Metastasis is considered to be the major cause of mortality in patients with cancer, and gastric cancer is a highly metastatic cancer. In the present study, the anti-metastatic activity of chitooligosaccharide (COS) in human gastric cancer cells and its underlying mechanism were investigated. It was found that COS significantly inhibited SGC-7901 cell proliferation and metastasis in a dose-dependent manner, as observed by MTT, wound-healing and Transwell assays. Quantitative real-time polymerase chain reaction and western blot analysis indicated that COS could decrease the expression of cluster of differentiation 147 (CD147) and subsequently reduce matrix metalloproteinase-2 (MMP-2) expression. A clear dose-dependent inhibition of MMP-2 activity was also observed in SGC-7901 cells following treatment with COS in gelatin zymography experiments. Furthermore, overexpression of CD147 (when transfected with pEGFP-C1 plasmid) in SGC-7901 cells partially protected against COS-induced inhibition of MMP-2. The results of the present study demonstrated the potential of COS in suppressing gastric cancer metastasis, and that the CD147/MMP-2 pathway may be involved as the key mechanism of its anti-metastatic effect.
Being a worldwide malignant liver tumor, gastric cancer ranks the fourth in frequency among common human solid tumors and the second leading cause of cancer-related mortality (
Matrix metalloproteinases (MMPs) have been regarded as major critical molecules that assist tumor cells during metastasis. MMPs, also designated matrixins, are proteinases that participate in extracellular matrix (ECM) degradation. They selectively cleave polypeptide bonds in ECM and remodel structural proteins that are essential for the maintenance of connective tissue integrity, such as collagens, aggrecan, fibronectin, proteoglycan and laminin. To date, 24 different vertebrate MMPs have been identified, 23 of which are found in humans (
Cluster of differentiation 147 (CD147), a 55-kDa transmembrane glycoprotein, is located on the surface of human tumor cells and normal keratinocytes (
Chitooligosaccharide (COS) is a natural alkaline polysaccharose, an oligosaccharide formed by 2–10 amino-glucoses through 1,4-glucosidic bond connection (
COS (1 kDa<MW<3 kDa) was obtained from the Dalian Institute of Chemical Physics, the Chinese Academy of Sciences (Dalian, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and Transwell chambers were purchased from Corning Inc. (Corning, NY, USA). All reagents used for cell culture were obtained from Gibco BRL (Grand Island, NY, USA). Monoclonal goat anti-human CD147, monoclonal rabbit anti-human MMP-2 and monoclonal mouse anti-human GADPH antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
The SGC-7901 human gastric cancer cell line was obtained from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). AGS and NCI-N87 human gastric cancer cells were purchased from American Type Culture Collection (Rockville, MD, USA). All the cells were grown in RPMI-1640 (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal calf serum at 37°C in a 5% CO2 incubator.
For CD147 overexpression, CD147 was constructed into a pEGFP-C1 eukaryotic expression vector (Clontech, Heidelberg, Germany). Transfection was carried out using Lipofectamine 2000 (Invitrogen Life Technologies), according to the manufacturer’s instructions. The cells were selected in the presence of 500 mg/l G418 (Invitrogen Life Technologies). Stable clones were selected for at least 4 weeks before single colonies were picked and analyzed for CD147 expression by western blotting. The empty vector pEGFP-C1 was also transfected into SGC7901 cells, which served as the control group.
Cells were seeded into 96-well plates at a density of 1×105 cells/100 μl/well. After 24 h growth, cells were treated with different concentrations of COS (50, 100, 250, 500 and 1,000 μg/ml) for 24, 48 and 72 h. Following the addition of 100 μl MTT (0.5 mg/ml; Sigma-Aldrich), cells were incubated at 37°C for 1 h. The formazan deposits that formed were solubilized in DMSO, and the absorbance of each well was measured at 570 nm in an EMax Precision microplate reader (Molecular Devices Instruments, Sunnyvale, CA, USA).
Cells were cultured in a 24-well plate with 100% confluency. A micro-pipette tip was used to scratch a line in the cell monolayer. The medium was removed and the monolayer was washed with warm phosphate-buffered saline three times. Then growth medium containing different concentrations of COS was added to each well. Following incubation for 48 h, cell migration was observed and photographs were taken under a light microscope (Olympus BX41; Olympus Corporation, Tokyo, Japan).
Cell migration through Matrigel-coated filters was measured by using Transwell chambers (Costar Corporation, Tewksbury, MA, USA) with 8-μm-pore polycarbonate filters coated with Matrigel matrix. SGC-7901 cells were seeded in the upper compartment of each invasion chamber and incubated in the presence of COS for 48 h. The lower well was filled to the top (500 μl) with RPMI-1640 containing 10% fetal calf serum (Hangzhou Sijiqing Bioengineering Material Co., Ltd., Hangzhou, China) as a chemoattractant. Subsequently, non-migrating cells on the upper surface of the membrane were removed by gently scrubbing with a cotton swab, and the invading cells on the lower surface were fixed with 100% methanol and stained with crystal violet (0.1%; Beyotime Institute of Biotechnology, Nantong, China). The number of cells was counted under a light microscope (Olympus BX41; Olympus Corporation) at a magnification of ×100. Images were captured and subjected to computer-assisted image analysis using a computer coupled to the Olympus BX41 light microscope (Olympus Corporation) using AnalySis software (Olympus Corporation).
Total RNA of cells with or without COS intervention was extracted using TRIzol reagent (Invitrogen Life Technologies). Subsequently, 2 μg of total RNA was reverse transcribed in a total volume of 20 μl containing 200 units of SuperScript II RNase H- Reverse Transcriptase (Invitrogen Life Technologies), 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 500 μM dNTPs (each), 500 ng oligo (dT) 23 primer and 40 units of RNaseOUT, at 42°C for 50 min. This was then followed by inactivating at 70°C for 15 min. qPCR was conducted by LightCycler 480 SYBR Green I Master (Roche Diagnostics, Indianapolis, IN, USA) with 1 mM primers. The expression of GADPH was used as endogenous control for the normalization of gene expression. All reactions were performed in duplicate. The forward and reverse primers were 5′-CCCCAAAACGGACAAAGAC-3′ and 5′-CTTCAGCACAAACAGGTTGC-3′, respectively, for human MMP-2; 5′-CCATGCTGGTCTGCAAGTCAG-3′ and 5′-CCGTTCATGAGGGCCTTGTC-3′, respectively, for human CD147; and 5′-CCAACCGCGAGAAGATGA-3′ and 5′-CCAGAGGCGTACAGGGATAG-3′, respectively, for human GADPH.
Whole cell lysates from cultured cells were harvested with cell lysis buffer. Equal amounts of protein (20 μg) were separated by SDS-PAGE produced in-house and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skimmed milk in Tris-buffered saline and then incubated with primary antibodies for 1 h at room temperature. Horseradish peroxidase-conjugated monoclonal goat anti-rabbit, goat anti-mouse and donkey anti-goat secondary antibodies and an enhanced chemiluminescence kit (all Beyotime Institute of Biotechnology) were used for detection.
Gelatin zymography was performed to determine the activity of MMP-2. Briefly, protein in medium was then separated in 10% SDS-PAGE gel containing 1 mg/ml gelatin (Sigma-Aldrich) at 4°C. After running, the gel was incubated in 2.5% Triton X-100 (Beyotime Institute of Biotechnology) in deionized water for renaturing with gentle agitation for 30 min at room temperature. Subsequently, the gel was incubated in developing buffer (50 mM Tris-HCl, 0.2 M NaCl, 5 mM CaCl2 and 0.02% Brij35) overnight with gentle shaking. The gel was stained with Coomassie blue R-250 (Beyotime Institute of Biotechnology) for 30 min and then washed. Gelatinolytic bands were observed as clear zones against the blue background and the intensity of the bands was evaluated using ImageMaster software (Amersham Pharmacia Bioscience, NJ, USA).
Data are expressed as the mean values ± standard deviation from at least three experiments. Statistical comparisons were based on Student’s t-test or analysis of variance. P<0.05 was considered to indicate a statistically significant difference.
To investigate the antiproliferative effect of COS on various human gastric cancer cells, an MTT assay was carried out following treatment with different concentrations of COS ranging from 50 to 1000 μg/ml. As shown in
In order to further assess the influence of COS on SGC-7901 cells, cell migration and invasion assays were employed to determine these two key factors of malignant progression and metastasis. The wound healing assay showed that treatment of SGC-7901 cells with increasing concentrations of COS after 48 h led to a concentration-dependent decrease in wound-healing cell migration (
Based on the abovementioned results, further studies were carried out to examine the inhibitory effect of COS on CD147 mRNA and protein expression in SGC-7901 cells. Total RNA of cells was isolated and qPCR was performed as described in Materials and methods. As shown in
Increased MMP-2 activity is considered to be important for the increased capability of gastric cancer cells to traverse the membrane and invade and metastasize to distant sites (
To further elucidate the mechanism by which COS mediates MMP-2 inhibition through CD147, exogenous CD147 was introduced into SGC-7901 cells. As shown in
Chitosan, which is a linear polysaccharide composed of randomly distributed β-(
Expression of various MMPs has been found to be upregulated in virtually every type of human cancer and correlates with advanced stage, invasive and metastatic properties and, in general, poor prognosis. Further upregulation of MMP expression, in particular the gelatinases, which can degrade basement membrane components, allows the tumor cells to invade into the adjacent stroma and to break down the basement membranes associated with capillaries and lymphatic vessels, allowing the tumor cells to enter the circulation (
As a tumor-associated antigen, CD147 forms homo-oligomers in both heterotypic and homotypic cell-cell interactions to induce production of MMPs. The functional importance of CD147 has been demonstrated to be associated with its ability to stimulate MMP expression. CD147 can induce the production of MMP-1, MMP-2, MMP-3, MMP-9, MMP-14, and MMP-15 (
As the regulation of gene expression of MMP-2 is controlled by CD147, the current study next investigated whether overexpression of CD147 could attenuate the effect of COS on MMP-2 inhibition. It was found that this effect could be effectively attenuated by CD147 overexpression, suggesting that the downregulating role of COS in MMP2 expression may be through the upregulation of CD147. Further characterization of the effect of COS on gastric cancer invasion and metastasis may lead to the identification of new diagnostic markers and therapeutic targets. We propose that these results may apply to a number of additional cancer types other than gastric cancer, as CD147 and MMP-2 are frequently upregulated in numerous other cancer types as well.
In conclusion, to the best of our knowledge, the present study is the first to demonstrate that COS exhibits an anti-metastatic effect in human gastric cancer. The effects of COS on cell invasion and migration ability may be achieved through the inhibition of MMP-2 expression by decreasing CD147. Overexpression of CD147 partially protected against COS-mediated inhibition of MMP-2. Overall, our findings have demonstrated the potential of COS in suppressing gastric cancer metastasis and that the CD147/MMP-2 pathway may be involved as the key mechanism of its anti-metastatic effect.
This study was supported by grants from the Young Talent Project of Hubei Provincial Education Department (Q200724004), Outstanding Youth Scientific Innovation Team of Hubei University of Medicine (2011CXG03), Scientific Research Foundation of Hubei University of Medicine (nos. 2010QDJ20 and 2010QDJ21).
Effects of different concentrations of COS on cell viability in cultured NCI-N87, AGS and SGC-7901 cells. The proliferation of three cell lines were quantified by the cell survival rate after 24, 48 and 72 h of incubation with concentrations of COS ranging from 50 to 1000 μg/ml, assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Data are presented as means of values ± SD from three independent experiments. *P<0.05 vs. 0 μg/ml COS treatment group. COS, chitooligosaccharide.
COS inhibits SGC-7901 cell migration and invasion. (A) COS markedly reduces SGC-7901 cell migration in dose-dependent manner. (B) Representative photographs of invasive SGC-7901 cells on the membrane (magnification, ×100). (C) Average number of invasive SGC-7901 cells. (*P<0.05, compared with the 0 μg/ml COS treatment group). COS, chitooligosaccharide.
COS represses the mRNA and protein expression of CD147 and MMP-2 in dose-dependent manner. SGC-7901 cells were treated for 48 h with COS and analyzed for the indicated (A) mRNA by quantitative real-time polymerase chain reaction and (B) protein by western blotting. The results represent the mean ±SD of experiments performed in triplicate. (*P<0.05, compared with the 0 μg/ml COS treatment group). COS, chitooligosaccharide; CD147, cluster of differentiation 147; MMP-2, matrix metalloproteinase-2.
COS treatment downregulates the activity of MMP-2 in SGC-7901 cells. (A) SGC-7901 cells were treated with various concentrations of COS for 48 h, and the activity of MMP-2 was evaluated by gelatin zymography. (B) Areas and relative intensities of gelatin-digested bands by MMP-2 were quantified by densitometry and expressed as relative activity. The results represent mean ± SD of experiments performed in triplicate. (*P<0.05, compared with the 0 μg/ml COS treatment group). COS, chitooligosaccharide; MMP-2, matrix metalloproteinase-2.
CD147 overexpression could attenuate the effect of COS on MMP-2 inhibition. SGC-7901 cells were treated for 48 h with 1000 μg/ml COS and analyzed for the indicated protein by western blotting. GADPH was used as the internal control. All assays were performed in triplicate. CD147, cluster of differentiation 147; COS, chitooligosaccharide; MMP-2, matrix metalloproteinase-2.