Oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities
Introduction
Angiogenesis, new blood vessel formation, is an indispensable biological event for a variety of physiological or pathological processes [1], [2]. Highly regulated and transient angiogenesis is necessary for embryonic development, wound healing, and corpus luteum formation. However, uncontrolled and persistent angiogenesis occurs in several pathological states such as diabetic retinopathy, rheumatoid arthritis, and tumor progression. Tumor growth requires angiogenesis to supply nutrients and oxygen [3]. In addition, it utilizes the newly formed blood vessels as conduits to disseminate invasive tumor cells. Because the growth and metastasis of malignant tumors are dependent upon angiogenesis, a novel anticancer treatment has been developed in which tumors are regressed by prolonged inhibition of angiogenesis. Thus, a variety of anti-angiogenic agents are currently undergoing clinical trails for dormancy therapy of tumors [4].
Tumor-induced angiogenesis is regulated by cell-produced factors that have mitogenetic and chemotactic effects on vascular endothelial cells. Several endothelial growth factors such as fibroblast growth factors, transforming growth factor-β, platelet-derived growth factor, and VEGF have been identified as angiogenic factors [5], [6], [7]. In particular, the expression of VEGF in tumor cells is considered to play a major role in tumor-induced angiogenesis [8], [9]. As a high expression of VEGF and its receptors in the tumor is closely correlated to its vascularity, progression, and metastasis, targeting of VEGF and/or VEGF receptors is thought to be an effective strategy of anti-angiogenic therapy [10], [11]. Several sulfated polysaccharides have been shown to modulate the proliferation and migration of vascular endothelial cells by altering the binding of endothelial growth factors to their cell surface receptors [12], [13], [14].
Fucoidan, which is a sulfated polysaccharide present in brown marine algae, has been reported to exhibit antitumor and antimetastatic activities in xenograft mouse models [15], [16]. However, the molecular mechanism by which fucoidan suppresses tumor growth and metastasis has not been clarified. We previously showed that NF efficiently suppresses FGF-2-induced migration and tube formation of HUVEC, and that the potency of the agent can be increased by chemical sulfation [17]. We have also shown that OSF inhibits the migration and tube forming ability of HUVEC on Matrigel made from murine Engelbreth-Holm-Swarm sarcoma [18]. From these data, we hypothesized that fucoidan might exert its antitumor activity through the inhibition of tumor-induced angiogenesis.
In the present study, we provide evidence that both NF and OSF suppress the growth of tumor cells implanted in mice by preventing tumor-induced angiogenesis. We also investigated the possible effects of NF and OSF on the activities of the most specific angiogenic growth factor, VEGF. Our data demonstrated that both fucoidans prevent the VEGF-induced phosphorylation of VEGFR-2 by their binding to VEGF itself, and that the degree of sulfation in the fucoidan molecule plays an important role in interfering with VEGF binding to VEGFR-2.
Section snippets
Materials
The following materials were commercially obtained: Fucus vesiculosus fucoidan from the Sigma Chemical Co.; recombinant human VEGF165 from Oncogene Research Products; DMEM and FBS from GIBCO BRL, Life Technologies, Inc.; and porcine Type IV collagen from the Nitta Gelatin Co.
Purification and oversulfation of fucoidan
NF (200 mg) was purified on a Sephadex G-100 column (2.5×36 cm) equilibrated with 0.5 M NaCl. The content of fucoidan in each fraction was determined by the anthrone-H2SO4 method. Fractions corresponding to a 100–130 kDa
Effects of NF and OSF on the VEGF165-induced proliferation and migration of HUVEC
VEGF is a potent selective mitogenic cytokine for vascular endothelial cells [8], [20], [21]. Five isoforms of human VEGF mRNA, which encode VEGF proteins of 121, 145, 165, 189, and 206 amino acids are produced from a single gene, as the result of alternative splicing [22]. The best characterized VEGF is the 165-amino acid form, VEGF165, which induces angiogenesis and blood vessel permeability in vivo and displays a mitogenic activity restricted to vascular endothelial cells [23]. As shown in
Acknowledgements
This study was supported, in part, by a Grant-in-Aid for Encouragement of Young Scientists from the Japan Society for the Promotion of Science (S.K., 13771448).
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