Fucoidan inhibits epithelial-to-mesenchymal transition via regulation of the HIF-1α pathway in mammary cancer cells under hypoxia

Abstract

This study examined the effects of fucoidan on epithelial-to-mesenchymal transition (EMT) in a human triple-negative breast cancer (TNBC) cell line in a hypoxic microenvironment. Transwell and wound-healing assays were performed to analyze the invasion and migration of MDA-MB-231 human mammary cancer cells, respectively. The expression levels of EMT markers and hypoxia-inducible factor-1α (HIF-1α) were detected through western blotting. Under hypoxia, fucoidan treatment inhibited proliferation of breast cancer cells. Fucoidan also suppressed the invasion and migration of MDA-MB-231 cells. Western blotting revealed that fucoidan treatment significantly reduced the protein expression levels of HIF-1α and HIF-1 target genes. Furthermore, the nuclear translocation and activity of HIF-1α were reduced. Fucoidan treatment significantly downregulated the expression levels of mesenchymal markers (N-cadherin and vimentin), but upregulated the expression levels of the epithelial markers zonula occludens-1 and E-cadherin. In addition, overexpression of HIF1-α protected cells from fucoidan-mediated suppression of migration and invasion. These data suggested that fucoidan may inhibit EMT in human TNBC cells via downregulation of the HIF1-α signaling pathway.

Keywords: epithelial-to-mesenchymal transition; fucoidan; hypoxia-inducible factor-1α; triple-negative breast cancer.

Figure 1.

Sequence information of the overexpression vector HIF-1α-pcDNA3.0. HIF-1α (NCBI Reference Sequence, NM_001530.4) was inserted between the EcoRI and XbaI restriction sites. HIF-1α, hypoxia-inducible factor-1α.

Figure 2.

Effect of fucoidan treatment on the proliferation of MDA-MB-231 cells under hypoxia. The effects of fucoidan on the proliferation of breast cancer cells under hypoxia were detected via MTT assay. Fucoidan treatment inhibited the proliferation of MDA-MB-231 cells (P<0.05). Following treatment with 25 µg/ml fucoidan for 72 h, cell proliferation declined by 53.2%. Data are presented as the means ± standard deviation. *P<0.05 vs. control (0 µg/ml fucoidan); ^#P<0.05 vs. 6.25 µg/ml fucoidan.

Figure 3.

Fucoidan inhibits the migration and invasion of breast cancer cells. (A) A scratch assay was used to determine the migratory ability of breast cancer cells. Under a microscope, the relative wound closure was detected and images were captured (magnification, ×100). Cell motility was quantified based on the wound width. Fucoidan treatment inhibited the migratory ability of breast cancer cells (P<0.001). (B) Transwell assays were used to determine the invasive ability of MDA-MB-231 cells. Representative fields showing invasive cells on the membrane are presented (magnification, ×200). Following treatment with 12.5 or 25 µg/ml fucoidan, cell migration was inhibited. All experiments were repeated three times. Data are presented as the means ± standard deviation. *P<0.05 vs. control; ^#P<0.05 vs. 6.25 µg/ml fucoidan.

Figure 4.

Effects of fucoidan on the expression levels of EMT markers. Following treatment with fucoidan, membrane and cytosolic extracts were prepared to measure the expression levels of EMT markers by western blot analysis. Fucoidan treatment significantly upregulated the expression levels of the epithelial markers ZO-1 and E-cadherin, but downregulated the expression levels of mesenchymal markers (N-cadherin and vimentin). All experiments were repeated three times. Data are presented as the means ± standard deviation. *P<0.05 vs. control; ^#P<0.05 vs. 6.25 µg/ml fucoidan; ^ΔP<0.05 vs. 12.5 µg/ml fucoidan. ZO-1, zonula occludens-1.

Figure 5.

Fucoidan inhibits HIF-1α protein activation and nuclear accumulation. (A) Effects of fucoidan on the expression and nuclear translocation of HIF-1α under hypoxic conditions were evaluated by western blotting. Fucoidan treatment inhibited the nuclear translocation of HIF-1α. (B) Activation of HIF-1α was detected. Fucoidan treatment decreased the activation of HIF-1α. All experiments were repeated three times. Histone H3 was used as a reference for nuclear protein determination. Data are presented as the means ± standard deviation. *P<0.05 vs. control; ^#P<0.05 vs. 6.25 µg/ml fucoidan. HIF-1α, hypoxia-inducible factor-1α.

Figure 6.

Fucoidan downregulates the protein expression levels of hypoxia-inducible factor-1α target genes. Western blotting suggested that fucoidan treatment inhibited the expression levels of TWIST, Snail, CAIX and GLUT-1. Data are presented as the means ± standard deviation. *P<0.05 vs. control; ^#P<0.05 vs. 6.25 µg/ml fucoidan. CAIX, carbonic anhydrase IX; GLUT-1, glucose transporter protein type-1.

Figure 7.

Overexpression of HIF-1α attenuates fucoidan-mediated suppression of cell invasion and migration. (A) Breast cancer cells were transfected with HIF-1α overexpression plasmid (HIF-1α-pcDNA3.0). Cell lysates from different groups were prepared and probed for HIF-1α and its target genes, including TWIST and Snail, through western blotting. (B) HIF-1α mRNA expression in breast cancer cells was measured by reverse transcription-quantitative polymerase chain reaction. GAPDH was used as s reference. Post-transfection with HIF-1α-pcDNA3.0, the mRNA expression levels of HIF-1α were increased. Data are presented as the means ± standard deviation. ^aP<0.05 vs. empty plasmid. (C) Scratch assay (magnification, ×100). (D) Transwell assay (magnification, ×200). Breast cancer cells were transfected with HIF-1α-pcDNA3.0 and further treated with 25 µg/ml fucoidan. Migration and invasion of cancer cells were detected after 24 h. Data are presented as the means ± standard deviation. *P<0.05 vs. fucoidan + empty plasmid. CAIX, carbonic anhydrase IX; GLUT-1, glucose transporter protein type-1; HIF-1α, hypoxia-inducible factor-1α.