Fucoxanthin inhibits tumour-related lymphangiogenesis and growth of breast cancer

Abstract

Tumour lymphangiogenesis plays an important role in promoting the growth and lymphatic metastasis of tumours. The process is associated with cell proliferation, migration and tube-like structure formation in lymphatic endothelial cells (LEC), but no antilymphangiogenic agent is currently used in clinical practice. Fucoxanthin is a material found in brown algae that holds promise in the context of drug development. Fucoxanthin is a carotenoid with variety of pharmacological functions, including antitumour and anti-inflammatory effects. The ability of fucoxanthin to inhibit lymphangiogenesis remains unclear. The results of experiments performed as part of this study show that fucoxanthin, extracted from Undaria pinnatifida (Wakame), inhibits proliferation, migration and formation of tube-like structures in human LEC (HLEC). In this study, fucoxanthin also suppressed the malignant phenotype in human breast cancer MDA-MB-231 cells and decreased tumour-induced lymphangiogenesis when used in combination with a conditional medium culture system. Fucoxanthin significantly decreased levels of vascular endothelial growth factor (VEGF)-C, VEGF receptor-3, nuclear factor kappa B, phospho-Akt and phospho-PI3K in HLEC. Fucoxanthin also decreased micro-lymphatic vascular density (micro-LVD) in a MDA-MB-231 nude mouse model of breast cancer. These findings suggest that fucoxanthin inhibits tumour-induced lymphangiogenesis in vitro and in vivo, highlighting its potential use as an antilymphangiogenic agent for antitumour metastatic comprehensive therapy in patients with breast cancer.

Keywords: MDA-MB-231 cells; fucoxanthin; lymphatic metastasis; tumour-induced lymphangiogenesis.

Figure 1

Effect of fucoxanthin on viability and cell cycle distribution in human lymphatic endothelial cells. A, Chemical structure of fucoxanthin. B, Cell viability after 12, 24 or 48 h in culture. C, Flow cytometry histograms and (D) cell cycle distribution as assessed via flow cytometry. After 24 h, fucoxanthin treatment arrested cells in the S phase and significantly decreased length of the G0/G1 phase. Values are mean ± SD. *P < 0.05 and **P < 0.01 vs untreated control (one‐way ANOVA)

Figure 2

Fucoxanthin inhibits lymphatic tube formation in human lymphatic endothelial cells (HLEC). Cells were incubated for 24 h in medium with or without fucoxanthin (25, 50, 100 μM). A, Photomicrographs of HLEC tube formation (scale bar =50 μm). Complete tubular structures are marked with asterisks. B, Quantitative analysis of the rate of tube formation. C, Expression of vascular endothelial growth factor (VEGF)‐C and VEGF receptor‐3 (VEGFR‐3), as determined by Western blot. D, mRNA levels of VEGF‐C and VEGFR‐3, as determined by Real‐time qPCR. E, p‐VEGFR3 levels in fucoxanthin‐treated HLEC and controls. *P < 0.05 and **P < 0.01, compared with controls (one‐way ANOVA)

Figure 3

Fucoxanthin inhibits human lymphatic endothelial cells (HLEC) migration in vitro. Human lymphatic endothelial cells were incubated in medium for 24 h with or without fucoxanthin (25, 50, 100 μmol/L). A, Microscopic images (scale bar = 50 μm) and (C) number of migrated cells, as determined by Transwell assay. Averages of five fields were calculated. Data are presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, compared with control group (one‐way ANOVA). B, Microfilaments and nuclei were stained with phalloidin (green) and 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue) respectively. D, Levels of phospho‐PI3K, phospho‐Akt and NF‐κB in HLEC treated with fucoxanthin (25, 50, 100 μmol/L), as determined by western blot. *P < 0.05, **P < 0.01, compared with controls (one‐way ANOVA)

Figure 4

Effect of fucoxanthin on malignant phenotypes in MDA‐MB‐231 cells. Cells were treated with fucoxanthin (25, 50, 100 μmol/L) for 12‐48 h. A, Decreased cell viability in cells treated with fucoxanthin for 12‐48 h. B, Decreased mRNA and protein levels of vascular endothelial growth factor‐C after fucoxanthin treatment, as determined with real‐time qPCR and ELISA. C, Microscopic images of migration and (D) invasion in MDA‐MB‐231 cells (scale bar = 50 μm). Averages were calculated from five fields. Data are presented as mean ± SD; **P < 0.01 vs controls (one‐way ANOVA). The protein expression levels of matrix metalloproteinase‐2 (MMP‐2), matrix metalloproteinase‐9 (MMP‐9) and tissue inhibitor of metalloproteinase‐1 were measured by western blot analysis. The secretion levels of MMP‐2 and MMP‐9 were measured by ELISA.

Figure 5

Fucoxanthin inhibits tumour‐induced lymphangiogenesis in vitro. Human lymphatic endothelial cells (HLEC) were incubated in conditional medium with or without fucoxanthin (25, 50, 100 μmol/L) for 24 h. A, Cell viability, (B) migration and (D) tube formation are decreased in HLEC. C, Results of ELISA showing fucoxanthin decreases phosphorylation of vascular endothelial growth factor receptor‐3. NC, negative control. Data are shown as mean ± SD; *P < 0.05 vs NC; *P < 0.05, **P < 0.01 vs controls (one‐way ANOVA)

Figure 6

Fucoxanthin inhibited tumour growth in the MDA‐MB‐231 xenograft model of breast cancer. Female nu/nu mice were inoculated subcutaneously with MDA‐MB‐231 cells. Five days after inoculation, NS or fucoxanthin (100, 500 μmol/L; 100 μL/per mouse) was injected at the tumour's edge every day for 26 days. Tumour length and width were measured individually every 4 days. A, Representative images obtained on day 26 of tumours implanted subcutaneously. B, Tumour growth curves and (C) tumour weight. Data are presented as mean ± SD. *P < 0.05, **P < 0.01 vs controls (one‐way ANOVA)

Figure 7

Fucoxanthin inhibits lymphangiogenesis in vivo. Top: Immunofluorescent images of tumour lymphatic vessels stained with antibodies to vascular endothelial growth factor receptor‐3 (green) and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE‐1) (red), from mice inoculated with MDA‐MB‐231 cells. Bottom: Quantitative assessment of tumour lymphatic vascular density. Data are presented as mean ± SD. **P < 0.01 vs controls (one‐way ANOVA)