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Dextran sulfate inhibition on human gastric cancer cells invasion, migration and epithelial-mesenchymal transformation

The objective of the present study was to observe the influence of dextran sulfate (DS) on the proliferation, invasion and migration of AGS, BGC-23, GES-1, MGC-803 and SGC-7901 cells. Additionally, the possible inhibition mechanism of DS on BGC-823 cells epithelial-mesenchymal transition (EMT) was explored. The cells in the control and experimental group were treated with PBS and DS respectively. The effect of DS on the invasion and migration of these five types of cells were investigated using Transwell invasion and migration assays. Immunocytochemistry, western blotting and reverse transcription-polymerase chain reaction (RT-PCR) assays were used to measure gene and protein expression of hypoxia-inducible factor 1α (HIF1-a) and EMT associated factors [Twist, E-cadherin, N-cadherin and β-catenin] of BGC-823 cells. According to the results of CCK-8, DS significantly decreased the proliferation of AGS, SGC-7901 and BGC-823 cells to different extents, but there were no notable differences for MGC-803 cells. Transwell migration and invasion results demonstrated that, compared with the control group, DS reduced the migration and invasion of every types of cells to different extents, and the inhibition to BGC-823 cells invasion is the most notably. Immunofluorescence, RT-PCR and western blot analysis results indicated that HIF-1α, Twist and N-cad expressions levels had different degrees of reduction in the experimental group following DS treatment; however, the expression level of E-cad had increased. In conclusion, DS inhibited the proliferation of AGS, BGC-823, SGC-7901 and GES-1 cells, the inhibition degree may be associated with the differentiation degree of every cancer cell, the higher the differentiation degree, the stronger the inhibition. DS inhibited migration and invasion of the five types of gastric cancer cells in different degree. DS may inhibit EMT of BGC-823 by inhibiting Wnt signaling.


Introduction
Morbidity rate and mortality rate of gastric cancer are the highest for digestive tract tumors globally, and the results indicate a younger trend (1,2). Drug therapy is the main treatment option for intra-abdominal planting transfer of gastric cancer; however, thus far the curative effect is still not sufficient. Dextran sulfate (DS) is a macromolecule Dextran with a molecular weight of 500,000. Previous studies have demonstrated that DS can inhibit cancer cells adhesion, cell cycle progression and gene expression (3,4), DS can reduce of expression of gastric cancer cells in terms of vascular endothelial growth factor (VEGF) and integrin β1 in vivo and in vitro, and there was a significant inhibitory effect on gastric cancer cell adhesion and angiogenesis (5), but the inhibiting mechanism of DS on the metastasis of gastric cancer still requires investigating.
Peritoneal transfer of gastric cancer is a multi-factor, multi-phase continuous complex process. In this process, epithelial-mesenchymal transformation (EMT) serves a key role in promoting the invasion and metastasis of tumor cells (6). EMT mechanism is a complicated process, and hypoxia is one of the most important initiating factors to EMT (7), In a hypoxic microenvironment, cancer cells adapt to the hypoxic microenvironment through highly expressing hypoxia-inducible factor 1α (HIF-1α) (8), HIF-1α high expression then promote: i) EMT markers, including Twist, E-cad and N-cad; ii) the regulation of angiogenesis, including vascular endothelial growth factor (VEGF); iii) cell adhesion, including ITGβ1 (9,10). At present, the examination of EMT, invasion and migration mechanisms is the notable basis for the development in the diagnosis and treatment of gastric

Dextran sulfate inhibition on human gastric cancer cells invasion, migration and epithelial-mesenchymal transformation
cancer, and additionally helps to provide potential intervention targets for the treatment of a malignant tumor (11). The present study investigated the inhibiting effect of DS on EMT by measuring the expression of EMT associated factors [Twist, E-cadherin (E-cad), N-cadherin (N-cad) and β-catenin], proliferation, invasion and migration of 4 types of different degree of differentiate gastric cancer cells in the process of peritoneal metastasis, and then suggested a theoretical basis for discovering a potential drug in the treatment of peritoneal metastasis of gastric cancer. Following an incubation at 37˚C for 0.5 h, DAPI was added in the dark, the film was sealed and observed using a fluorescence microscope with a magnification of x100. Cumulative density demonstrated the expression quantity of the factors, which was analyzed by Student's t-test using GraphPad Prism 7 plotting software (GraphPad Software, Inc., La Jolla, CA, USA).   Following prolonged culture in oxygen, the control group HIF-1α expression increased gradually after 8 h, and at 8, 12 and 24 h, the expression significantly decreased in the experimental group, compared with the control group. In the control cells after 12 h, E-cad had a significantly lower expression, N-cad was significantly upregulated, DS significantly slowed down in the reduction of E-cad, and there was an increase of N-cad at 12 and 24 h. β-catenin expression in control cells increased gradually after 8 h, with a gradual increase in the cytoplasm and nuclei, and relative decrease in the nucleus and cytoplasm of experimental group, at 8, 12 and 24 h. Additionally, DS significantly reduced the β-catenin expression of the BGC-823 cells, as depicted in Fig. 3.

RT-PCR detection of HIF-1α and EMT associated factors mRNA expression in BGC-823 cells.
When hypoxia time was extended, in the control group the cell HIF-1α, Twist and N-cad, β-catenin mRNA level gradually increased, whereas E-cad mRNA level gradually reduced. At 2, 8, 12 and 24 h, the mRNA level of HIF-1α and Twist in the experimental group was significantly lower, compared with the control group, and the experimental group E-cad mRNA level was significantly increased, compared with the control group. At 2, 8 and 12 h, the N-cad and β-catenin mRNA level of the experimental group increased significantly, compared with the control group.
Following prolonged culture in oxygen, in the control group cells the mRNA level of HIF-1α, Twist, N-cad and β-catenin all demonstrated an increasing trend, whereas the E-cad mRNA level gradually reduced. At 8, 12 and 24 h, the mRNA levels of HIF-1α, Twist and β-catenin in the experimental group were significantly decreased, compared with the control group, and the experimental group E-cad mRNA level was significantly increased, compared with the control group. At 12 and 24 h, the N-cad mRNA level of the experimental group was significantly increased, compared with the control group, as depicted in Figs. 4 and 5.

Western blot detection of EMT associated factors protein expression in BGC-823 cells.
In hypoxia environment, as the duration of hypoxia increased, HIF-1α, Twist, N-cad and β-catenin expression gradually increased in the control group, and HIF-1α, N-cad and β-catenin expression of the experimental group was significantly lower, compared with the control group, at 2, 8, 12 and 24 h. At 8, 12 and 24 h, Twist  expression of the experimental group was significantly lower, compared with the control group, and the expression of E-cad increased significantly, compared with the control group.
In normoxia environment, as the duration of normoxia increased, in the control group HIF-1α, Twist, β-catenin and N-cad expression gradually increased, E-cad decreased at the time point of 12 h. At 8, 12 and 24 h, HIF-1α, Twist and β-catenin expression in the experimental group was significantly lower, compared with the control group. At 12 and 24 h, the experimental group N-cad expression was significantly lower than the control group, and the expression of E-cad increased significantly, compared with the control group, as depicted in Figs. 6 and 7.

Discussion
The prevention and treatment of proliferation, invasion and metastasis of gastric cancer is one of the problems that require further study.
The present study used the gastric cancer cell lines AGS, BGC-823, MGC-803 and SGC-803 and normal gastric mucosa cells GES-1 to detect the effects of DS on proliferation, invasion and migration. The cell lines MGC-803 (undifferentiated), BGC-823 (low differentiation) and AGS (high differentiated) are all derived from gastric cancer tumors, and the SGC-7901 cell line was derived from lymph node metastasis (moderately differentiated). GES-1 is derived from human fetal gastric mucosa epithelial cells (immortalized) (12).
One in vitro study has demonstrated that DS can inhibit cancer cell adhesion, cell cycle progression and gene expression (3). In the present study, using a CCK-8 proliferation test, it was determined that at 6     inhibitory effect on MGC-803 cells. It may be safely concluded that DS inhibited the highly differentiated gastric cancer cell AGS most, as the reduction of the AGS cell number was the most significant, whilst it had no significant proliferation inhibition effect on the undifferentiated gastric cancer cell line MGC-803; Therefore, it was speculated that gastric cancer cell proliferation inhibition by DS may be associated with differentiation degree.
Tumor cells with different degrees of differentiation have varying invasion and migration abilities (13). Research has indicated that gastric cancer cell line BGC-823 has an improved invasion ability, compared with the cell lines AGS, SGC-7901 and GES-1 (14). The Transwell migration experiment in the present study demonstrated that migrated numbers of the four gastric cancer cell lines were notably more than the GES-1, the four gastric cancer cells lines had an improved ability to migrate, compared with the than normal gastric mucosa cells. Calculating the average gastric cancer cells that migrated, the undifferentiated gastric cancer cells MGC-803 had the highest, and then, in turn, the low differentiated gastric cancer cells BGC-823, moderately differentiated gastric cancer cells SGC-7901 and high differentiated gastric cancer cell AGS. This demonstrated that the lower degree of differentiation, the more cells that migrated across the membrane, indicating an improved migration ability. Significantly, DS reduced all five cell lines migration count, compared with the control group. The cell line with the most notable inhibitory effect was AGS, followed by SGC-7901, MGC-803, BGC-823 and GES-1. So it was speculated that DS may have an inhibitory effect on migration ability of all five types of cell lines, which may be associated with the degree of differentiation.
The primary difference between the invasion and migration experiment was the layer of Matrigel. Cells must penetrate the matrix firstly, then through the chamber on the underside of the membrane filter. The invasion experiment indicated that the average invaded cells was greatest for the undifferentiated MGC-803 gastric cancer cells, and then, in turn, the poorly differentiated gastric cancer cells BGC-823, moderately differentiated gastric cancer cells SGC-7901 and high differentiated gastric cancer cell AGS; therefore, the lower degree of differentiation, the stronger the cell invasion ability. Compared with control group, DS significantly reduced the invasion of all five types of cells in the experimental group, and it has the most notable inhibiting effect on BGC-823 cells, followed by the AGS, MGC-803, SGC-7901 and GES-1. It was determined that the strength of inhibition had no notable association with cell differentiation. A previous study demonstrated that matrix metalloproteinases secreted by tumor cells is the main component of degrading basement membrane and mesenchyme (15). The strength of inhibition by DS had a different order of cell lines for invasion and migration, which may be due to different abilities of these cells secreting matrix metalloproteinases and degrading extracellular matrix; in addition, this process could be influenced by DS in some degree for different cells, but the specific inhibition mechanism still requires further study. β-catenin serves an important role in the process of invasion and migration of tumor cells, connecting adhesion molecules outside the cell to the frame within cells, through mediating the interaction of E-cad and β-catenin involved in the cell adhesion, migration and transfer process (16). β-catenin is highly expressed in gastric cancer cells AGS, HGC-27, MGC-803 and BGC-823. By contrast, it has a lower expression in normal gastric mucosa cells (17). Abnormal expression of E-cad is closely associated with tumor invasion, metastasis and diffusion (18). E-cad and β-catenin expression in different degrees of differentiation of gastric cancer cells, to a certain extent, could reflect the ability of invasion and migration.
β-catenin signaling pathway, also known as the Wnt signaling pathway, contains β-catenin, which has a key role in this pathway. It combines with the E-cad intracellular area, producing the β-catenin/E-cad complex, then connects to the actin cytoskeleton, mediates adhesion between cells, thus regulating the invasion and metastasis of tumor cells (19). The loss of combined E-cad leads to the accumulation of intracellular β-catenin, causing an increasing in β-catenin within nuclear localization in diffuse-gastric cancer, and at the same time an immunohistochemical staining study has demonstrated the reduction in E-cad on membrane (20). Accordingly, immunofluorescence staining was conducted to observe the expression changes; the effect of DS on MGC-803 is not particularly evident using CCK-8, therefore DS effective cells (BGC-823) was selected for further study. The results indicated that, with time, the expression of HIF-1α, Twist and N-cad in the control group cells gradually increased, whereas the E-cad expression gradually reduced. Simultaneously, it was considered that the β-catenin expression gradually increased, including in the nuclei; however, DS significantly reduced the expression of β-catenin in cell nucleus. As a necessary endogenous signal, Wnt signaling pathways can also directly control the ability of HIF-1α to induce the occurrence of EMT (21); therefore, it was speculated that DS may use the Wnt pathway to inhibit the expression of HIF-1α.
In a hypoxic microenvironment, HIF-1α overexpression in cancer cells accelerates tumor growth and metastasis by inducing tumor cells secretion of VEGF, regulating angiogenesis and promoting EMT (22,23). A previous study confirmed that HIF-1α induced tumor cell EMT through regulating the transcription factor Twist, which inhibits the expression of E-cad and promotes the expression of N-cad in hypoxia (24). Another study demonstrated that DS can inhibit the HIF-1α expression on the greater momentum of the mice (5). In the present study, gastric cancer cell line BGC-823 was treated with DS and PBS, cultured in vitro in normoxia and hypoxia conditions, checked at various time points, observed for cell morphology changes. RT-PCR and western blot analysis were also conducted for further detection, the results demonstrated that DS have different degrees of inhibition on HIF-1α, Twist and N-cad and β-catenin, as well as stimulation of E-cad expression; therefore, DS may affect Twist and E-cad through HIF-1α, inhibiting the occurrence of EMT.
In conclusion, DS has different degrees of inhibition on the proliferation of cell lines AGS, BGC-823, SGC-7901 and GES-1, which may be associated with the degree of differentiation. DS has a different degree of inhibition effect on migration and invasion ability of all five types of cells. Furthermore, DS may inhibit HIF-1α through the Wnt signaling pathway, reducing the gene and protein level of Twist, increasing the epithelial marker E-cad and reducing the expression of mesenchymal marker N-cad, thus inhibiting EMT of gastric cancer cell line BGC-823. In order to further study the anticancer mechanism of DS in human gastric cancer metastasis, experiments in vivo will be carried out in the subsequent tests.