Malignant tumor metastasis, the process by which cancer spreads, is a crucial aspect of the disease and accounts for the vast majority of deaths attributed to cancer. Tumor metastasis via lymphatic vessels is considered one of the fundamental steps in the metastatic dissemination of tumors. The development of effective new cancer treatments thus requires the inhibition of lymphangiogenesis and the blocking of lymphatic metastasis.
Fucoidan, however, is a naturally occurring sulfated polysaccharide with demonstrated antimetastatic effects against multiple cancers in recent studies. Several reports have shown that fucoidan exhibits anti-angiogenic activity through VEGF-regulating signal pathways. However, the anti-lymphangiogenic activity of fucoidan has not been investigated to date, along with its direct effects on lymphatic endothelial cells (LECs).
In this blog, I would like to share the following study, “Fucoidan inhibits lymphangiogenesis by downregulating the expression of VEGFR3 and PROX1 in human lymphatic endothelial cells” by Yazong Yang et al. In an in vitro study, fucoidan extracted from the sporophyll of Undaria pinnatifida showed a significant ability to inhibit several key processes in lymphatic endothelial cells (HLECs). Specifically, it suppressed their proliferation and migration, it blocked the formation of their characteristic tubular structures, and it demonstrated an inhibitory effect on tumor-induced lymphangiogenesis.
The effect of fucoidan (0, 100, 200, 400 μg/ml) on HLEC viability was initially assessed via MTT assay across a time course. It was shown that fucoidan significantly reduced cell proliferation after 48 and 72 h of treatment. To establish the mechanism of proliferation inhibition, human lymphatic endothelial cells (HLECs) were treated with fucoidan (0, 100, 200, 400 μg/ml) for 48 h, and flow cytometry was performed to show that fucoidan induced an increase in cells in G0/G1 phase and a decrease in cells in S phase (Figure 1A and 1B). In addition to this, the results showed that fucoidan reduced the expression of CDK4 and cyclin D1 by western blotting, as shown in Figure 2C. This data suggests a mechanism by which fucoidan reduces cell proliferation: downregulation of CDK4 and cyclin D1, and subsequent G1 cell cycle blockage.
Lymphatic metastasis is a complex, multi-step process in malignant tumors, and lymphangiogenesis is essential to this process, which involves the migration of lymphatic endothelial cells (LECs). In vitro, transwell and wound scratch assays were employed to assess fucoidan’s ability to inhibit HLEC migration. Fucoidan-treated cells showed significantly less migration compared to control cells in the transwell assay. Visual inspection of wound scratch assays revealed a significant difference in cell migration distances following fucoidan treatment. After 24 hours, treated cells demonstrated reduced migration compared to controls.
To investigate the anti-lymphangiogenic activity of fucoidan, we assessed tube formation in HLECs in vitro. Control cells cultured on Matrigel for 24 h formed complete tubular structures, whereas fucoidan-treated HLECs did not. Furthermore, the control group showed a substantially greater number of tubes than the fucoidan-treated groups. Thus, fucoidan treatment effectively reduced tube formation in HLECs in a dose-dependent manner.
Cell migration and tube formation are inextricably linked to the cytoskeleton. This cellular structure is crucial for maintaining cell shape and resisting deformation, and plays an active role in cell migration by generating pseudopodia. Green fluorescent phalloidin staining of HLECs allowed the experts to investigate the effects of fucoidan on microfilament distribution. In fucoidan-treated HLECs, the distribution of microfilaments was affected and cell polarity was altered. It is possible that fucoidan inhibited cell movement by controlling the rearrangement of the cytoskeleton.
VEGFR3 and PROX1 are pivotal proteins present during migration and lymphangiogenesis of LECs. Immunofluorescence and Western blotting were used to determine if fucoidan inhibits metastasis and lymphangiogenesis by downregulating VEGFR3 and PROX1 expression. As shown in Figures 2A and 2B, the mRNA and protein levels of VEGFR3 and PROX1 were decreased with increasing concentrations of fucoidan. Fucoidan at concentrations of 200 and 400 μg/ml significantly reduced the expression of VEGFR3, and fucoidan at concentrations of 200 and 400 μg/ml significantly reduced the expression of PROX1. Western blot analysis was performed to investigate whether fucoidan has an inhibitory effect on the activation of VEGFR3-induced signaling pathways. The results showed that the phosphorylation of PI3K and Akt and the protein levels of NF-κB were all decreased by fucoidan in a concentration-dependent manner (Fig. 2C).
Lymphatic vessel structure was assessed in a co-culture system to explore fucoidan’s inhibitory effect on tumor lymphangiogenesis. In the control group of co-culture, more tubular structures were formed, and the lumens were closely connected. The results of the experiment clearly showed that the tubular structures produced in the co-culture system displayed a significantly improved ability compared to those in the HLEC monoculture. In the co-culture system, treatment with fucoidan effectively reduced the tube formation of HLEC in a dose-dependent manner (Fig. 3B).
They inoculated mice with mouse hepatocellular carcinoma cells (Hca-F cells) with high lymphatic metastasis activity to evaluate the antitumor and anti-lymphangiogenesis activities of fucoidan in vivo. Double immunofluorescence staining of tumor tissue with LEC markers VEGFR3 and LYVE-1 revealed that fucoidan treatment (120 mg/kg) reduced the average tumor micro-lymphatic vessel density (micro-LVD). The micro-LVD of mice treated with fucoidan was significantly decreased compared with the saline control group. The mean tumor weight of mice treated with fucoidan was also significantly lower compared with the control group. The analysis demonstrated fucoidan’s ability to suppress tumor growth and lymphangiogenesis within the tumor itself.
In vitro, fucoidan was shown to inhibit lymphangiogenesis, while in vivo tumor models demonstrated its antitumor and antilymphangiogenic properties. Emerging research suggests that fucoidan holds considerable promise as a novel antitumor agent, offering a potential new avenue for clinical therapies against cancer.
Figure. 1) Effect of fucoidan on the cell cycle distribution in HLECs
Source: Oncotarget. 2016 Jun 21; 7(25): 38025–38035. doi: 10.18632/oncotarget.9443