Fucoidan inhibits lymphangiogenesis by downregulating the expression of VEGFR3 and PROX1 in human lymphatic endothelial cells

Abstract

Lymphangiogenesis is one of the promoters of tumor lymphatic metastasis. Fucoidan which is a fucose-enriched sulfated polysaccharide has effect on various pharmacological activities including anti-metastasis activity. However, the inhibitory effect of fucoidan on lymphangiogenesis remains unclear. Here, fucoidan extracted from U. pinnatifida sporophylls suppressed HLECs proliferation, migration and tube-like structure formation, and had inhibitory effect of tumor-induced lymphangiogenesis in vitro. Additionally, we found that fucoidan had a dose-dependent depressive effect on the expressions of PROX1, vascular endothelial growth factor receptor 3 (VEGFR3), NF-κB, phospho-PI3K and phospho-Akt in HLECs. Moreover, anti-lymphangiogenesis effect of fucoidan was assessed by using mouse tumor model. In summary, fucoidan inhibit tumor lymphangiogenesis and lymphatic metastasis by suppressing the NF-κB/PI3K/Akt signaling pathway through reduced levels of PROX1 and VEGFR3.

Keywords: PROX1; fucoidan; lymphangiogenesis; lymphatic endothelial cells; lymphatic metastasis.

Figure 1. Effects of fucoidan on viability of HLECs

Cell viability was detected in each group after 48 and 72 h in culture. Values are the mean ± SD from three independent experiments. **p < 0.01 versus the untreated control (two-way ANOVA).

Figure 2. Effect of fucoidan on the cell cycle distribution in HLECs

(A) Flow cytometry histograms and (B) cell cycle distributions for the G0/G1, S and G2/M phases assessed via flow cytometry, showing the G0/G1 phase was blocked by fucoidan after 48 h, inducing a decrease of cells in the S phase. (C) Effect of fucoidan on the protein expression levels of CDK4 and cyclin D1. Values are mean ± SD. *p < 0.05 and **p < 0.01 versus the untreated control (two-way ANOVA).

Figure 3. Fucoidan inhibits HLEC migration in vitro

(A) Microscopy images (scale bar is 50 μm) and (C) number of migrated cells from the Transwell assay (migrated cells were stained and counted). The average value was calculated from five fields. Mean ± SD is shown; **p < 0.01 versus the untreated control (one-way ANOVA). (B) Quantification of wound healing areas was performed by using an ImageJ program. The graphs represent the means ± SD of triplicate experiments. (D) Wound scratch assays visually show the changes in cell migration with different concentrations of fucoidan over time (scale bar is 100 μm).

Figure 4. Fucoidan inhibits lymphatic tube formation in HLECs on matrigel and regulates cytoskeleton polarization of HLECs

HLECs were incubated for 24 h in medium with or without fucoidan (0, 100, 200 and 400 μg/ml). (A) Photomicrographs of tube formation in HLECs (scale bar is 50 μm). The symbols (_*) on the figure represents complete tubular structures. (B) Quantitative analysis of the rate from the number of tubes. Data represented as mean ± SD; **p < 0.01 versus the untreated control (one-way ANOVA). (C) Cells were incubated in medium with or without fucoidan (0, 100, 200 and 400 μg/ml) for 24 h. Cells were stained with phalloidin (green) and DAPI (blue), and observed (magnification, ×100).

Figure 5. Fucoidan downregulates the expression levels of VEGFR-3 and PROX1 and VEGFR3-related signaling pathways

(A) Microscopy images (scale bar is 50 μm) showing the immunofluorescence staining, which was performed with TRITC-labeled anti-VEGFR3 and anti-PROX1 (red) and DAPI (blue). (B) The protein expression levels of PROX1 and VEGFR3 were measured by western blot analysis; The transcription levels of PROX1 and VEGFR3 measured by RT-PCR. (C) Protein levels of p-PI3K, p-Akt and NF-κB in fucoidan-treated or control groups.

Figure 6. Fucoidan inhibits tumor-induced lymphangiogenesis in vitro

(A) The ideograph of transwell co-culture system. MHCC97H cells and HLECs were cultured in insert wells and lower chambers, respectively. (B) Photomicrographs of tube formation in HLECs and the quantitative analysis shown on the right. NC stands for the negative control which HLECs cultured alone. Mean ± SD is shown; ^#p < 0.05 versus the NC group and **p < 0.01 versus the co-culture control (one-way ANOVA).

Figure 7. Fucoidan inhibits lymphangiogenesis in vivo

Top: Immunofluorescence images of tumor lymphatic vessels from mice inoculated with Hca-F cancer cells. VEGFR3 antibody (green) and LYVE-1 antibody (red) were used in the assay. Bottom: Quantitative assessment of tumor LVD and tumor weight. Mean ± SD is shown; *p < 0.05 and **p < 0.01 versus the control.