Introduction
According to the 2012 Chinese cancer registration report and statistics of the World Health Organization, the Chinese population presents a high incidence of gastric carcinoma (1). Gastric carcinoma presented the second largest incidence of malignant tumors in China and the third highest rate of mortality; cases of gastric carcinoma and associated mortality account for half the malignant tumor cases worldwide (2). Gastric cancer in China presents high morbidity and mortality rates, and most cases are diagnosed at the advanced stage. The incidence rate of gastric carcinoma in patients under the age of 30 increased from 1.7% in the 1970s to 3.3% at present in China (3).
Nuclear factor (NF)-κB is a transcription factor observed in various cell types that serves a role in physiological and pathological processes through the NF-κB-inducing kinase (NIK)/IκB kinase (IKK)/NF-κB signaling pathway (4,5). Previous studies have demonstrated that NF-κB is associated with proliferation, differentiation, apoptosis, invasion and metastasis of tumor cells (6-8). In addition, aberrant activation of NF-κB was demonstrated in gastric cancer cells and pathological tissues (9).
X-linked inhibitor of apoptosis protein (XIAP) is an effective caspase inhibitor and the most investigated molecular structure of the inhibitor of apoptosis protein (IAP) family. XIAP selectively binds to caspases-3, -7 or -9 to inhibit their activity and prevent cell apoptosis (10). Embelin is a small-molecule inhibitor of XIAP that binds to the baculoviral IAP repeat 3 structural domain of XIAP and prevents binding to caspases-3, -7 or -9, thus inducing cell apoptosis (11). However, the anticancer effect of embelin in human gastric carcinoma cells and the mechanisms underlying it are poorly understood. The present study hypothesizes that the anticancer effect of embelin induces cell apoptosis in human gastric carcinoma cells through the p38 mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways.
Materials and methods
Chemical reagents
Invitrogen RPMI-1640 and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). MTT was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). DAPI staining assays (cat. no. C1005) and caspase-3 activation commercial kits (cat. no. C1116) were obtained from Beyotime Institute of Biotechnology (Haimen, China). A Pierce BCA Protein Assay kit was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China). An Annexin V-FITC/PI Apoptosis Detection kit (cat. no. KGA101) was obtained from KeyGen Biotech Co., Ltd. (Nanjing, China). An NF-κB p65 colorimetric assay kit was obtained from Elabscience Biotechnology Co., Ltd. (Wuhan, China; cat. no. E-EL-H1388c).
Cell culture and cell proliferation assay
The human gastric carcinoma cell line SGC7901 was acquired from the experimental center of the Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) and maintained in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA), in a 5% CO2 atmosphere at 37°C. SGC7901 cells were seeded in 96-well plates and treated with embelin (5, 10 or 15 µM; purity ≥98%; Sigma-Aldrich) for 1, 3 and 5 days as previously described (12). Cell proliferation was determined using the MTT assay. Briefly, MTT solution (20 µl; 5 mg/l; Sangon Biotech Co., Ltd.) was added to each well for a 4-h incubation in a 5% CO2 atmosphere, at 37°C. Following incubation, 150 µl dimethyl sulfoxide was added to each well and shaken for 20 min at room temperature. The optical density was determined using a 96-well multiscanner at 570 nm (ELx808; Bio-Tek Instruments, Inc., Winooski, VT, USA).
Analysis of caspase-3 activity
SGC7901 cells were seeded in 6-well plates and treated with embelin (5, 10 or 15 µM) for 5 days. Following treatment, caspase-3 activity was assessed using caspase-3 activation commercial kits. Briefly, cells were prepared in cell lysis buffer for 30–60 min at 4°C and centrifuged at 12,000 × g for 10 min at 4°C. The protein concentrations in the cell lysates were measured with the Pierce BCA Protein Assay kit according to the manufacturer's instructions. Equal amounts of protein were mixed with the Ac-DEVD-pNA reaction buffer and incubated at 37°C for 2 h in the dark. Following incubation, absorbance was measured at 405 nm with the XL-818 instrument.
Analysis of cell apoptosis
SGC7901 cells were seeded in 6-well plates and treated with embelin (5, 10 or 15 µM) for 5 days. Cells were washed twice with ice-cold phosphate-buffered saline (PBS), collected and resuspended in annexin V binding buffer from the kit according to the manufacturer's instructions. Following resuspension, 5 µl annexin V-FITC and 5 µl propidium iodide were added to the cells, and incubated for 10 min on ice in the dark. Cell apoptosis was immediately detected using a flow cytometer (Epics Altra; Beckman Coulter, Inc., Brea, CA, USA) to identify annexin V- and/or PI-positive cells.
DAPI staining assay
SGC7901 cells were seeded in 6-well plates and cultured with embelin (5, 10 or 15 µM) for 5 days. SGC7901 cells were washed twice with ice-cold PBS and fixed using 0.5 ml paraformaldehyde (4%; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 30 min at 4°C. Cells were then washed twice with PBS and incubated with sodium citrate (0.1%; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) containing 0.1% Triton X-100 (Sinopharm Chemical Reagent Co., Ltd.) for 5 min, at 4°C. Cells were incubated with the DAPI staining assay for 10–15 min at 4°C in the dark. DAPI was excited by ultraviolet light to indicate nuclear apoptosis and images were captured with an Axio Observer A1 fluorescence microscope (Zeiss AG, Oberkochen, Germany) at the excitation wavelength of 340 nm.
Analysis of NF-κB p65 activation
SGC7901 cells were seeded in 96-well plates and cultured with embelin (5, 10 or 15 µM) for 5 days. NF-κB p65 activation was measured using the NF-κB p65 colorimetric assay kit, according to the manufacturer's instructions.
Western blotting
SGC7901 cells were seeded in 6-well plates and cultured with embelin (5, 10 or 15 µM) for 5 days. Following treatment, cells were prepared in cell lysis buffer for 30–60 min at 4°C and centrifuged at 12,000 × g for 10 min at 4°C. The protein concentrations in cell lysates were measured with the Pierce BCA Protein Assay kit according to the manufacturer's protocol. Equal volumes of proteins were resolved in 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated overnight at 4°C with mouse anti-phosphorylated (p)-p38 MAPK monoclonal antibody (1:1,000; cat. no. sc-7973; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-p-IκBα monoclonal antibody (1:1,000; cat. no. ab133462; Abcam, Cambridge, MA, USA), anti-p-IKKα/β polyclonal antibody (1:1,000; cat. no. sc-21661; Santa Cruz Biotechnology, Inc.) and anti-β-actin polyclonal antibody (1:500; cat. no. D110007; Sangon Biotech Co., Ltd., Shanghai, China). Following washing with Tris-buffered saline supplemented with Tween-20, the membranes were incubated with a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:1,000; cat. no. sc-2380; Santa Cruz Biotechnology, Inc.). for 2 h. Proteins were visualized using enhanced chemiluminescence (Santa Cruz Biotechnology, Inc.) and detected using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Statistical analysis
Data are presented as the mean ± standard deviation of at least three independent experiments and analyzed with SPSS software, version 19.0 (IBM SPSS, Armonk, NY, USA). Differences between groups were analyzed by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.
Results
Effect of embelin on tumor growth in gastric carcinoma cells
The chemical structure of embelin is presented in Fig. 1. To investigate the anticancer effect of embelin on tumor growth, SGC7901 cells were treated with embelin (5, 10 or 15 µM) for 1, 3 or 5 days, and the levels of cell proliferation were determined using the MTT assay. As demonstrated in Fig. 2, 10 and 15 µM embelin significantly suppressed cell proliferation following 5-days of culture, compared with the control group (P<0.01). Therefore, further assays were performed on SGC7901 cells treated with the various concentrations of embelin for 5 days.
Embelin induces caspase-3 activity in gastric carcinoma cells
To investigate the therapeutic effect of embelin on cell apoptosis, SGC7901 cells were treated with embelin (5, 10 and 15 µM) for 4 days, and caspase-3 activity was measured. As demonstrated in Fig. 3, 10 or 15 µM embelin significantly increased caspase-3 activity compared with the control group (P<0.01).
Embelin induces cellular apoptosis in gastric carcinoma cells
In order to examine the effect of embelin on cell apoptosis, SGC7901 cells were treated with embelin (5, 10 or 15 µM) for 4 days, and the cellular apoptosis was determined. As demonstrated in Fig. 4, 10 and 15 µM embelin significantly increased the percentage of cellular apoptosis compared with the control group (P<0.01).
Effect of embelin induces nuclear apoptosis in gastric carcinoma cells
In order to investigate the anticancer effect of embelin on nuclear apoptosis, SGC7901 cells were treated with 5, 10 or 15 µM embelin for 4 days, and nuclear apoptosis was evaluated with the DAPI staining assay. As demonstrated in Fig. 5, nuclear apoptosis was observed following embelin treatment at all concentrations.
Embelin induces p-p38 MAPK protein expression levels in gastric carcinoma cells
SGC7901 cells were treated with embelin (5, 10 or 15 µM) for 4 days, and the p-p38 MAPK protein expression levels were measured using western blotting. The results demonstrated that administration of embelin treatment (10 or 15 µM) significantly upregulated the relative p-p38 MAPK protein expression levels compared with those of the control group (P<0.01; Fig. 6).
Embelin inhibits NF-κB p65 activity in gastric carcinoma cells
SGC7901 cells were treated with embelin (5, 10 or 15 µM) for 4 days, and the NF-κB p65 activity was measured. As demonstrated in Fig. 7, embelin treatment (10 or 15 µM) significantly reduced NF-κB p65 activity compared with the control group (P<0.01).
Embelin inhibits the phosphorylation of IκBα in gastric carcinoma cells
SGC7901 cells were treated with embelin (5, 10 or 15 µM) for 4 days, and the p-IκBα expression levels relative to β-actin were determined with western blotting. As demonstrated in Fig. 8, embelin treatment (10 or 15 µM) significantly suppressed p-IκBα protein expression compared with the control group (P<0.01).
Embelin inhibits the phosphorylation of the IKKα/β in gastric carcinoma cells
To investigate the anticancer therapeutic effect of embelin treatment on IKKα/β protein expression, SGC7901 cells were treated with embelin (5, 10 or 15 µM) for 4 days, and the p-IKKα/β protein expression levels relative to β-actin were determined with western blotting. As demonstrated in Fig. 9, embelin treatment (10 or 15 µM) significantly reduced the relative p-IKKα/β protein expression levels compared with the control group (P<0.01).
Discussion
Malignant tumors are the second leading cause of mortality in China, and stomach cancer presents the highest rate of mortality associated with these tumors (13). China has the highest rate of gastric carcinoma worldwide (14) and the rate has risen over the past 20 years (15). In the present study, embelin treatment significantly suppressed cell proliferation, induced caspase-3 activation, and increased cell and nuclear apoptosis in the SGC7901 cells. Marsh et al (16) suggested that embelin suppresses the growth of pancreatic cancer in A549 non-small cell lung cancer (17) and brain glioma cells (18). Embelin is considered to be a potential drug for the treatment of carcinoma.
MAPKs are Ser/Thr protein kinases that mediate the cellular response and apoptosis, react to various cell growth and mitosis promotion factors, and transduct outer signals into cells (19). The signal transmission of the MAPK pathway is completed by the continuous phosphorylation of MAPKKK, MAPKK and MAPK (20). There are five activators of the MAPK pathways; ERK, p38, JNK, ERK3/ERK4 and ERK5 (21). Extracellular stimuli result in different biological responses depending on the MAPK pathway activated (22). In the present study, administration of embelin significantly upregulated the relative p38 MAPK protein expression in human gastric carcinoma cells. Wang et al (23) indicated that embelin reduced cell viability in gastric cancer cells via p38 MAPK pathway activation. Avisetti et al (24) indicated that embelin induced apoptosis in lung cancer cells through the activation of the p38/JNK pathway. Activation of the p38/JNK pathway may be a potential target for embelin treatment in human gastric carcinoma cells.
The occurrence and development of a tumor is a complex and multistep process that involves a series of genetic changes, including metastasis of cells deriving from the primary tumor into the blood and lymphatic vessels, through adhesion to endothelial cells, resulting in the metastasis of the tumor (25). A previous study demonstrated that the upregulation of NF-κB leads to the occurrence of tumors, and NF-κB mediates one of the main mechanisms by which tumor cells resist apoptosis during tumor development (26). Upon activation, it produces anti-apoptotic signals to aid the development of the tumor. Royuela et al (27) demonstrated that NF-κB induces anti-apoptosis genes, including IAG, cl-2 like factor, TRAF1, TRAF2 and A20D. Similarly, NF-κB promotes tumor formation via a non-apoptotic pathway, activating the proto-oncogenes c-myc and cyclin D1 (28). Yang et al (28) demonstrated that cyclin D1 was the target gene of NF-κB, and that NF-κB initiated the transcription of cyclin D1, promoting the transit from phase G1/G0 to phase S, leading to the cell proliferation, malignant transformation and cancerization (29). In the present study, embelin significantly reduced the NF-κB activity in human gastric carcinoma cells. Ahn et al (30) indicated that embelin is a potential suppressor of tumorigenesis, as it suppresses NF-κB-regulated anti-apoptosis. Reuter et al (12) demonstrated that embelin suppresses osteoclastogenesis through the inhibition of the NF-κB cell signaling pathway. Therefore, the suppression of NF-κB may be a marker for embelin treatment in human gastric carcinoma cells.
Pathological classification of the tumor may indicate its invasiveness, thus, NF-κB p65 activity may have an effect on tumor metastasis (31). NF-κB serves a role in the activation of the immune system, cell apoptosis and inflammatory cell chemotaxis associated with gene transcription. As a gene encoding inflammatory molecules, NF-κB regulates and controls the expression of numerous inflammation-mediating genes, and NF-κB p65 activation was previously associated with cell infiltration (32,33). In the present study, a significant reduction in the relative p-IκBα and p-IKKα/β protein expression levels was observed following embelin treatment (10 or 15 µM). Park et al (34) indicated that the administration of embelin induced apoptosis in human glioma cells through the degradation of p-IκBα and p-IKKα/β. This may have been due to the decrease in p-IκBα and p-IKKα/β protein expression induced by embelin treatment.
In conclusion, the results of the current study suggest that embelin suppresses cell proliferation and induces levels of apoptosis in human gastric carcinoma cells through the inhibition of the NF-κB signaling pathway. These results indicate that the suppression of the NF-κB signaling pathway, due to embelin administration, is a potential therapeutic target for the treatment of gastric carcinoma.