Antimetastatic Effects of Celastrus orbiculatus on Human Gastric Adenocarcinoma by Inhibiting Epithelial–Mesenchymal Transition and NF-κB/Snail Signaling Pathway

Abstract

Aim of the Study. Celastrus orbiculatus has been used as a folk medicine in China for the treatment of many diseases. In the laboratory, the ethyl acetate extract of Celastrus orbiculatus (COE) displays a wide range of anticancer functions. However, the inhibition of the metastasis mechanism of COE in gastric cancer cells has not been investigated so far. The present study was undertaken to determine if the antimetastatic effects of COE were involved in inhibition of the epithelial–mesenchymal transition (EMT) of human gastric adenocarcinoma SGC-7901 cells. Methods. The adhesion, invasion, and migration of SGC-7901 cells were determined by COE treatment in vitro, using Matrigel-coated plate, transwell membrane chamber, and wound healing models, respectively. In vivo, the growth-inhibiting and antimetastatic effects of COE on the nude mice model of gastric cancer were tested and the mechanisms were explored. The expression of EMT markers and nuclear factor κB (NF-κB)/Snail signaling pathway were evaluated by using western blotting and immunohistochemistry. Results. Treatment with COE dose-dependently inhibited the proliferation, adhesion, invasion, and migration of SGC-7901 cells in vitro, which was realized by enhancing the expression of E-cadherin and reducing N-cadherin and vimentin expression. Moreover, COE suppressed the activation of NF-κB/Snail signaling pathway induced by tumor necrosis factor-α. In addition, COE effectively suppressed tumor growth and metastasis in the nude mice model due to reduced expression of N-cadherin, vimentin, NF-κB p65, and Snail and increased expression of E-cadherin in the tumor tissues. Conclusion. Our findings provided new evidence that COE is an effective inhibitor of metastatic potential of SGC-7901 cells through suppression of EMT and NF-κB/Snail signal pathway. Based on these findings, COE may be considered a novel anticancer agent for the treatment of metastasis in gastric cancer.

Introduction

Gastric cancer is the most common gastrointestinal cancer originating from the epithelium and is a serious threat to human health. ¹ Most patients are diagnosed at an advanced stage, with metastasis and poor prognosis. ² Metastasis of gastric cancer is not only a sign of deterioration but also the major cause of treatment failure and death. ³ Therefore, effective therapeutic agents inhibiting cancer metastasis are significant for gastric cancer treatment.

Epithelial–mesenchymal transition (EMT) is a process in which epithelial cells lose their cell–cell contacts and undergo remodeling of the cytoskeleton to form a migratory phenotype. ⁴ The expression of epithelial marker proteins, such as E-cadherin and Keratin, are downregulated, while the expression of mesenchymal markers, such as vimentin and N-cadherin, are upregulated with EMT. ⁵ Therefore, it is widely accepted that EMT plays an important role in cancer metastasis of several human malignancies including gastric cancer. ⁶ The inhibition of EMT may prevent or restrain the metastasis of gastric cancer, so EMT could be a very promising therapeutic target.

Snail, an important transcription factor, has been reported to be involved in the regulation of EMT by repressing the expression of the E-cadherin gene. ⁷ Recent study has demonstrated that the nuclear factor κB (NF-κB)-related cascade modulates Snail expression that leads to EMT in various cell types.^8,9 On the basis of these facts, it was hypothesized that NF-κB/Snail signaling pathway was critically involved in the acquisition of EMT in gastric cancer cells.

In recent years, bioactive components extracted from plants used in traditional Asian medicine have been found to be effective to prevent potential cancers and to be therapeutic agents.^{10 -12} Celastrus orbiculatus (Celastraceae) has been used as a folk medicine in China for the treatment of many diseases, including arthritis and other inflammatory diseases. It was found that the ethyl acetate extract of Celastrus orbiculatus (COE) displays anticancer effects in vitro and in vivo through the inhibition of proliferation, angiogenesis, invasion, and metastasis.^{13 -15} Although COE has been discussed in various bioactivity research studies, the molecular mechanism by which COE inhibits the metastasis of gastric cancer cell in human has not been fully elucidated so far.

In this study, we investigated the effects of COE on cellular metastasis of gastric cancer in vitro and in vivo through various experiments and explored the underlying molecular mechanism. The results indicated that COE inhibited tumor cell metastasis by inhibiting EMT development and NF-κB/Snail signal pathway in SGC-7901 cells. Therefore, the present findings suggest that COE might have potential therapeutic effect against gastric cancer.

Materials and Methods

Materials and Chemicals

RPMI-1640 and fetal bovine serum (FBS) were acquired from Gibco-BRL (Gaithersburg, MD). 3-(4,5-Dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Sigma Chemical Co (St Louis, MO). Recombinant TNF-α was obtained from R&D systems (Minneapolis, MN). Matrigel was purchased from BD Biosciences. Antibodies against E-cadherin, N-cadherin, vimentin, Snail, NF-κB (p65), IκBα, phosphorylation of IκBα, Histone H3, and β-actin were purchased from Cell Signaling Technology (Beverly, MA). SABC immunohistochemical kit was from Wuhan Boster Bioengineering Inc (Wuhan, China). Other chemicals of analytical grade used were from commercial sources.

Preparation of COE

The plant materials were purchased from Guangzhou Zhixin Pharmaceutical Co Ltd (Guangzhou, China) in 2007. The COE was prepared at the Department of Chinese Materia Medica Analysis, China Pharmaceutical University (Nanjing, China). The preparation procedure has been described previously. ¹⁶ Briefly, dried stems of Celastrus orbiculatus were minced, extracted with 95% ethanol, filtered, and evaporated to dryness. The extract was evacuated with a membrane pump to remove residual solvent. The aqueous layer was then partitioned from the ethanol extract with ethyl acetate. Finally, the ethyl acetate extract was condensed and lyophilized into powder and stored at 4°C. The chemical constituents from the stems of Celastrus orbiculatus were investigated and compounds were isolated as previously described.^17,18 23-Hydroxybetulonic acid, 23-hydroxy-3-oxoolean-12-en-28-oic acid, oleanolic acid, 3-oxo-24-norolean-12-en-28-oic acid, and wiforlide B were confirmed to be included in the extract by high performance liquid chromatography (HPLC) assay (Supplementary Figure S1, available online at http://ict.sagepub.com/content/by/supplemental-data). The resultant COE micropowder was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co, St Louis, MO) and diluted to different concentrations before use.

Cell Culture

The human gastric carcinoma cell line SGC-7901 was obtained from the Cell Bank of Chinese Academy of Sciences Shanghai Institute of Cell Biology (Shanghai, China). SGC-7901 cells were cultured in RPMI1640 containing 10% FBS and maintained at 37°C in a humidified incubator in an atmosphere of 5% CO₂.

Cell Viability Assay

To evaluate the effect of COE on cell viability, the SGC-7901 cells were seeded in 96-well plates and treated with COE at various concentrations (20, 40, 80, 160 µg/mL) in triplicate. After 24 and 48 hours of incubation, cell viability was determined by being incubated with medium containing MTT for 4 hours, followed by dissolving the formazan crystals with DMSO. The absorbance at 490 nm was determined by a microplate reader and presented as relative cell viability. The tests were performed at least 3 independent times.

Cell Adhesion Assay

Each well of a 96-well plate dry-coated with Matrigel was blocked with 1% bovine serum albumin for nonspecific binding for 1 hour at room temperature. SGC-7901 cells, pretreated with COE (5, 10, and 20 µg/mL) for 24 hours, were allowed to adhere to the Matrigel-coated well for 1 hour at 37°C. Nonadherent cells were removed by washing with cold phosphate-buffered saline (PBS). The percentage of adherent cells was determined using absorbance by the MTT assay colorimetric method as previously described.

Cell Invasion Assay

According to manufacturer’s instruction, cell invasion assay was performed by using a transwell membrane (Corning Costar Corporation, Cambridge, MA). Matrigel was applied to the upper chamber. After treated with various concentrations of COE (5, 10, and 20 µg/mL) for 24 hours, cells were added to the upper chamber in serum-free medium. Medium containing 10% FBS was applied to the lower chamber as a chemoattractant. The chamber was incubated for 8 hours at 37°C. After the incubation, the cells on the upper surface of the membrane were removed by cotton swabs, and cells that had invaded across the Matrigel to the lower surface of the membrane were fixed with methanol and stained with crystal violet. Images were obtained under a microscope at 400× magnification (Nikon, Chiyoda-Ku, Tokyo, Japan), and invading cells were quantified by manual counting. Each experiment was repeated 3 times.

Wound Healing Assay

Cells were plated in 6-well plates and grew to confluence. After serum starvation overnight, a wound was created by scratching cells with a sterile 10 µL pipette tip. The cells were washed twice with PBS to remove the floating cells, and then a serum-free medium was added with or without various concentrations of COE (5, 10, 20 µg/mL) for 24 hours. Wound closure was measured in 10 random fields at 100× magnification using Image-Pro Express software and a Nikon inverted microscope. All experiments were performed 3 times. The distance between groups was considered that the difference was statistically significant when P < .05.

Western Blot Analysis

Total cell lysates of equal protein contents from the control and COE treated SGC-7901 cells were resolved by SDS–PAGE and transferred into nitrocellulose membranes (Millipore, Bedford, MA). The blot was blocked in blocking buffer (5% notfat dry milk and 1% Tween-20 in PBS) for 2 hours at room temperature, and then incubated with appropriate primary antibodies in blocking buffer overnight at 4°C. Subsequently, the membranes were washed and incubated with secondary antibodies for 2 hours at room temperature. Enhanced chemiluminescence was used to detect a signal using the Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL) on a Molecular Imager Chemi Doc XRS System (Bio-Rad). The experiments was replicated 3 times. The bands from western blotting were quantified by Quantity One analysis software (Bio-Rad).

Experimental Animals

Male athymic nude BALB/c mice (6 weeks old and weighing 18-22 g) were obtained from the Comparative Medicine Laboratory Animal Center (License No. scxk (SU) 2012-0004) of Yangzhou University (Jiangsu, China). The animals were fed standard rat chow and tap water ad libitum, and maintained with a 12 hours dark/light cycle at 21°C. The experiments were carried out in accordance with internationally accepted guidelines on the use of laboratory animals, and the protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Yangzhou University. The mice were randomly divided into blank control group, negative control group (1% DMSO), positive control group (capecitabine), and low-dose, medium-dose, and high-dose COE groups (10, 20, and 40 mg/kg, respectively). Each group contained 7 mice.

In Vivo Tumor Xenograft Growth Assay

For in vivo xenograft specimens, SGC-7901 cells (2 × 10⁶) mixed with PBS (200 µL/mouse) were injected into the right flank of each mouse. After the implantation of tumor cells lasted for 10 days and tumors reached 50 mm³, COE was given by intragastric administration at doses of 10, 20, 40 mg/kg/day for 21 days and capecitabine (267 mg/kg) was given for 1 to 14 consecutive days with 1 week rest. The tumor dimensions were measured every 3 days by a digital caliper and tumor volume was calculated using the following formula: V = length × width ² × 0.5. The body weight of each mouse was measured every 3 days after treatment. After 21 days of treatment, the mice were killed by cervical dislocation, and their tumors were excised and weighed. Tumors were collected, fixed with 4% formaldehyde, embedded in paraffin, and sectioned for hematoxylin and eosin (H&E) staining according to standard histological procedures.

Experimental Tumor Metastasis Model

To evaluate the tumor metastasis, cultured human gastric cancer SGC-7901 cells (2 × 10⁶) were inoculated into the peritoneal cavity of nude mice. Peritoneal tumors in nude mice were established for 14 days after the injection of gastric cancer cells. Then, mice were intragastrically given COE (10, 20, and 40 mg/kg/day, respectively) for 21 days; capecitabine (267 mg/kg) was given for 1 to 14 consecutive days with 1 week rest. At the end of treatment, all the mice were sacrificed by cervical dislocation and laparotomy was performed. Peritoneal dissemination, liver metastasis, lung metastasis, and ascites formation were examined. The number of mesentery nodules larger than 1 mm in diameter was also determined. The peritoneal nodules, liver, and lungs were paraffin-embedded, sectioned, and subjected to H&E staining for histological analysis.

Immunohistochemistry

Sections (4 µm) of the tumor tissues were subjected to immunohistochemical staining as follows. The slides were baked in an oven at 60°C for 1 hour, washed in xylene, and hydrated in different concentrations of alcohol. Subsequently, endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 minutes. To unmask the antigen, the sections were submerged in citrate buffer at 95°C for 5 minutes. The sections were blocked for 20 minutes with normal goat serum blocking solution. Primary antibodies were incubated at 4°C overnight. Afterwards, the tissues were washed in PBS and incubated with the appropriate secondary antibody for 20 minutes. The tissues were washed in PBS again and incubated with SABC working solution. The tissues were washed 3 times in PBS and visualized with 3,3-diaminobenzidine (DAB). Finally, the slides were counterstained with hematoxylin, dehydrated, and mounted for imaging.

Statistical Analysis

All values were expressed as the mean ± SD for at least 3 separate experiments, unless otherwise stated. Results from different groups were analyzed by one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test or Student’s t test. Statistical analysis of the incidences of peritoneal dissemination and organ metastasis were analyzed by χ² test or Fisher’s exact probability test. All statistical analysis was performed using SPSS for Windows, version 10. P values below .05 were considered significant.

Results

COE Inhibits Proliferation of SGC-7901 Cells

The effect of COE on the viability of SGC-7901 cells was assessed with the MTT assay. As shown in Figure 1A, SGC-7901 cells were treated with COE at various concentration levels (10, 20, 40, 80, and 160 µg/mL) for 24 and 48 hours. It was observed that COE inhibited proliferation of SGC-7901 cells in time- and dose-dependent patterns. No obvious inhibitory effects on proliferation were obtained in 10, 20 µg/mL groups in 24 hours. Therefore, low toxicity concentrations (5, 10, 20 µg/mL) of COE was selected in the following studies in order to confirm that the effect of COE on invasion and metastasis of tumor cell were not due to direct cytotoxicity.

Figure 1.

COE inhibits metastasis and EMT in SGC-7901 cells.

COE Suppresses Metastasis via Inhibition of EMT in SGC-7901 Cells

Adhesion of tumor cells to basement membranes, cell invasion, and migration activity were considered to be the vital steps for metastatic tumor cells. ¹⁹ To evaluate the antimetastatic potential of COE in SGC-7901 cells, we performed the transwell assay, wound healing assay, and adhesion assay after treatment with 5, 10, and 20 µg/mL of COE. It was found that COE obviously decreased the invasion and migration of SGC-7901 cells in a dose-dependent manner (Figure 1B and C). However, COE only caused a slight reduction in cell adhesion even at the concentration of 20 µg/mL (Figure 1D). The slight reduction in adhesion may be associated with the significant decreases in invasion and migration by COE treatment. In addition, the changes in epithelial and mesenchymal markers in COE treatments were examined. As shown in Figure 1E, western blot analysis showed that COE increased the E-cadherin protein level, but decreased N-cadherin and vimentin protein levels in SGC-7901 cells. These results demonstrated that COE was able to suppress gastric cancer cell metastasis in vitro by inhibiting the EMT program.

COE Inhibits the Activation of NF-κB/Snail Signal Pathway in SGC-7901 Cells

To explore whether the above effect of COE is associated with the inhibition of NF-κB-Snail activation, the expression level of the NF-κB p65 subunit, the active form of NF-κB and Snail protein were measured in gastric cancer cells by western blot analysis. Our results demonstrated that the protein expression of NF-κB and Snail decreased in a time-dependent manner after COE treatment (Figure 2A). It was also reported that phosphorylation and degradation of IκB played a critical role in the process of NF-κB activation. ²⁰ On the basis of these facts, we assessed the effect of COE on the status of IκB. As shown in Figure 2A, COE decreased the phosphorylation of IκB and increased the amount of IκB in SGC-7901 cells, which suggested that COE affected NF-κB by decreasing IκB phosphorylation.

Figure 2.

COE inhibits NF-κB/Snail signal pathway in SGC-7901 cells.

Inflammatory factors such as TNF-α are thought to be major causes of elevated NF-κB in cancer cells. ²¹ Therefore, we investigated whether COE also inhibited TNF-α-mediated NF-κB translocation. We found that treatment of SGC-7901 cells with TNF-α (10 ng/mL) increased the translocation of NF-κB, but this effect was abrogated by COE (Figure 2B). These results supported previous hypothesis that COE might inhibit EMT via the regulation of the NF-κB/Snail pathway.

COE Inhibits Growth of SGC-7901 Cells in In Vivo Xenograft Model

To investigate the effects of COE on tumor growth in vivo, we used different concentrations of COE in a xenograft model. After being inoculated with SGC-7901 cells, the mice were given oral doses of COE (10, 20, 40 mg/kg/day) for 21 days. As shown in Figure 3A, compared with the control group, the COE-treated group showed a significant inhibition of tumor growth. The difference between the body weights among the different groups of mice was not significant, which showed that COE at the curative dose was not toxic (data not shown). At the end of the experiment, all the mice were dead and tumors were removed and weighed. It was found that COE significantly decreased the tumor weight compared with the control group (Figure 3B). Furthermore, light microscopy revealed that tumor tissue in mice receiving COE (40 mg/kg) displayed more severe necrosis than that in the control group, which was similar to the effect of capecitabine (Figure 3C). In conclusion, results from the current study demonstrated that COE had potent antitumor effects in gastric cancer xenograft model.

Figure 3.

COE inhibits tumor growth in SGC-7901 xenograft model.

COE Suppresses Metastasis of SGC-7901 Cells in Experimental Metastasis Model

To evaluate the impact of COE on the peritoneal dissemination and organ metastasis of gastric cancer, an experimental metastasis model was utilized in vivo. Nude mice received intraperitoneal injections of SGC-7901 cells, and 2 weeks later COE at different dosages (10, 20, 40 mg/kg/day) was gavaged for 21 days. Then, we examined whether COE resulted in inhibition of peritoneal dissemination by counting the macroscopic nodules of peritoneal dissemination at the end of the experiment. The results showed that COE had the capability of reducing the peritoneal dissemination, since the number of macroscopic nodules in COE groups was much lower than that in the control group (Figure 4A). The H&E stain of peritoneal nodules showed that the degree of tumor necrosis in the high-dose COE group was higher than that in the control group (Figure 4B). In order to test the effect of COE on organ metastasis, histological evaluation of the liver and lung sections from each mouse were performed. As shown in Figure 4C, the metastatic lesions were distinguishable on H&E-stained sections from the liver and lung tissue (some marked by arrows). We observed that COE treatment markedly reduced liver and lung metastases and also suppressed ascites formation and dissemination to other tissues including omentum, parietal peritoneum, and diaphragm, compared with the control group (Table 1). Together, these results support the role of COE in suppression of gastric cancer metastasis.

Figure 4.

COE suppresses the peritoneal dissemination and organ metastasis of gastric cancer in vivo.

Table 1.

Effect of COE on the Incidences of Peritoneal Dissemination and Organ Metastasis in SGC-7901 Cells.

Treatment	Ascites	Metastasis
		Omentum	Mesentery	Parietal Peritoneum	Diaphragm	Liver	Lung
Control	5/7 (71%)	7/7 (100%)	7/7 (100%)	5/7 (71%)	4/7 (57%)	4/7 (57%)	2/7 (29%)
COE (10 mg/kg)	3/7 (43%) a	5/7 (71%) a	5/7 (71%) a	2/7 (29%) a	2/7 (29%) a	2/7 (29%) a	1/7 (14%) a
COE (20 mg/kg)	1/7 (14%) b	3/7 (43%) a	4/7 (57%) a	1/7 (14%) b	1/7 (14%) b	1/7 (14%) b	0/7 (0%) b
COE (40 mg/kg)	0/7 (0%) b	1/7 (14%) b	3/7 (43%) a	0/7 (0%) b	0/7 (0%) b	0/7 (0%) b	0/7 (0%) b
Xeloda (267 mg/kg)	0/7 (0%) b	1/7 (14%) b	2/7 (29%) b	0/7 (0%) b	0/7 (0%) b	0/7 (0%) b	0/7 (0%) b

Abbreviation: COE, ethyl acetate extract of Celastrus orbiculatus.

a

P < .01 versus control group.

b

P < .001 versus control group.

COE Suppresses EMT Properties and NF-κB/Snail Signal Pathway In Vivo

To better understand the molecular mechanism of COE on tumor growth and metastasis in vivo, we examined the expression levels of E-cadherin, N-cadherin, vimentin, NF-κB p65, and Snail in the tumor tissue in subcutaneous and peritoneal dissemination tumor models by immunohistochemisty analysis. The results revealed that COE could increase the expression E-cadherin and reduce the expression of N-cadherin and vimentin in subcutaneous tumor models (Figure 5A). Furthermore, it was also observed that the targeted proteins of NF-κB/Snail signaling were also diminished in COE-treated tumors compared with solvent-treated animals (Figure 5A). In addition, a similar result was observed in peritoneal dissemination tumor model (Figure 5B). A novel mechanism of COE that suppressed EMT and inhibited NF-κB/Snail signaling pathway during treatment of gastric cancer was revealed by these data.

Figure 5.

COE suppresses EMT properties and NF-κB/Snail signal pathway in vivo.

Discussion

COE is the product extracted from the plant of Celastrus orbiculatus, a traditional Chinese herb. Recent studies reveal that COE exhibits anticarcinogenic potentials, such as the inhibition of cell proliferation, induction of cell apoptosis, inhibition of angiogenesis.^13,16 COE was also found to inhibit invasion and migration of human colorectal carcinoma cells, ²² suggesting that COE might possess antimetastasis potential. However, the inhibition of the metastasis mechanism of COE in gastric cancer cells has not been investigated so far. In this research, an unknown mechanism of EMT that probably explained the COE-induced suppressive effect on metastasis of gastric cancer was observed and explored.

Cancer metastasis is a multistep process, initiated by attachment of cancer cells to basement membrane, secretion of matrix-degrading enzymes, and movement through the basement membrane and the underlying matrix. ²³ Tumor cell adhesion, invasion, and migration are important factors during this sequential process of metastasis. ²⁴ In this study, we explore the effects of COE on the metastatic activity of SGC-7901 cells. Our results demonstrated that COE suppressed proliferation in a dose-dependent manner. When SGC-7901 cells were treated with COE under low-cytotoxic concentrations, adhesion, invasion, and migration were inhibited in a dose-dependent manner. Thus, the research findings provide evidence that COE inhibits the metastasis of SGC-7901 cells in vitro, which suggests that COE has an antimetastatic property.

Many studies have reported that EMT has a potential mechanism for the metastasis of cancer cells. ⁵ EMT is a biological process in which epithelial cells undergo from phenotypic conversion to mesenchymal cells. ²⁵ During EMT, the ability of an epithelial cell to change its morphological characteristics to a mesenchymal cell is a fundamental process in tumorigenesis. ²⁶ The reduction of E-cadherin expression is representative characteristic of EMT and an important part of the metastatic process. ²⁷

The evidence that COE inhibited EMT in SGC-7901 cells was shown by the increase in expression of an epithelial phenotype marker (E-cadherin) and decrease of the mesenchymal phenotype markers (N-cadherin, vimentin). Therefore, COE may inhibit gastric cancer cell metastasis by suppressing EMT development.

The NF-κB signaling pathway is involved in the pathogenesis of various cancers, including gastric cancer. ²⁸ Continuous activity of NF-κB in cancer cells is highly metastatic, so inhibition of activity of NF-κB in the cells could greatly decrease their invasiveness. ⁸ NF-κB activity was inhibited by IκB by which NF-κB in the cytoplasm was blocked. ²⁹ It is reported that stimulus-induced phosphorylation and ubiquitination of IκB by the IκB kinase complex could result in proteasome-mediated degradation, which in turn causes nuclear translocation and DNA binding of NF-κB. ³⁰ Thus, several agents able to suppress NF-κB activation have the potential to suppress cancer metastasis and have shown therapeutic potential. Li et al demonstrated that celastrol, a triterpene extracted from Celastrus orbiculatus, inhibited the invasion and migration of human hepatocellular carcinoma cells by inhibiting the NF-κB signaling pathway. ³¹ Downregulation of NF-κB activity was also found to be involved in celastrol-induced apoptosis in human gastric adenocarcinoma cells. ³² In the present study, the observation that TNF-α increases transcriptional activity of NF-κB is confirmed by western blot assay. However, administration of COE can markedly inhibit TNF-α-induced NF-κB transcriptional activity in SGC-7901 cells. Furthermore, the inhibitory effect of COE on NF-κB is associated with the decrease of IκB phosphorylation and degradation.

Snail, as the transcription factor, is one of the target genes of NF-κB and a key regulatory factor in EMT. ³³ Snail transcriptionally suppresses the adherent junction protein, E-cadherin, by binding to E2-box type elements within its promoter, resulting in EMT. In this study, we found that COE had significant inhibitory effect on Snail expression, and the blockage of NF-κB was consistent. ³⁴ These results suggested that the mechanism of COE to inhibit EMT process may relate to the suppression of NF-κB/Snail signal pathway.

The in vitro results prompted us to investigate the role of COE in gastric cancer growth and metastasis in vivo using the nude mice model, which provides a scientific rationale to treat patients with gastric cancer. Our results showed that COE conspicuously reduced tumor volume, peritoneal dissemination, and organ metastasis in nude mice model. Moreover, immunohistochemical assessment of tumor tissues provides strong molecular evidence to support our hypothesis that COE-mediated tumor growth and metastasis were controlled by suppressing EMT and inhibiting NF-κB/Snail signaling pathway.

In this study, COE had growth-inhibiting and antimetastatic effects in nude mice models, and the antitumor activity of COE in inhibiting the EMT process of the human gastric cancer cell line was satisfactory. Thus, COE may become a potential therapy against metastasis. Further studies are needed to identify the metabolism and pharmacokinetics in animal models. In addition, we also need to confirm whether COE and the molecular mechanism identified in the present study has the same function in other tumor types.

In conclusion, COE is a new candidate anticancer agent that inhibits tumor cell invasion and metastasis by inhibiting EMT and NF-κB/Snail signal pathway of human gastric cancer. These findings reveal that application of COE in treating metastatic gastric cancer boasts a promising prospect and may become a potential therapy all over the world.