Dextran Sulfate Effects EMT of Human Gastric Cancer Cells by Reducing HIF-1α/ TGF-β

Abstract

The peritoneal implant metastasis is one of the main pathway and main cause for high mortality for gastric cancer metastasis. Researchs show that epithelial-mesenchymal transition (EMT) playing essential role in modulating gastric cancer metastasis, and the expression of hypoxia inducible factor-1α (HIF-1α) can promote EMT in tumor cells. This research aims to explore the influence and mechanism of Dextran Sulfate (DS) affecting EMT of human gastric cancer. In the present study, we found that DS can enter into the cytoplasm and function in it. Inhibition of HIF-1α or DS significantly inhibit the migration and invasion of human gastric cancer cells, and decrease the mRNA and protein expressions of HIF-1α, matrix metalloproteinase-2 (MMP-2), transforming growth factor-β (TGF-β), Twist and N-cadherin (N-cad), rise E-cadherin (E-cad) expression, DS with HIF-1α knockdown has a stronger effect. In vivo studies indicated that compared with using DS or HIF-1α knockdown alone, DS with HIF-1α knockdown can better suppress the volume and number of metastatic tumors, and reduce the mRNA and protein expressions of HIF-1α, MMP-2, TGF-β, Twist and N-cad in metastatic tumor tissues of nude mice. We further demonstrated that the expression of HIF-1α, MMP-2, TGF-β , Twist and N-cad were higher in well and poorly differentiated gastric cancer than paracancerous tissue, and poorly differentiated gastric cancer were even higher, while E-cad expression was opposite. Taken together, this study shows that DS can interfere the expression of HIF-1α, thereby inhibiting TGF-β-mediated EMT of gastric cancer cells, and demonstrated a promising application of DS in gastric cancer therapy.

Keywords: Dextran sulfate; EMT.; HIF-1α; Human gastric cancer.

Figure 1

(A) Western blot shows the HIF-1α expression level in gastric cancer cells and normal gastric epithelial cell (*P<0.001 compared with GES-1 group). (B) Fluorescence microscopy observed that DS entered the cytoplasm of human gastric cancer cells (HGC-27) 19 h and 24 h after DS intervention. (C) Western blot showed that the HIF-1α was successfully knockdown in human gastric cancer cells (ns, **P<0.01, ***P<0.001 compared with Control group).

Figure 2

Transwell experiment detect the ability of cell invasion and metastasis in each group (**P<0.01, ***P<0.001, ****P<0.0001 compared with Control or shNC group).

Figure 3

QRT-PCR detect the mRNA expression of HIF-1α, MMP-2, TGF-β, Twist, N-cad, E-cad in each group of cells (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with Control group; #p<0.05, ##p<0.01, ###p<0.001, ###p<0.0001 compared with shHIF-1α group).

Figure 4

Western blot detect the protein expression of HIF-1α, TGF-β, MMP-2, Twist, N-cad, E-cad in each group of cells (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with Control group; #p<0.05, ##p<0.01, ###p<0.001, ###p<0.0001 compared with shHIF-1α group).

Figure 5

(A) Observation of the abdominal cavity metastasis in nude mice in each group. (B) Immunohistochemistry detect the expression of HIF-1α, MMP-2, TGF-β, Twist, N-cad, and E-cad in metastatic tumor tissues of nude mice (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with Control group; ##p<0.01 compared with shHIF-1α group).

Figure 6

(A) QRT-PCR detect mRNA expression of HIF-1α, MMP-2, TGF-β, Twist, N-cad, E-cad in metastatic tumor tissues of nude mice (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with Control group; #p<0.05, ##p<0.01, ###p<0,0001 compared with shHIF-1α group). (B) Western blot detect the expression of HIF-1α, MMP-2, TGF-β, Twist, N-cad, and E-cad proteins in metastatic tumor tissues of nude mice (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with Control group; #p<0.05, ##p<0.01, ###p<0.0001 compared with shHIF-1α group).

Figure 7

Immunohistochemistry detect the expression of HIF-1α, MMP-2, TGF-β, Twist, N-cad, and E-cad in gastric cancer tissues (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).