collected and the protein concentrations were determined using BCA Protein Quantification kit (cat. no. 23227; Thermo Fisher Scientific, Inc.). A total of 20 µg protein samples were loaded in each well and separated by electrophoresis in a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Subsequent to blocking with 5% milk in TBS containing 0.1% Tween-20 (TBST) at room temperature for 2 h, the membrane was incubated with primary antibodies against Beclin-1 (1:1,000; cat. no. ab210498; Abcam), light chain (LC) 3 II/I (1:500; cat. no. ab128025; Abcam), B-cell lymphoma-extra large (Bcl-xL; 1:800; cat. no. ab2568; Abcam) and β-actin (1:4,000; cat. no. A5441; Sigma-Aldrich; Thermo Fisher Scientific, Inc.). Following washing, membranes were incubated with a 1:3,000 solution of goat anti-rabbit IgG HRP-conjugated secondary antibody (cat. no. 7074; Cell Signaling Technology, Inc., Danvers, MA, USA) for 2 h at room temperature. The antigen-antibody complexes were detected using Western Lightening Plus enhanced chemiluminescence reagent (cat. no. NEL103E001EA; PerkinElmer, Inc., Waltham, MA, USA) with β-actin as the internal control protein. For immunoblotting quantification, the band intensity of the target protein was normalized to the internal control protein band from the same lane (ImageJ software, version 1.6; National Institutes of Health, Bethesda, MD, USA). Data were presented as the mean ± standard deviation of ≥3 replications for each group.

At 48 h post-transfection, the cells were washed twice with cold PBS and dual staining was performed using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Staining/Detection kit (cat. no. 55654; BD Biosciences, Franklin Lanes, NJ, USA). Briefly, cells were resuspended in 1X binding buffer at a concentration of 1×10⁶ cells/ml. The 100 µl solution was transferred to a 5-ml culture tube. A total of 5 µl Annexin V-FITC and 5 µl propidium iodide were added to the tube and incubated for 15 min at 37°C. A total of 400 µl 1X binding buffer was added to each tube. The cells were evaluated by flow cytometer (BD Biosciences) and data were analyzed by FCS Express software (version 3; De Novo Software, Los Angeles, CA, USA).

Following transfection, the cells were collected and resuspended in serum-free DMEM at a concentration of 1×10⁵ cells/ml. The lower chambers of Transwells (8 µm pore size; Corning Incorporated, Corning, NY, USA) were filled with 800 µl DMEM with 10% FBS, and 400 µl cell suspension was added to the upper chamber. Subsequent to incubation at 37°C with 5% CO₂ in air for 48 h, cells on the lower surface were fixed with 75% ethanol for 30 min at room temperature and stained with 0.1% crystal violet for 2 min at room temperature. The migrated cells were counted at magnification, ×60 using a light microscope (BX60; Olympus Corporation, Tokyo, Japan). Cells were counted in 3–5 fields of interest, which were randomly selected on each membrane, and the average number of cells was calculated.

Statistical analyses were performed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA) and GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference. The results were analyzed with χ² tests.

The immunohistochemical results demonstrated that positive Beclin-1 staining was primarily localized in the cytoplasm and occasionally in the nuclei (Fig. 1). Beclin-1 protein expression was positive in the majority of para-carcinoma tissue samples, with a positive expression rate of 76.67% (46/60). Gastric cancer tissues were primarily negative or weakly positive for Beclin-1, with a positive expression rate of 36.67% (22/60). The frequency of positive Beclin-1 expression in gastric carcinoma tissue samples was significantly lower compared with that in para-carcinoma samples (P<0.01; Table I).

Western blot analysis indicated that Beclin-1 protein expression in para-carcinoma tissue samples was markedly higher than in tumor tissue (Fig. 2; 1.024±0.097 vs. 0.572±0.102; P<0.01).

Based on the Beclin-1 protein expression levels detected by immunohistochemical staining, the gastric carcinoma specimens were divided into a low expression group (−, +) and a high expression group (++, +++). The clinical data of 60 cases were reviewed, and it was demonstrated that Beclin-1 protein expression was not associated with age, sex, tumor size, differentiation or lymph node metastasis, whereas it was associated with TNM stage (P=0.008) and invasion status (Table II; P=0.035).

As Beclin-1 expression was low in the gastric carcinoma tissues and correlated with the TNM stage and invasion status of patients, the present study aimed to characterize the role of Beclin-1 in gastric cancer. A previous study indicated that Beclin-1 expression was associated with tumor cell apoptosis and migration (6); therefore, we hypothesized that Beclin-1 acted through a similar mechanism in gastric cancer. Beclin-1-overexpressing gastric cancer cells were produced by the plasmid transfection of MKN-45 gastric cancer cells. As indicated in Fig. 3A, the apoptosis rate was increased in the overexpression group (22.6±3.1%) compared with the control transfection group (8.4±1.4%) at 48 h post-transfection. A significant difference in the apoptosis rate between the groups was observed (Fig. 3B; P<0.01).

A Transwell assay was employed to evaluate the effect of Beclin-1 on MKN-45 cell invasion. The numbers of migrating cells in the Beclin-1 overexpression and control groups were 28.4±5.1 and 75.9±6.9, respectively, which was demonstrated to be a statistically significant difference (Fig. 3C; P<0.01).

To further explore the mechanisms for Beclin-1-regulated apoptosis resistance, alterations to the protein expression levels of the downstream autophagy effector LC 3 II/I and the pro-apoptotic factor Bcl-xL were examined by western blotting. Beclin-1 overexpression was associated with the increased expression of the autophagy effector LC 3 II/I and the reduced expression of the anti-apoptotic factor Bcl-xL (Fig. 3D). The data indicated that Beclin-1 promoted autophagy and apoptosis.