The result was shown in Fig. 4. Increase in the Caspase-3 activity that arises after the cleavage of pro-caspase-3 is a hallmark of induced apoptosis and is considered to be a point of no return. These data demonstrated that fucoidan induced caspase-3- dependent apoptotic pathway in 4T1 cells. Fucoidan down-regulated VEGF expression In order to investigate whether fucoidan has potent anti- angiogenic activity, we examined the VEGF expression in 4T1 cells after treatment with fucoidan by Western-blotting. The expression of VEGF protein was significantly suppressed by fucoidan (P,0.05) (Fig. 4). Fucoidan inhibits 4T1 tumor growth in vivo The prominent inhibitory effect of fucoidan on 4T1 cell proliferation in vitro suggested that it might suppress tumor growth in vivo. To verify this hypothesis, we s.c. inoculated female Balb/c mice with 4T1 cells. After 10 days, visible tumors had developed at the injection sites (8,10 mm2 in size). Then 4T1 tumor bearing Balb/c mice were treated by intraperitoneal injection of 5 and 10 mg/kg.bw fucoidan and 0.1 ml NS alone (as blank control), and in each treatment seven animals were used. The whole process were repeated every 2 days and were conducted for totally ten times. As shown in Fig. 5, fucoidan reduced the tumor volume and the tumor weight in Balb/c mice. Figure 5. Antitumor effects of fucoidan on breast cancer in vivo. Fucoidan reduced the tumor volume and the tumor weight in Balb/c mice. A) Tumor growth curves. Each point in the curves represents the mean 6 SD (n = 7 tumors). The therapy started when the 3 tumors reached a volume of 0.1cm . The fucoidan inhibited tumor growth, *P,0.05 vs Control. B) Weight of the tumors. Each bar represents the mean 6 SD (n = 7 tumors). Mean weights of the tumors are 2.77g, 2.15g, and 1.19g, for the control group, 5 mg/kg.bw and 10mg/kg.bw fucoidan group respectively. *P,0.05 vs Control. doi:10.1371/journal.pone.0043483.g005 Anticancer Effect of Fucoidan on Breast Cancer Fucoidan induced apoptosis in tumor tissue To further confirm the ability of fucoidan to elicit apoptosis in vivo, in situ TUNEL staining was carried out on tissue sections of tumors excised on the 20th day since 4T1 cell-implanted mice were treated with various doses of fucoidan. As illustrated in Fig. 6, fucoidan treatment caused a significantly higher percentage of TUNEL-positive apoptotic cells(P,0.01). These results indicated that fucoidan could exert a strong antitumor effect on breast cancer model and is a potent apoptosis-inducing agent in vivo. Fucoidan inhibited angiogenesis in tumor tissue To investigate the effect of fucoidan on angiogenesis in vivo, we evaluated microvessel density in the harvested tumors. As shown in Fig. 7, the microvessel number was apparently reduced in the tumors belonging to the mice treated with fucoidan(P,0.05). Moreover, we harness the tumors for immunohistochemistry and ELISA assay to measure the expression of VEGF. As shown in Fig. 8A, we analyzed the gross distribution of immunoreactive VEGF in the tumors and observed a general decrease of VEGF staining in the tumors belonging to the mice treated with fucoidan, whereas the tumors of control group exhibited significantly more VEGF staining(P,0.05). Consistently, the ELISA assay showed that fucoidan caused a significant reduction in intratumoral VEGF expression compared with control group (P,0.05), as shown in Fig. 8B. Fucoidan suppressed lung metastasis of breast cancer 4T1 cell is a highly invasive breast cancer cell lines. To investigate the effect of fucoidan on invasiveness of 4T1 tumor, mice were euthanized, and the lungs harvested for enumeration of lung metastatic nodules metastases after fucoidan treatment. The results are shown in Fig. 9. Compared with control mice, and who had an average of 34 nodules per mouse, fucoidan resulted in significantly fewer lung metastases (P,0.01). Discussion Fucoidan is a complex sulfated polysaccharide obtained from naturally occurring edible brown seaweeds, which has been consumed as a dietary fiber in many Asian countries for centuries. Fucoidan has been shown to inhibit the growth of a wide variety of tumor cells [5,18]. Therefore, fucoidan has become a focus of great interest because of its anticancerous activity and is expected to be a new candidate for low toxic cancer therapy. In this study, we used a commercial available fucoidan purified from F. vesicu- losus. We demonstrated that fucoidan strongly presented apoptosis induction and angiogenesis inhibition on mouse breast cancer in vitro and in vivo. However, fucoidan did not affect proliferation of normal cells. It has also been reported that fucoidan inhibited the growth of peripheral blood mononuclear cells (PBMCs) from adult T-cell leukemia patients but not that of normal PBMC [19]. Therefore, fucoidan may be a good chemopreventive and antitumor candidate without toxic effects on normal cells. In this study we investigated in detail the molecular pathways induced by fucoidan on breast cancer. Apoptosis is a biological process requiring activation of several signaling cascades. It is widely accepted that mitochondrial pathway is one of apoptosis pathways. Pro-survival Bcl-2 and its helpers compete with Bax and other pro-apoptosis proteins to regulate the release of proteins, such as c-Jun amino-terminal kinase(JNK), and mitogen-activated protein kinase (MAPK)/extracellular signal regulated kina- se(ERK), and cytochrome C from mitochondria, which in turn activate caspases [20]. The Bcl-2 family consists of both antiapoptotic and proapoptotic members that are important PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43483