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.

material found in brown algae that holds promise in the context of drug development. Fucoxanthin prevents the proliferation of tumour cells through classical pathways involved in metastasis and the cell cycle, including the PI3K/Akt and nuclear factor kappa B (NF-κB) pathways 4 . Although fucoxanthin has been found to play an important role in human health, specific effects on tumour lymphatic metastasis remain to be elucidated. Here, we explore the effects of fucoxanthin on lymphangiogenesis induced by MDA-MB-231 breast cancer cells.
Lymphatic metastasis, a process in which tumour cells migrate from primary tumours to lymph nodes via lymphatic vessels, facilitates metastasis of malignant tumours. Lymphatic vessel formation is observed in diverse types of tumour tissue, including tumour vessels, tumour cells, tumour-associated fibroblasts and inflammatory cells. Lymphangiogenesis involves the proliferation and migration of lymphatic endothelial cells (LEC) in lymphatic vessels surrounding the tumour; this process is promoted by the secretion of lymphangiogenic factors from inflammatory and tumour cells.
Lymphangiogenic factors such as vascular endothelial growth factor (VEGF)-C, which binds to VEGF receptor-3 (VEGFR-3), increase the ability of lymphatic vessels to invade tumours 6 . Expression of VEGF-C was found to correlate with metastasis in human cancer 7,8 .
In animal models, inhibition of lymphangiogenesis through blockade of VEGF-C or VEGFR-3 has been shown to prevent lymphatic metastasis. Ongoing studies are investigating the role of VEGFR-3 and VEGF-C/-D in tumour lymphangiogenesis, using soluble VEGFR-3 or antibodies to VEGFR-3 to inhibit tumour metastasis and lymphangiogenesis 9 .
Nuclear factor kappa B is another molecule that plays an important role in lymphangiogenesis. Nuclear factor kappa B is a transcription element located in the cytoplasm. Before release is triggered by a stimulus, NF-κB remains bound in an inert NF-kappa-B inhibitor (IκB) complex. On stimulation, a nuclear localization signal regulates transport of the activated NF-κB complex into the nucleus to induce related gene expression, in particular, VEGF-C, Cyclin D1, matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Nuclear factor kappa B may be activated by numerous growth factors, cytokines and receptor families associated with tumour necrosis factor. Nuclear factor kappa B is also triggered by other signalling pathways, such as PI3K/Akt and Ras/MAPK, to induce antiapoptotic effects 10 . PI3K/Akt signalling contributes significantly to tumour angiogenesis and carcinogenesis 11 . Excitation of the PI3K/Akt/NF-кB signalling pathway may promote tumour-induced lymphangiogenesis, which is involved in cell proliferation and migration in LEC.
Inhibition of lymphangiogenesis represents a novel strategy for the treatment of breast cancer. Inhibition of lymphangiogenesis is expected to help control primary tumours and pre-metastatic, tumour-conditioned regional lymph nodes. Growth of MDA-MB-231 cells and tumour-conditioned lymph nodes was mechanistically delayed by VEGFR2/3 and NRP1/2 complex formation in the presence of VEGF-A/C 12 . During the process of tumour metastasis, cellular basement membrane was prominently degraded by malignant cells, and cells spread to distant organs, resulting in the formation of secondary tumours. Various proteases, including matrix metalloproteinases (MMPs), degrade extracellular matrix (ECM) and basement membrane. Matrix metalloproteinase-2, MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1), the endogenous inhibitor of MMP, are highly associated with local tissue invasion, which is clearly aided by the degradation of ECM 13 .
We hypothesized that ECM degradation might be of special con-

| Preparation of fucoxanthin
Fucoxanthin was isolated from U pinnatifida and the preparation method as previously reported 14 .

| Cell culture
Human LEC were obtained from Sciencell Research Laboratories
The resulting cell pellet was collected via centrifugation at 107 g for 5 minutes. Prior to incubation, 100 μL RNase A was added. Cell preparations were incubated for 30 minutes at 37°C. DNA staining was performed with propidium iodide (400 μL). Progression through the cell cycle was analysed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).   An inverse microscope (NOVEL, Nanjing, China) was used to obtain images ≥3 fields per well. The rate of tube formation was calculated as follows:

Cell viability
Formation rate (%) = Tube quantity treated Tube quantity control

| Immunohistochemistry
To stain the cytoskeleton, cells were fixed in 3.7% methy-

| Western blot
To examine protein expression levels in HLEC after treatment with fu-

| Quantitative real-time PCR (Real-time qPCR)
Cells treated with fucoxanthin were cultured for 24 hours. TRIzol

| Conditional culture
Briefly, MDA-MB-231 cells were treated with fucoxanthin (25, 50, 100 μmol/L) for 24 hours and cultured with serum-free medium for 24 hours. Supernatant was centrifuged and collected. Human LEC were cultured with 40% conditional medium for assays of tube formation.

| Statistical analysis
Data are expressed as mean ± SD. Significant differences were evaluated by one-way ANOVA using spss and Prism 5 (Version 5.04, GraphPad Software, Inc, La Jolla, CA). Statistical significance was defined as P < 0.05 and P < 0.01. All experiments were performed at least three times for quantitative comparison.
Our data, as shown in Figure 1B

| Fucoxanthin inhibits tube formation in HLEC
Tube formation of HLEC was assessed to investigate the antilymphangiogenic effect of fucoxanthin. Tube formation capacity was inhibited in HLEC treated with fucoxanthin for 24 hours (Figure 2A Treatment with fucoxanthin decreased levels of phospho-VEGFR3 in a concentration-dependent manner ( Figure 2E).

| Fucoxanthin inhibits HLEC migration
To evaluate the effects of fucoxanthin on HLEC migration, we performed Transwell assays as well as cytoskeletal and nuclear staining.
Cells treated with fucoxanthin for 24 hours migrated significantly less than control cells. This effect was concentration dependent ( Figure 3A,C). Increasing concentrations of fucoxanthin affected the distribution of microfilaments within the cell, with effects on cell morphology and cell polarity ( Figure 3B). These results indicate that fucoxanthin regulates the rearrangement of microfilaments to inhibit cell motility.

| Fucoxanthin inhibits tumour-induced lymphangiogenesis in vitro
Cell growth, migration and tube formation were investigated using a 24-hour conditional culture system in order to further analyse whether fucoxanthin inhibited tumour-induced lymphangiogenesis.
Cell viability, migration and tube formation were significantly in-

| Inhibition of tumour growth and lymphangiogenesis in a mouse model
Fucoxanthin-mediated inhibition of tumour growth and lymphangiogenesis were investigated in a MDA-MB-231 breast cancer xenograft model using Balb/c nude mice. The results showed that fucoxanthin significantly decreased tumour volume ( Figure 6A,B) and weight ( Figure 6C).

| D ISCUSS I ON
Metastasis is a feature used for tumour classification as well as a common cause of cancer-related death. Metastasis via lymphatic vessels is an important initial event. Tumour-induced lymphangiogenesis in regional lymph nodes or local tumours has been associated with formation of various types of tumours, including breast cancers 15  In this study, LVD was significantly lower in nude mice treated with fucoxanthin, compared to those treated with saline. The lymphatic vessels observed were also smaller and less func-

CO N FLI C T O F I NTE R E S T S
The authors declare no conflict of interest.