Skip to main content
NCBI home page
Journal of Zhejiang University. Science. B logoLink to Journal of Zhejiang University. Science. B
. 2011 Jul;12(7):534–544. doi: 10.1631/jzus.B1000217

Ds-echinoside A, a new triterpene glycoside derived from sea cucumber, exhibits antimetastatic activity via the inhibition of NF-κB-dependent MMP-9 and VEGF expressions*

Qin Zhao 1, Zhi-dong Liu 1, Yong Xue 1, Jing-feng Wang 1,†,‡, Hui Li 1, Qing-juan Tang 1, Yu-ming Wang 1, Ping Dong 1, Chang-hu Xue 1,†,‡
PMCID: PMC3134607  PMID: 21726060

Abstract

Ds-echinoside A (DSEA), a non-sulfated triterpene glycoside, was isolated from the sea cucumber Pearsonothuria graeffei. In vitro and in vivo investigations were conducted on the effects of DSEA on tumor cell adhesion, migration, invasion, and angiogenesis. In this study, we found that DSEA inhibited the proliferation of human hepatocellular liver carcinoma cells Hep G2, with a half-maximal inhibitory concentration (IC50) of 2.65 μmol/L, and suppressed Hep G2 cell adhesion, migration, and invasion in a dose-dependent manner. DSEA also reduced tube formation of human endothelial cells ECV-304 on matrigel in vitro and attenuated neovascularization in the chick embryo chorioallantoic membrane (CAM) assay in vivo. Immunocytochemical analysis revealed that DSEA significantly decreased the expression of matrix metalloproteinase-9 (MMP-9), which plays an important role in the degradation of basement membrane in tumor metastasis and angiogenesis. DSEA also increased the protein expression level of tissue inhibitor of metalloproteinase-1 (TIMP-1), an important regulator of MMP-9 activation. From the results of Western blotting, the expressions of nuclear factor-kappa B (NF-κB) and vascular endothelial growth factor (VEGF) were found to be remarkably reduced by DSEA. These findings suggest that DSEA exhibits a significant anti-metastatic activity through the specific inhibition of NF-κB-dependent MMP-9 and VEGF expressions.

Keywords: Triterpene glycoside, Ds-echinoside A (DSEA), Metastasis, Angiogenesis, Nuclear factor-κB (NF-κB), Matrix metalloproteinase-9 (MMP-9), Vascular endothelial growth factor (VEGF)

1. Introduction

Metastasis, which refers to the spread of cancer cells from primary neoplasms to distant sites, is often proven to be the most damaging aspect of cancer, and is one of the major causes of mortality in cancer patients. Tumor metastasis, which includes cell proliferation, proteolytic digestion of extracellular matrix (ECM), cell migration to the circulatory system, and tumor growth at metastatic sites, is a multistep and complex process (Kleiner and Stetler-Stevenson, 1999; Chambers et al., 2002; Steeg, 2006; Imanaka et al., 2008). When tumor cells metastasize, various proteolytic enzymes contribute to the degradation of ECM and basement membrane (BM) (Pasco et al., 2005). Matrix metalloproteinases (MMPs) play a crucial role in ECM degradation associated with both metastasis and angiogenesis of cancer (Westermarck and Kähäri, 1999; McCawley and Matrisian, 2000). Both the transcriptional and post-transcriptional levels of MMPs are closely related. MMP expression is controlled by various cytokines and growth factors at the transcriptional level. At the post-transcriptional level, MMP activity is controlled by proteolytic activation of latent proenzymes and by interactions with specific inhibitors (Rao, 2003). MMP-9, a member of the MMP family, has been suggested to play a key role in tumor invasion and angiogenesis (Nabeshima et al., 2002) and is overexpressed in many human cancers with invasive and metastatic capabilities (Sanceau et al., 2003). The expression of MMP-9 can be regulated by nuclear factor-kappa B (NF-κB) (Nagao et al., 2000; Choi et al., 2010). NF-κB activation has also been reported to be associated with metastatic phenotypes (La Porta and Comolli, 1998), and inhibition of NF-κB activation has been shown to suppress MMP-9 and increase tumor invasion (Alaniz et al., 2004).

Angiogenesis, which is required to sustain the growth of primary tumors and metastases, is mediated by an increased production of various angiogenic molecules released by both the tumor itself and neighboring host cells (Folkman, 2006). Among the known angiogenic growth factors and cytokines, vascular endothelial growth factor (VEGF) and its corresponding receptors are indispensable in regulating multiple facets of angiogenic processes (Leung et al., 1989). VEGF is an NF-κB-inducible protein that is regulated by activated NF-κB (Dai et al., 2009). Recently, several studies have reported that VEGF production and gene expression are compromised by NF-κB inhibition (Shibata et al., 2002; Ko et al., 2006; Dai et al., 2009).

The sea cucumber Pearsonothuria graeffei (P. graeffei), which is of little food value, is widely distributed in Taiwan, the Nansha Islands, Madagascar, the Maldives, the Philippines, Guam, and Fiji. It contains a high content of triterpene glycosides, up to 3.5% of the body wall dry matter. Triterpene glycosides with a wide spectrum of biological effects (Chludil et al., 2002; Liu et al., 2007; Zhang et al., 2007) are the predominant secondary metabolites of holothurians. Recently, the anti-tumor and anti-angiogenic activities of triterpene glycosides have attracted considerable attention. Different species of the sea cucumber show differences in the content and structure of triterpene glycosides, and differ according to the growing environment and food intake. Both frondoside A and cucumarioside A2-2, which are sulfated triterpene glycosides, isolated from sea cucumbers Cucumaria frondosa and Cucumaria japonica, respectively, possessed cell apoptosis-inducing capability in a caspase-dependent or -independent manner, depending on the holothurian structure (Jin et al., 2009). Another two sulfated triterpene glycosides, philinopside A (PA) and philinopside E (PE), derived from the sea cucumber Pentacta quadrangulari, were both found to exhibit dual anti-angiogenic and anti-tumor effects through receptor tyrosine kinases (RTKs) or VEGF-induced kinase insert domain-containing receptor (KDR) signaling pathways, respectively (Tian et al., 2005; Tong et al., 2005). Ds-echinoside A (DSEA), a non-sulfated triterpene glycoside, was derived from the desulfurization reaction of echinoside A by Kitagawa in 1980, and so far, the only report on the bioactivity of DSEA has been about its antifungal effects (Kitagawa et al., 1980). To our knowledge, there is no report on the separation of DSEA from a natural source directly, and no information is available about the anticancer potential and related molecular mechanisms of nonsulfated triterpene glycosides. Here, for the first time, we directly isolated DSEA from the sea cucumber P. graeffei and demonstrated that DSEA exhibited a significant anti-metastatic activity both in vitro and in vivo. Regarding the underlying mechanism, the bioactivity of DSEA may involve the modulation of NF-κB, which, in turn, reduced the expressions of VEGF and MMP-9.

2. Materials and methods

2.1. Triterpene glycoside

DSEA was isolated and purified using the procedure described by Dong et al. (2008). Briefly, an ethanolic extract of P. graeffei was sequentially submitted to macroporous resin, Si-gel, and reversed Si-gel chromatographies, and the final purification of DSEA was achieved by preparative high-performance liquid chromatography (HPLC). The chemical structure of DSEA was determined by spectroscopic analysis (infrared spectroscopy, nuclear magnetic resonance spectroscopy, and electrospray ionization mass spectrometry). The molecular weight of DSEA was 1 104 Da, and the molecular formula was deduced to be C54H88O23 (Fig. 1). DSEA was dissolved in dimethyl sulfoxide (DMSO) and diluted to the desired concentrations before use. The final concentration of DMSO in the culture media was below 0.05% (v/v), and this concentration of DMSO showed no effect in the assay systems.

Fig. 1.

Fig. 1

Open in a new tab

Chemical structure of ds-echinoside A (DSEA)

2.2. Cell lines and cell culture

Human hepatocellular liver carcinoma cells Hep G2 and human umbilical vein endothelial cells ECV-304 were obtained from Shanghai Cell Bank (Shanghai, China) and grown in RPMI-1640 medium or Dulbecco’s modified eagle medium (DMEM), supplemented with 10% (v/v) newborn calf serum (NCS), 100 μg/ml streptomycin, and 100 U/ml penicillin at 37 °C in a humidified atmosphere containing 5% CO2. All experiments were repeated three times to ensure the reproducibility.

2.3. Cell proliferation assay

Hep G2 cells (8×103 well−1) were seeded in a 96-well plate and incubated for 24 h. Then, the medium was replaced with fresh RPMI-1640 medium containing different concentrations of DSEA. After incubation for 6–24 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (0.5 mg/ml in RPMI-1640 medium) was added and further incubated for 4 h. Cell viability was determined by the MTT assay (Skehan et al., 1990).

2.4. Cell adhesion assay

Cell adhesion efficiency was determined by measuring the number of cells that adhered to a given substrate, as described by Liaw et al. (1994). Matrigel diluted to 50 μg/ml with DMEM was applied to 96-well plates and allowed to polymerize in a humidified incubator at 37 °C for 1 h. Hep G2 cells pretreated with various concentrations of DSEA (0, 1.35, and 2.70 μmol/L) for 12–24 h were collected and suspended at a final concentration of 2×105 cells/ml in serum-free medium; 100 µl of the cell suspension was seeded into each well and allowed to adhere at 37 °C for 1.5 h. Non-adherent cells were carefully rinsed off with phosphate-buffered saline (PBS) and the remaining cells were measured using the MTT assay.

2.5. Wound migration assay

Hep G2 cells (1.5×105 well−1) were seeded into a 24-well plate for 24 h. The confluent monolayer was starved using serum-free medium for 8 h and wounded by scratching with a 1-ml pipette tip. After washed three times with PBS, cells were incubated in serum-free medium containing various concentrations of DSEA (0, 1.35, and 2.70 μmol/L). Photographs were taken at 0, 12, and 24 h after wounding. The width of the wound was measured using the Image Pro Plus 5 software.

2.6. Cell invasion assay

The cell invasion assay was performed using a Transwell Boyden chamber (diameter 6.5 mm) with polycarbonate filter (pore size 8 µm). Briefly, the upper culture chamber was coated with a uniform layer of matrigel (1:20, diluted in RPMI-1640), and 750 µl RPMI-1640 medium containing 20% (v/v) fetal bovine serum (FBS) was placed in each lower well. Hep G2 cells (5×104 cells) were loaded into each upper well in 100 µl RPMI-1640 along with various concentrations of DSEA (0, 1.35, and 2.70 μmol/L). After incubation at 37 °C for 12 h, nonmigrating cells on the upper surface of the filter were removed with a cotton swab. The filters were then fixed with ethanol and stained with 10 g/L crystal violet. The cells were visualized using an inverted microscope (IX51; Olympus, Japan) and the images were analyzed using the Image Pro Plus 5 software. Five random fields were counted for each filter. The rate of invasion was calculated as migrated cells of the treated/migrated cells of the control.

2.7. Tube formation assay

A tube formation assay was carried out to determine the effect of DSEA on angiogenesis in vitro. A 96-well plate coated with 50 μl of matrigel (1:4, diluted in DMEM) per well was allowed to solidify at 37 °C for 1 h. Each well was seeded with 1×104 ECV-304 cells, resuspended in DMEM with 2% (v/v) NCS, and cultured in a medium containing various concentrations of DSEA (0, 2.26, and 4.53 μmol/L) for 24 h. The networks of enclosed tubes were photographed from five randomly chosen fields under an inverted phase contrast microscope (IX51; Olympus, Japan). Images were captured under an Olympus DP72 microscope digital camera system using the Image Pro Plus 5 software.

2.8. Chicken embryo chorioallantoic membrane (CAM) assay

Inhibition of angiogenesis in vivo was determined using a modified CAM assay (Tan et al., 2001). Fertilized chicken eggs were allowed to grow for 9 d in an egg incubator. Then, a small hole was punched into the broad side of the egg and a 1-cm2 window was carefully created in the eggshell. The shell membrane was removed to expose the CAM. Sterile filter-paper discs (diameter 5 mm) saturated with various concentrations of DSEA (0, 0.23, and 0.46 nmol/L per egg) were placed on the CAM. After further incubated for 24 h, the neovascular zones under the disks were photographed under a stereomicroscope (SZ61; Olympus, Japan) at 10× magnification.

2.9. Western blotting analysis

Hep G2 cells were plated onto 6-well plates and allowed to grow to confluence (1×106 cells). Dilutions of DSEA (0, 1.35, and 2.70 μmol/L) were added and incubated for 12 h at 37 °C. The cells were then lysed with lysis buffer (20 mmol/L Tris/HCl (pH 8.0), 2 mmol/L EDTA, 137 mmol/L NaCl, 1 mmol/L Na3VO4, 10% (v/v) glycerol, 1% (v/v) Triton X-100, and 1 mmol/L phenylmethylsulfonyl fluoride). Lysates were clarified by centrifugation (12 000×g, 10 min), separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk overnight at 4 °C and incubated with primary antibodies against NF-κB p65, VEGF, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for 1.5 h at room temperature. Then, the membranes were washed (Tris-buffered saline and Tween 20 (TBS/T), three times, 5 min per time), incubated (1.5 h, room temperature) with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000), and washed three times with TBS/T. Antigen reactivity was detected using a Tiangen KC-420 chemiluminescent substrate kit (Beijing, China). Each experiment was repeated at least three times; representative blots are presented.

2.10. Immunocytochemical assay

Hep G2 cells (5×105 well−1) were seeded in 6-well plates with sterilized coverslips and treated with various concentrations of DSEA (0, 1.35, and 2.70 μmol/L) for 12 h. Then the cells were washed in PBS and fixed with 4% (v/v) paraformaldehyde at 4 °C. The streptavidin biotin complex (SABC) immunohistochemistry kit was used for immunocytochemical analysis. Cells were incubated overnight with polyclonal MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) antibodies at 4 °C. After washing three times with PBS, cells were incubated with biotinylated goat anti-rabbit IgG for 40 min at 37 °C. Then cells were incubated with SABC at 37 °C for 40 min and colored with diaminobenzidine (DAB) for 15 min. Finally, the slides were counterstained with hematoxylin, dehydrated, cleared in xylene, and mounted. Stained cells were examined and photographed using a light microscopy (BX41; Olympus, Japan). Data were analyzed using the Image Pro Plus 5 software.

2.11. Statistical analysis

Data are presented as mean±standard deviation (SD). Statistical significance was determined by one-way analysis of variance (ANOVA) and the least significant difference (LSD) test.

3. Results

3.1. DSEA inhibits proliferation of Hep G2 cells

To determine the cytotoxicity of DSEA, its inhibitory effect on the proliferation of Hep G2 cells was measured by the MTT assay. As shown in Fig. 2, DSEA had a dose-dependent inhibitory effect on the viability of Hep G2 cells, with an average half-maximal inhibitory concentration (IC50) of 2.65 μmol/L. These results indicate that DSEA was strongly cytotoxic to hepatocellular carcinoma cells.

Fig. 2.

Fig. 2

Open in a new tab

Effect of DSEA on the proliferation of Hep G2 cells

Hep G2 cells were treated with various concentrations of DSEA (0, 0.44, 0.88, 1.75, 2.63, 3.51, and 4.39 μmol/L) for 6, 9, 12, and 24 h. Cell viability was estimated by MTT assay. Data represent the mean±SD of three independent experiments

3.2. DSEA suppresses Hep G2 cell adhesion

Cell adhesion is a prerequisite for tumor cell invasion and metastasis formation. Thus, we investigated whether DSEA affected the adhesion of tumor cells to matrigel, a common analog of ECM. After attaching efficiently to matrigel for 1.5 h, DSEA-treated Hep G2 cells showed a dose- and time-dependent decrease in cell adhesion. As shown in Fig. 3, DSEA decreased the average adhesion rate of Hep G2 cells to 67.6% and 39.5% at 1.35 and 2.70 μmol/L, respectively (P<0.01).

Fig. 3.

Fig. 3

Open in a new tab

Effect of DSEA on the adhesion of Hep G2 cells

Hep G2 cells were seeded on matrigel-coated wells with the indicated concentrations of DSEA for 1.5 h. The relative number of attached cells was assessed using the MTT assay. Data represent the mean±SD of three independent experiments. * P<0.01 vs. control (0 μmol/L)

3.3. DSEA inhibits Hep G2 cell migration

As tumor cell migration is an essential process in cancer development and metastasis, we explored the effects of DSEA on Hep G2 cell motility using a scratch wound assay. As shown in Fig. 4, in the absence of DSEA, Hep G2 cells migrated along the edges of the wound and showed a large-scale migration, whereas a dose- and time-dependent inhibition of cell flattening and spreading was observed in the presence of DSEA. At 1.35 and 2.70 µmol/L, DSEA decreased the average migration rates of Hep G2 cells to 80.9% and 36.4%, respectively.

Fig. 4.

Fig. 4

Open in a new tab

Effect of DSEA on the migration of Hep G2 cells

Microphotographs of Hep G2 cells (a) and quantitative analysis (b) of the wound migration assay. Hep G2 cells were seeded in a 24-well plate and the confluent monolayers were wounded and then incubated in serum-free medium with 0, 1.35, and 2.70 μmol/L DSEA. At 0, 12, and 24 h after wounding, the cells were photographed under an inverted microscope. Migration rate was expressed as a percentage of control (0 μmol/L). Data represent the mean±SD of three independent experiments. * P<0.01 vs. control (0 μmol/L)

3.4. DSEA hinders Hep G2 cell invasion

The effects of DSEA on Hep G2 cell migration were analyzed in a Transwell Boyden chamber assay. As shown in Fig. 5, incubation of control Hep G2 cells in the chamber for 12 h resulted in large-scale migration of cancer cells to the lower side of the filter. In contrast, treatment with DSEA caused a dose-dependent inhibition of Hep G2 invasion, with DSEA concentrations of 1.35 and 2.70 µmol/L yielding inhibition rates of 60.5% and 81.4%, respectively.

Fig. 5.

Fig. 5

Open in a new tab

Effect of DSEA on the invasion of Hep G2 cells

Microphotographs of filters (a) and quantitative analysis (b) of the matrigel chamber invasion assay are shown. Hep G2 cells seeded in Transwell Boyden chambers were incubated for 24 h with 0, 1.35, and 2.70 μmol/L DSEA. Invasion rate was expressed as a percentage of control (0 μmol/L). Data represent the mean±SD of three independent experiments. * P<0.01 vs. control (0 μmol/L)

3.5. DSEA disrupts microvascular tube formation of endothelial cells

As capillary tube formation of endothelial cells is an essential process in angiogenesis, we studied the effect of DSEA on the formation of tube-like structures in the matrigel tube formation assay. As shown in Fig. 6, control ECV-304 cells differentiate into extensive networks of enclosed tubes. In contrast, the tube-forming ability of DSEA-treated ECV-304 cells decreased significantly. DSEA treatment disrupted tube structure, leading to the development of incomplete tube morphologies, in a dose-dependent manner.

Fig. 6.

Fig. 6

Open in a new tab

Effect of DSEA on the tube formation ability of ECV-304 cells

ECV-304 cells seeded in matrigel-coated 96-well plates were incubated for 24 h with DSEA at the concentrations of 0, 2.26, and 4.53 μmol/L. The enclosed networks of tubes were photographed using an inverted microscope

3.6. DSEA inhibits angiogenesis in the CAM assay

To investigate the effect of DSEA on angiogenesis in vivo, a modified CAM assay was carried out. The results indicated that in control eggs, blood vessels formed dense branching vascular networks. In contrast, CAM neovascularization, presented as blood vessel numbers and branching patterns, was dose-dependently suppressed by DSEA (Fig. 7).

Fig. 7.

Fig. 7

Open in a new tab

Effect of DSEA on the angiogenesis of CAM

Various concentrations of DSEA (0, 0.23 and 0.46 nmol/L per egg) were loaded on the CAMs. After 24 h incubation, 10% (v/v) formaldehyde was added onto the surface of CAMs to fix the blood. The disc and surrounding CAMs were harvested and photographed at 10× magnification

3.7. DSEA suppresses the expression level of MMP-9 via enhancement of TIMP-1 expression

MMP-9 is known to play an important role in cancer cell invasion by mediating the degradation of ECM proteins. Thus, we examined the effects of DSEA on the expression levels of MMP-9 and TIMP-1 by using immunocytochemical methods. As shown in Fig. 8, the expression of MMP-9 was decreased significantly by DSEA. At the concentration of 2.70 μmol/L, the expression of MMP-9 in DSEA-treated cells was reduced to 87.9%.

Fig. 8.

Fig. 8

Open in a new tab

Effect of DSEA on the protein expression of MMP-9

Hep G2 cells were treated with various concentrations of DSEA for 12 h. The protein expression level of MMP-9 was measured by immunocytochemical analysis. Microphotographs of Hep G2 cells (a) and quantitative analysis (b) of the MMP-9 protein expression are shown. Data represent the mean±SD of three independent experiments. * P<0.05 vs. control (0 μmol/L)

The protein expression level of TIMP-1 in Hep G2 cells treated with different concentrations of DSEA for 12 h is shown in Fig. 9. DSEA increased TIMP-1 expression in Hep G2 cells by 534.0% and 613.3% at 1.35 and 2.70 μmol/L, respectively.

Fig. 9.

Fig. 9

Open in a new tab

Effect of DSEA on the protein expression of TIMP-1

Hep G2 cells were treated with various concentrations of DSEA (0, 1.35, and 2.70 μmol/L) for 12 h. The protein expression levels of TIMP-1 were measured by immunocytochemical analysis. Data represent the mean±SD of three independent experiments. * P<0.01 vs. control (0 μmol/L)

3.8. DSEA down-regulates the protein expression level of VEGF in Hep G2 cells

VEGF plays pivotal roles in angiogenesis and subsequent cancer growth and progression. After exposure to DSEA, the expression of VEGF protein was determined by Western blotting; the result is shown in Fig. 10a. DSEA dose-dependently inhibited VEGF expression in Hep G2 cells. At 1.35 and 2.70 μmol/L, DSEA reduced VEGF expression in Hep G2 cells by 44.1% and 72.9%, respectively.

Fig. 10.

Fig. 10

Open in a new tab

Effect of DSEA on the protein expression of VEGF (a) and NF-κB (b)

Hep G2 cells were incubated with various concentrations of DSEA for 12 h at 37 °C. The levels of VEGF and NF-κB were measured by Western blotting. Densitometry ratios of VEGF and NF-κB were normalized to GAPDH. Data represent the mean±SD of three independent experiments. * P<0.01 vs. control (0 μmol/L)

3.9. DSEA inhibits the protein expression level of NF-κB in Hep G2 cells

It is accepted that the expressions of MMP-9 and VEGF are regulated by NF-κB (Farina et al., 1999; Dai et al., 2009). We further examined the effects of DSEA on the expression level of NF-κB p65 by Western blotting analysis. As shown in Fig. 10b, the expression of NF-κB p65 was significantly suppressed by DSEA. After treatment with 2.70 μmol/L DSEA for 12 h, the expression of NF-κB p65 was decreased to 5.77% in Hep G2 cells.

4. Discussion

Metastasis is among the last great frontiers in cancer research, and preventing or eliminating metastasis is one of the biggest challenges for therapeutic intervention (Machesky, 2008). The discovery of novel natural products and their components to block cancer metastasis has attracted researchers’ interests and attentions (Lee et al., 2008). Marine natural products contain a variety of chemotherapeutic compounds that have been shown to prevent the development of malignancies, and several marine-derived molecules are currently in or are entering clinical trials in cancer therapy (Tian et al., 2005). Here, we found that DSEA, a novel marine natural non-sulfated triterpene glycoside, is capable of inhibiting the main steps involved in metastasis, including suppressing Hep G2 cell migration, adhesion, and invasion, and blocking endothelial cell tube formation and angiogenesis in an in vivo CAM model. Our findings demonstrate that DSEA has a potent anti-metastatic activity both in vitro and in vivo.

MMPs, which belong to a multigene family of zinc-dependent endopeptidases and are principal enzymes in ECM degradation, play a major role in promoting angiogenesis and tumor metastasis (McCawley and Matrisian, 2000). Among the MMPs, MMP-2 and MMP-9 are key enzymes in the process of tumor metastasis as a result of ECM degradation (Chang and Werb, 2001). Both MMP-2 and MMP-9 have been correlated with an aggressive, advanced invasive or metastatic tumor phenotype (Liotta et al., 1991). It has been demonstrated that MMP-9 is over-expressed in many human solid and hematological malignancies (Sanceau et al., 2003). Thus, an inhibitory effect on MMP-9 expression is important for anti-metastasis. Our data demonstrate that DSEA suppressed MMP-9 expression levels in a dose-dependent manner in Hep G2 cells. The levels of cancer invasiveness and malignancy are primarily determined by a sensitive balance between MMPs and their endogenous inhibitors, TIMPs (Hornebeck et al., 2005). TIMPs may display distinct influences on tumor progression. It is reported that up-regulation of TIMPs inhibits tumor growth, invasion, and metastasis (Jiang et al., 2002). Generally, regulation of MMP-9 is controlled by proenzyme activation and inhibition by TIMP-1 (Watanabe et al., 1993). Down-regulation of TIMP-1 was demonstrated to correlate with high cancer invasiveness (Zhang et al., 2003; Chan et al., 2005). In this study, we found that DSEA enhanced TIMP-1 expression significantly in Hep G2 cells. These results suggest that DSEA directly inhibits the metastatic ability of Hep G2 cells by depressing MMP-9 expression, with augmented TIMP-1 expression.

Two crucial events in tumor progression, tissue invasion and metastasis, are regulated by NF-κB-dependent genes, including MMPs (Farina et al., 1999). It has been reported that the MMP-9 promoter contains DNA-binding sites for NF-κB, and that the activation of NF-κB is required to up-regulate the expression of MMP-9 (Nagao et al., 2000). In normal resting cells, NF-κB is sequestered in the cytoplasm by binding to a member of inhibitory proteins of the IκB family. NF-κB activation involves its liberation from IκB and its subsequent translocation to the nucleus, where it binds to specific sequences within the promoter region of target genes, and induces gene expression (Pahl, 1999). NF-κB activation has also been found to induce resistance to various chemotherapeutic agents. Thus, NF-κB may be a good target for the anticancer drug development (Yu et al., 2005). Our results showed that the expression of NF-κB in Hep G2 cells was significantly decreased by DSEA. These findings suggest that DSEA suppresses the expression of MMP-9 by blocking NF-κB activation in Hep G2 cells.

NF-κB is also involved in the regulation of angiogenesis, by which tumor cells promote neovascularization for their growth and invasiveness. VEGF is one of the most potent and specific angiogenic factors of tumor-induced angiogenesis (Leung et al., 1989). Numerous reports have demonstrated that the metastatic potential of tumor cells is directly correlated with the VEGF expression level, and tumor growth and invasion were significantly suppressed through inhibition of VEGF-induced angiogenesis both in vitro and in vivo (Alferez et al., 2008; Wang et al., 2008). Here we showed that DSEA treatment markedly suppressed the expression of VEGF. VEGF is under transcriptional control by NF-κB (Kiriakidis et al., 2003). Although the VEGF promoter does not contain an NF-κB binding site (Forsythe et al., 1996), we and others have demonstrated that VEGF expression is inhibited by NF-κB inhibition (Shibata et al., 2002; Ko et al., 2006; Dai et al., 2009). These results suggest that DSEA down-regulates VEGF expression, in part through the NF-κB signaling pathway. There are various signal transduction pathways involved in the regulation of MMP-9 expression in tumor cells. Mitogen-activated protein kinases (MAPKs) are suggested to be intricately involved in the expression of the components involved in the induction of the promoter for MMP-9 via NF-κB (Kaomongkolgit et al., 2008). Further in-depth investigations are needed to better understand the complex mechanism involved.

In conclusion, the present study provides evidence that DSEA possesses marked anti-metastatic activity. We have shown that DSEA plays a role in the inhibition of Hep G2 cell migration, invasion, and angiogenesis through suppression of VEGF and MMP-9 and enhancement of TIMP-1 protein expression by blocking the NF-κB signaling pathway. To our knowledge, this is the first report demonstrating the anti-metastatic activity of a nonsulfated triterpene glycoside isolated from P. graeffei, and providing a scientific basis for its application in therapeutic intervention against tumor progression.

Footnotes

*

Project supported by the National High-Tech R & D Program (863) of China (No. 2007AA091805), and the National Natural Science Foundation of China (Nos. 30972284 and 30871944)

References

  • 1.Alaniz L, García M, Cabrera P, Arnaiz M, Cavaliere V, Blanco G, Alvarez E, Hajos S. Modulation of matrix metalloproteinase-9 activity by hyaluronan is dependent on NF-κB activity in lymphoma cell lines with dissimilar invasive behavior. Biochem Biophys Res Commun. 2004;324(2):736–743. doi: 10.1016/j.bbrc.2004.09.120. [DOI] [PubMed] [Google Scholar]
  • 2.Alferez D, Wilkinson R, Watkins J, Poulsom R, Mandir N, Wedge S, Pyrah I, Smith N, Jackson L, Ryan A. Dual inhibition of VEGFR and EGFR signaling reduces the incidence and size of intestinal adenomas in Apc Min/+ mice. Mol Cancer Ther. 2008;7(3):590–598. doi: 10.1158/1535-7163.MCT-07-0433. [DOI] [PubMed] [Google Scholar]
  • 3.Chambers A, Groom A, MacDonald I. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer. 2002;2(8):563–572. doi: 10.1038/nrc865. [DOI] [PubMed] [Google Scholar]
  • 4.Chan V, Chan M, Leung W, Leung P, Sung J, Chan F. Intestinal trefoil factor promotes invasion in non-tumorigenic Rat-2 fibroblast cell. Regul Pept. 2005;127(1-3):87–94. doi: 10.1016/j.regpep.2004.10.016. [DOI] [PubMed] [Google Scholar]
  • 5.Chang C, Werb Z. The many faces of metalloproteases: cell growth, invasion, angiogenesis and metastasis. Trends Cell Biol. 2001;11(11):S37–43. doi: 10.1016/S0962-8924(01)02122-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Chludil HD, Muniain CC, Seldes AM, Maier MS. Cytotoxic and antifungal triterpene glycosides from the Patagonian sea cucumber Hemoiedema spectabilis . J Nat Prod. 2002;65(6):860–865. doi: 10.1021/np0106236. [DOI] [PubMed] [Google Scholar]
  • 7.Choi JH, Han EH, Hwang YP, Choi JM, Choi CY, Chung YC, Seo JK, Jeong HG. Suppression of PMA-induced tumor cell invasion and metastasis by aqueous extract isolated from Prunella vulgaris via the inhibition of NF-κB-dependent MMP-9 expression. Food Chem Toxicol. 2010;48(2):564–571. doi: 10.1016/j.fct.2009.11.033. [DOI] [PubMed] [Google Scholar]
  • 8.Dai L, Gu L, Ding C, Qiu L, Di W. TWEAK promotes ovarian cancer cell metastasis via NF-κB pathway activation and VEGF expression. Cancer Lett. 2009;283(2):159–167. doi: 10.1016/j.canlet.2009.03.036. [DOI] [PubMed] [Google Scholar]
  • 9.Dong P, Xue C, Yu L, Xu J, Chen S. Determination of triterpene glycosides in sea cucumber (Stichopus japonicus) and its related products by high-performance liquid chromatography. J Agric Food Chem. 2008;56(13):4937–4942. doi: 10.1021/jf800893r. [DOI] [PubMed] [Google Scholar]
  • 10.Farina A, Tacconelli A, Vacca A, Maroder M, Gulino A, Mackay A. Transcriptional up-regulation of matrix metalloproteinase-9 expression during spontaneous epithelial to neuroblast phenotype conversion by SK-N-SH neuroblastoma cells, involved in enhanced invasivity, depends upon GT-box and nuclear factor κB elements. Cell Growth Differ. 1999;10(5):353–367. [PubMed] [Google Scholar]
  • 11.Folkman J. Angiogenesis. Annu Rev Med. 2006;57(1):1–18. doi: 10.1146/annurev.med.57.121304.131306. [DOI] [PubMed] [Google Scholar]
  • 12.Forsythe J, Jiang B, Iyer N, Agani F, Leung S, Koos R, Semenza G. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol. 1996;16(9):4604–4613. doi: 10.1128/mcb.16.9.4604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Hornebeck W, Lambert E, Petitfrère E, Bernard P. Beneficial and detrimental influences of tissue inhibitor of metalloproteinase-1 (TIMP-1) in tumor progression. Biochimie. 2005;87(3-4):377–383. doi: 10.1016/j.biochi.2004.09.022. [DOI] [PubMed] [Google Scholar]
  • 14.Imanaka H, Koide H, Shimizu K, Asai T, Kinouchi Shimizu N, Ishikado A, Makino T, Oku N. Chemoprevention of tumor metastasis by liposomal β-sitosterol intake. Biol Pharm Bull. 2008;31(3):400–404. doi: 10.1248/bpb.31.400. [DOI] [PubMed] [Google Scholar]
  • 15.Jiang Y, Goldberg I, Shi Y. Complex roles of tissue inhibitors of metalloproteinases in cancer. Oncogene. 2002;21(14):2245–2252. doi: 10.1038/sj.onc.1205291. [DOI] [PubMed] [Google Scholar]
  • 16.Jin JO, Shastina VV, Shin SW, Xu Q, Park JI, Rasskazov VA, Avilov SA, Fedorov SN, Stonik VA, Kwak JY. Differential effects of triterpene glycosides, frondoside A and cucumarioside A2-2 isolated from sea cucumbers on caspase activation and apoptosis of human leukemia cells. FEBS Lett. 2009;583(4):697–702. doi: 10.1016/j.febslet.2009.01.010. [DOI] [PubMed] [Google Scholar]
  • 17.Kaomongkolgit R, Cheepsunthorn P, Pavasant P, Sanchavanakit N. Iron increases MMP-9 expression through activation of AP-1 via ERK/Akt pathway in human head and neck squamous carcinoma cells. Oral Oncol. 2008;44(6):587–594. doi: 10.1016/j.oraloncology.2007.08.005. [DOI] [PubMed] [Google Scholar]
  • 18.Kiriakidis S, Andreakos E, Monaco C, Foxwell B, Feldmann M, Paleolog E. VEGF expression in human macrophages is NF-κB-dependent: studies using adenoviruses expressing the endogenous NF-κB inhibitor IκBα and a kinase-defective form of the IκB kinase 2. J Cell Sci. 2003;116(4):665–674. doi: 10.1242/jcs.00286. [DOI] [PubMed] [Google Scholar]
  • 19.Kitagawa I, Inamoto T, Fuchida M, Okada S, Kobayashi M, Nishino T, Kyogoku Y. Structures of Echinoside A and B, two antifungal oligoglycosides from the sea cucumber Actinopyga echinites (Jaeger) Chem Pharm Bull. 1980;28(5):1651–1653. doi: 10.1248/cpb.33.5214. [DOI] [PubMed] [Google Scholar]
  • 20.Kleiner D, Stetler-Stevenson W. Matrix metalloproteinases and metastasis. Cancer Chemoth Pharm. 1999;43(7):S42–S51. doi: 10.1007/s002800051097. [DOI] [PubMed] [Google Scholar]
  • 21.Ko H, Jung H, Seo K, Kang Y, Kim H, Park S, Lee H, Im S. Platelet-activating factor-induced NF-κB activation enhances VEGF expression through a decrease in p53 activity. FEBS Lett. 2006;580(13):3006–3012. doi: 10.1016/j.febslet.2006.04.042. [DOI] [PubMed] [Google Scholar]
  • 22.La Porta C, Comolli R. PKC-dependent modulation of IκBα-NF-κB pathway in low metastatic B16F1 murine melanoma cells and in highly metastatic BL6 cells. Anticancer Res. 1998;18(4A):2591–2597. [PubMed] [Google Scholar]
  • 23.Lee K, Hwang S, Choi J, Jeong H. Saponins derived from the roots of Platycodon grandiflorum inhibit HT-1080 cell invasion and MMPs activities: regulation of NF-κB activation via ROS signal pathway. Cancer Lett. 2008;268(2):233–243. doi: 10.1016/j.canlet.2008.03.058. [DOI] [PubMed] [Google Scholar]
  • 24.Leung D, Cachianes G, Kuang W, Goeddel D, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989;246(4935):1306–1309. doi: 10.1126/science.2479986. [DOI] [PubMed] [Google Scholar]
  • 25.Liaw L, Almeida M, Hart C, Schwartz S, Giachelli C. Osteopontin promotes vascular cell adhesion and spreading and is chemotactic for smooth muscle cells in vitro. Circ Res. 1994;74(2):214–224. doi: 10.1161/01.res.74.2.214. [DOI] [PubMed] [Google Scholar]
  • 26.Liotta L, Steeg P, Stetler-Stevenson W. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell. 1991;64(2):327–336. doi: 10.1016/0092-8674(91)90642-C. [DOI] [PubMed] [Google Scholar]
  • 27.Liu B, Yi Y, Li L, Zhang S, Han H, Weng Y, Pan M. Arguside A: a new cytotoxic triterpene glycoside from the sea cucumber Bohadschia argus Jaeger. Chem Biodivers. 2007;4(12):2845–2851. doi: 10.1002/cbdv.200790234. [DOI] [PubMed] [Google Scholar]
  • 28.Machesky L. Lamellipodia and filopodia in metastasis and invasion. FEBS Lett. 2008;582(14):2102–2111. doi: 10.1016/j.febslet.2008.03.039. [DOI] [PubMed] [Google Scholar]
  • 29.McCawley L, Matrisian L. Matrix metalloproteinases: multifunctional contributors to tumor progression. Mol Med Today. 2000;6(4):149–156. doi: 10.1016/S1357-4310(00)01686-5. [DOI] [PubMed] [Google Scholar]
  • 30.Nabeshima K, Inoue T, Shimao Y, Sameshima T. Matrix metalloproteinases in tumor invasion: role for cell migration. Pathol Int. 2002;52(4):255–264. doi: 10.1046/j.1440-1827.2002.01343.x. [DOI] [PubMed] [Google Scholar]
  • 31.Nagao N, Etoh T, Yamaoka S, Okamoto T, Miwa N. Enhanced invasion of Tax-expressing fibroblasts into the basement membrane is repressed by phosphorylated ascorbate with simultaneous decreases in intracellular oxidative stress and NF-κB activation. Antioxid Redox Sign. 2000;2(4):727–738. doi: 10.1089/ars.2000.2.4-727. [DOI] [PubMed] [Google Scholar]
  • 32.Pahl H. Activators and target genes of Rel/NF-κB transcription factors. Oncogene. 1999;18(49):6853–6866. doi: 10.1038/sj.onc.1203239. [DOI] [PubMed] [Google Scholar]
  • 33.Pasco S, Brassart B, Ramont L, Maquart F, Monboisse J. Control of melanoma cell invasion by type IV collagen. Cancer Detect Prev. 2005;29(3):260–266. doi: 10.1016/j.cdp.2004.09.003. [DOI] [PubMed] [Google Scholar]
  • 34.Rao J. Molecular mechanisms of glioma invasiveness: the role of proteases. Nat Rev Cancer. 2003;3(7):489–501. doi: 10.1038/nrc1121. [DOI] [PubMed] [Google Scholar]
  • 35.Sanceau J, Truchet S, Bauvois B. Matrix metalloproteinase-9 silencing by RNA interference triggers the migratory-adhesive switch in Ewing’s sarcoma cells. J Biol Chem. 2003;278(38):36537–36546. doi: 10.1074/jbc.M304300200. [DOI] [PubMed] [Google Scholar]
  • 36.Shibata A, Nagaya T, Imai T, Funahashi H, Nakao A, Seo H. Inhibition of NF-κB activity decreases the VEGF mRNA expression in MDA-MB-231 breast cancer cells. Breast Cancer Res Tr. 2002;73(3):237–243. doi: 10.1023/A:1015872531675. [DOI] [PubMed] [Google Scholar]
  • 37.Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst. 1990;82(13):1107–1112. doi: 10.1093/jnci/82.13.1107. [DOI] [PubMed] [Google Scholar]
  • 38.Steeg P. Tumor metastasis: mechanistic insights and clinical challenges. Nat Med. 2006;12(8):895–904. doi: 10.1038/nm1469. [DOI] [PubMed] [Google Scholar]
  • 39.Tan D, Kini R, Jois S, Lim D, Xin L, Ge R. A small peptide derived from Flt-1 (VEGFR-1) functions as an angiogenic inhibitor. FEBS Lett. 2001;494(3):150–156. doi: 10.1016/S0014-5793(01)02314-6. [DOI] [PubMed] [Google Scholar]
  • 40.Tian F, Zhang X, Tong Y, Yi Y, Zhang S, Li L, Sun P, Lin L, Ding J. PE, a new sulfated saponin from sea cucumber, exhibits anti-angiogenic and anti-tumor activities in vitro and in vivo. Cancer Biol Ther. 2005;4(8):874–882. doi: 10.4161/cbt.4.8.1917. [DOI] [PubMed] [Google Scholar]
  • 41.Tong Y, Zhang X, Tian F, Yi Y, Xu Q, Li L, Tong L, Lin L, Ding J. Philinopside A, a novel marine-derived compound possessing dual anti-angiogenic and anti-tumor effects. Int J Cancer. 2005;114(6):843–853. doi: 10.1002/ijc.20804. [DOI] [PubMed] [Google Scholar]
  • 42.Wang S, Liu H, Ren L, Pan Y, Zhang Y. Inhibiting colorectal carcinoma growth and metastasis by blocking the expression of VEGF using RNA interference. Neoplasia. 2008;10(4):399–409. doi: 10.1593/neo.07613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Watanabe H, Nakanishi I, Yamashita K, Hayakawa T, Okada Y. Matrix metalloproteinase-9 (92 kDa gelatinase/type IV collagenase) from U937 monoblastoid cells: correlation with cellular invasion. J Cell Sci. 1993;104(4):991–999. doi: 10.1242/jcs.104.4.991. [DOI] [PubMed] [Google Scholar]
  • 44.Westermarck J, Kähäri V. Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J. 1999;13(8):781–792. [PubMed] [Google Scholar]
  • 45.Yu Y, Li Q, Zhu Z. NF-κB as a molecular target in adjuvant therapy of gastrointestinal carcinomas. Eur J Surg Oncol. 2005;31(4):386–392. doi: 10.1016/j.ejso.2004.10.010. [DOI] [PubMed] [Google Scholar]
  • 46.Zhang S, Li L, Lin J, Lin H. Imbalance between expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in invasiveness and metastasis of human gastric carcinoma. World J Gastroenterol. 2003;9(5):899–904. doi: 10.3748/wjg.v9.i5.899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Zhang S, Tang H, Yi Y. Cytotoxic triterpene glycosides from the sea cucumber Pseudocolochirus violaceus . Fitoterapia. 2007;78(4):283–287. doi: 10.1016/j.fitote.2007.02.010. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Zhejiang University. Science. B are provided here courtesy of Zhejiang University Press

RESOURCES