Autophagy is a common phenomenon in cancer metabolism. However the mechanism and guiding significance of autophagy in the development of gastric cancer has remained to be elucidated. In the present study, 75 gastric cancer tissue specimens were collected at The China Japan Union Hospital of Jilin University (Changchun, China). Of these samples, 16 cases were stage 1, 40 stage 2 and 19 stage 3. Polymerase chain reaction and western blotting were used to detect the messenger RNA and protein expression of Beclin-1, a significant protein associated with cellular autophagy. It was found that expression of Beclin-1 in cancer tissues from stages 1 and 2 was higher, while in stage 3 cases levels were significantly lower than that of adjacent normal tissues. In addition, the infiltration of inflammatory cytokines was also increased in stage 1 and 2 cases.
The morbidity of gastric cancer is particularly high in China, where 23.9% of the total death toll resulting from tumors was attributed to patients with gastric cancer in 2012 (
Autophagy is a common occurrence in cell metabolism, and is involved in facilitating the cellular clearance of aggregation-prone proteins, thus exerting a cytoprotective function (
In the present study, the expression of autophagy-associated protein Beclin-1 and inflammatory cytokine interferon-γ (IFN-γ) were evaluated in clinical gastric cancer tissue samples and cell lines. The association between autophagy and inflammation was also examined. The present study aimed to elucidate the characteristics of autophagy in gastric cancer and the effects of inflammation on this process.
Between January 2010 and December 2014, clinical gastric cancer and adjacent noncancerous gastric mucosa tissue specimens were collected from 75 patients who had undergone surgeries for radical resection of gastric cancer at The China Japan Union Hospital of Jilin University (Changchun, China). The study protocol was approved by the institutional review board for human studies of The China Japan Union Hospital of Jilin University, and informed consent was obtained from all patients. Following collection, the tissues were divided into two parts, a large part (50–100 mg) and a small one (10 mg). The 50–100 mg samples were immediately frozen and stored in liquid nitrogen for western blot analysis and quantitative polymerase chain reaction (qPCR), while the 10 mg samples were washed with phosphate-buffered saline (PBS; Wuhan Boster Biological Technology, Ltd., Wuhan, China) and stored in 4% paraformaldehyde (Wuhan Boster Biological Technology, Ltd.) for further histological analysis. Details of the patients are listed in
Human gastric cancer cell lines BGC-823 and MKN-28 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), and human normal gastric mucosa epithelial cell line GES-1 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin and 100 g/ml streptomycin (Sangon Biotech, Shanghai, China). Cells were tested for authenticity by the Cell Bank of the Chinese Academy of Sciences prior to purchase. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2. Cells were plated in 6-well plates and experimental group cells were stimulated with 50 ng/ml IFN-γ (BioLegend, Inc., San Diego, CA, USA) for 12, 24 and 48 h (
Female BALB/c nude mice (18–20 g, 6 weeks of age) were purchased from Huafukang Co. Ltd. (Beijing, China). In total, 50 mice were used for the experiment, of which 10 were used for the tumor bearing assay and 40 were used for the xenograft assay, with 20 mice in the experimental group and 20 mice in the control group. The mice were bred at the animal center of Jilin University and were maintained according to the Jilin University animal management program. Mice were housed under specific pathogen-free conditions at a temperature of 23±1°C and relative humidity of 30–70%. Mice were maintained in a 12-h light-dark cycle, with free access to water and
A total of 53 clinical gastric cancer and adjacent noncancerous gastric mucosa tissue specimenscarcinomas tissues previously fixed in paraformaldehyde were used for immunohistochemistry study here. These samples were triformol-fixed, dehydrated in a graded alcohol series, embedded in paraffin and cut into sections. The aforementioned chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The sections were subsequently deparaffinized, blocked with 3% hydrogen peroxide for 10 min, washed with deionized water three times and sealed with serum (FBS; HyClone; GE Healthcare Life Sciences) for 30 min at room temperature. The sections were washed with deionized water and incubated for 30 min at 37°C with primary polyclonal rabbit anti-human Beclin-1 antibodies (1:500 in 1% BSA in PBS containing 0.1% Tween; cat no. ab62472; Abcam, Cambridge, MA, USA). Next, the sections were washed three times with PBS and incubated with a biotinylated goat anti-rabbit IgG secondary antibody (1:300 in 1% BSA in PBS containing 0.1% Tween; cat. no. ab64257; Abcam) for 20 min at room temperature. The sections were washed three times, followed by detection with the HRP Substrate Kit and ABC kit (SK-4100 and PK-6100, protocols provided in the kit. Vector Blue; Vector Laboratories Inc., Burlingame, CA, USA). and nuclei were counterstained with hematoxylin using the ULTRA Staining system (Ventana Medical Systems, Tucson, AZ, USA). Negative controls were obtained by substituting PBS for the primary antibody. The results were evaluated based on the proportion of stained cells and the staining intensity.
Tissues were interpreted as positive when a minimum of weak-to-moderate cytoplasmic staining was identified in >30% of the neoplastic cells. The rates of Beclin-1-positive cells were graded as ++ when ≥61% of the cells were positive, + when 30–60% were positive, and - when 0–29% were positive. Results were independently reviewed by two pathologists.
RNA in the clinical tissue samples was isolated using mechanical homogenization (LabGEN 700 Homogenizer; Cole-Parmer, Vernon Hills, IL, USA) and TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Complementary DNA was synthesized from the total RNA using SuperScriptIII RNase H-Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). The expression levels of the genes were quantified using TaqMan® Gene Expression assays (Applied Biosystems Life Technologies, Foster City, CA, USA). The results were expressed as relative expression, standardized against the expression of the gene encoding β-actin. β-actin was used as the cDNA loading control, and all qPCR read-outs were adjusted according to the β-actin results. The primers used were as follows: Beclin-1 forward, 5′-CAAGATCCTGGACCGTGTCA-3′ and reverse, 5′-TGGCACTTTCTGTGGACATCA-3′; β-actin forward, 5′-TGGCACCCAGCACAATGAA-3′ and reverse, 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′ (
Cytoplasmic proteins were extracted from gastric cancer cell lines and clinical samples using NE-PER Nuclear and Cytoplasmic Extractions reagents (Beyotime Institute of Biotechnology, Haimen, China). The lysates were resolved by 4–20% SDS-PAGE gradient gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Blots were blocked and incubated with rabbit anti-human polyclonal primary antibodies against Beclin-1 (cat no. ab62472), LC3 (cat no. ab128025) and GADPH (cat no. ab37168) at dilutions of 1:1,000 at 4°C overnight (Abcam). Blots were then incubated with a secondary goat anti-rabbit antibody (cat. no. sc-2004; 1:5,000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h. Blots were visualized using enhanced chemiluminescence detection reagents and then exposed to X-ray film (Thermo Fisher Scientific, Inc.) (
FACS requires a small number of high quality tissue samples and is expensive to run, therefore, only 5 of the 75 fresh clinical samples were selected for analysis. The samples were divided into sections and ground on a screen cloth. PBS was used to wash the ground tissues, filtering immune cells through the cloth by the wash buffer (PBS) and separating them from the cell debris. These cells were collected by centrifugation of the washing liquid and stained with phycoerythrin-CD3, allophycocyanin-CD4 and fluorescein isothiocyanate-IFN-γ monoclonal antibodies (BioLegend, Inc.). Data were analyzed using the BD FACS CantoII (BD Biosciences, Franklin Lakes, NJ, USA) (
Data are expressed as the mean ± standard error of the mean. Statistical significance was determined by the Student's t-test, Mann-Whiney U test or Fisher's exact test. P<0.05 was considered to indicate a statistically significant difference.
Immunohistochemistry and western blotting were used to analyze the expression levels of Beclin-1 in gastric cancer tissues and cell lines. As shown in
The expression of Beclin-1 in BGC-823, MKN-28 and normal gastric mucosa epithelial cell line GES-1 were analyzed by western blotting. BGC-823 is a poorly-differentiated gastric adenocarcinoma cell line, while MKN-28 is a well-differentiated adenocarcinoma cell line. It was observed that the expression of Beclin-1 in the poorly-differentiated BGC-823 cell line was significantly lower compared with that of the other cells (P<0.05). This result was in accordance with the results of the analysis of clinical tissue samples. However, significant differences in Beclin-1 expression were not observed between the well-differentiated MKN-28 and the normal GES-1 cell lines.
Immunopositivity for Beclin-1 was observed in 53/75 gastric adenocarcinoma patient samples analyzed in the present study. Expression of Beclin-1 was observed in 20.00% of the poorly-differentiated samples, 81.10% of the moderately-differentiated samples and 86.96% of the well-differentiated samples. There were significant differences between the poorly-differentiated group and the well- and moderately-differentiated groups (P<0.01; two-tailed Fisher's exact test). There were no significant differences in Beclin-1 expression with respect to the existence of lymph node metastasis. However, the expression of Beclin-1 was associated with the depth of invasion. Samples of early gastric cancer expressed higher levels of Beclin-1 than those of advanced gastric cancer. Details are presented in
FACS analysis was used to evaluate the percentage of immune cells secreting IFN-γ (
IFN-γ was subsequently used to induce autophagy in gastric cancer cells. Beclin-1 was originally highly expressed in MKN-28 cells, while rarely expressed in BGC-823 cells. The poorly-differentiated BGC-823 cell line was therefore selected for this
A rodent xenograft model was designed to detect whether autophagy could inhibit xenograft growth
Autophagy is the process of collecting and degrading intracellular proteins and organelles in lysosomes to preserve protein and organelle quality, as well as recycling certain materials to sustain metabolism and survival during starvation (
Beclin-1 is a mammalian ortholog of yeast Atg6, which has a critical role in the vesicle nucleation phase of autophagy (
Previous studies have revealed that autophagy is frequently activated in cancer cells following drug treatment (
In the present study, IFN-γ was used to directly stimulate BGC-823 cells, which originally expressed low levels of Beclin-1. It was demonstrated that following IFN-γ stimulation, the expression levels of LC3 and Beclin-1 were increased. As previous studies have confirmed that increasing expression of LC3 and Beclin-1 lead to increased numbers of autophagosomes (
The animal experiments utilized in the present study were designed to confirm the results of the
In conclusion, the results of the current study revealed that Beclin-1, an autophagy-associated protein, exhibited a complex expression pattern in gastric cancer tissues at various clinical stages. In contrast to other tumors, including breast or prostate cancer, the expression of Beclin-1 in well-differentiated gastric cancer tissues and cells was higher compared with that in adjacent tissues or normal cell lines. However, in poorly-differentiated tissues and cells the opposite effect was observed. The results also revealed that there was a decrease in the infiltration and secretion of IFN-γ associated with the progression of gastric cancer.
Expression of Beclin-1 in gastric cancer tissues and cell lines. (A) Western blot analysis of clinical samples of various stage gastric cancer and cell lines. (B) Expression levels of Beclin-1 in stage I and III cancer and adjacent tissues. (C) Expression of Beclin-1 mRNA in gastric cancer tissues and cells, evaluated by polymerase chain reaction. Values are expressed as the mean ± standard error of the mean, *P<0.05. (D) Visualization of Beclin-1 expression in gastric cancer tissues of various stages by immunohistochemistry (magnification, ×100). Bec-1, Beclin-1; mRNA, messenger RNA; GES, GES-1 cells; MKN, MKN-28 cells; BGC, BCG-823 cells.
Expression of IFN-γ in clinical gastric cancer tissue samples. (A) Fluorescence-activated cell sorting indicated that IFN-γ was secreted by CD3(+)CD4(+) cells, which indicated that these were Th1 cells. (B) Expression of IFN-γ mRNA and the percentage of IFN-γ-positive cells was significantly higher in stage I samples than those of other stages. Values are expressed as the mean ± standard error of the mean; *P<0.05, **P<0.01. (C) Expression of IFN-γ protein was significantly higher in tissues of stage I than that in other stages. mRNA, messenger RNA; IFN-γ, interferon-γ.
Details of patients included in the present study (n=75).
Parameter | Patients, n |
---|---|
Gender | |
Male | 47 |
Female | 28 |
Median age, years (range) | 54 (38–65) |
Median BMI, kg/m2 (range) | 24.3 (17.6–28.4) |
TNM stage | |
I | 16 |
II | 40 |
III | 19 |
BMI, body mass index; TNM, tumor/node/metastasis (
Beclin-1 expression in various types of gastric carcinoma.
Beclin-1 expression, n (%) | ||||
---|---|---|---|---|
Gastric carcinoma characteristic | − | + | ++ | Total positive |
Histological grade | ||||
Well | 3 | 9 | 11 | 20 (86.96) |
Moderate | 7 | 12 | 18 | 30 (81.10) |
Poor | 12 | 2 | 1 | 3 (20.00) |
Lymph node metastasis | ||||
No | 6 | 6 | 8 | 14 (65.00) |
Yes | 15 | 17 | 22 | 39 (70.90) |
Depth of invasion | ||||
EGC | 3 | 4 | 15 | 19 (83.63) |
AGC | 19 | 19 | 15 | 34 (64.15) |
EGC, early gastric cancer; AGC, advanced gastric cancer; -, 0–20% cells Beclin-1 positive; +, 30–60% cells Beclin-1 positive; ++, ≥61% cells Beclin-1 positive.
P<0.01 vs. well- and moderately-differentiated cancer
P<0.05 vs. AGC.