Anticancer Effect of Fucoidan on Breast Cancer Figure 8. Fucoidan down-regulated VEGF expression in vivo. A) Representative photographs of the tumor sections examined by immunohistochemical staining for VEGF (600 magnification). The assessment of VEGF was based on a cytoplasmic staining pattern. B) The tumors were harvested and assayed for VEGF protein by ELISA. The results are presented as mean 6 SD (n = 7 tumors). Fucoidan caused significant reduction in intratumoral VEGF expression compared with control group. *P,0.05 vs Control. doi:10.1371/journal.pone.0043483.g008 members involved in apoptosis. Bax can promote the release of cytochrome C into the cytosol from mitochondria. The anti- apoptotic proteins such as Bcl-2 preserve the integrity of the mitochondria. This blocks the release of cytochrome C that activates the effectors of apoptosis [21]. Since Bcl-2 functions by forming a heterodimer with its pro-apoptotic partner Bax, the Bcl- 2/Bax ratio is proportional to the relative sensitivity or resistance of the cells to various apoptotic stimuli [22]. Survivin is a unique member of the inhibitor of apoptosis (IAP) protein family and has been shown to be involved in both control of cell division and inhibition of apoptosis. Accumulating data indicate that altered expression and/or function of survivin in cancer cells are associated with cancer progression, drug/radiation resistance, poor prognosis, and short patient survival [23,24]. In the present study, the apoptotic induction of fucoidan was also accompanied by inhibition of extracellular signal-regulated kinases (ERKs) [25]. The ERKs pathway thus may play a major regulatory role for apoptosis. Zhang Z, et al [26] found that fucoidan extract could induce phosphorylation of ERK1/2 in MCF-7 cells. Our results indicated fucoidan reduced the Bcl-2 level, resulting in a decrease in Bcl-2/Bax ratio, down-regulation of survivin and the decrease of phosphorylated ERKs activity in 4T1 cells. These results revealed that the molecular events occurring during fucoidan-induced mitochondria-mediated apoptosis. Inhibition of the expression of anti-apoptotic factors will result in the release of cytochrome C from mitochondria and the activation of caspase-3, one of the key executioners of apoptosis. In this study, we found that treatment of fucoidan resulted in the release of cytochrome C, and finally increased expression of cleaved caspase-3. Cleaved caspase-3 results in increased apoptosis in 4T1 cells. This result was consistent with effect of fucoidan on human colon cancer cells [17]. Based on the results, we concluded that several critical links such as Bcl-2, Survivin, ERKs, cytochrome C and caspase-3 play very important roles in apoptosis of 4T1 cells induced by fucoidan. In addition, the results strongly supported the hypothesis of mitochondrial apoptotic pathway involvement. Furthermore, fucoidan exhibited potent anti-angiogenic activity and suppressed lung metastasis of breast cancer in vivo. We observed down-regulation of VEGF proteins both in vitro and in vivo. Suppression of VEGF proteins induced by fucoidan is likely caused by interference of ERKs signaling. It has previously been demonstrated that activation of the p38 and p42/44 MAPK up- regulates VEGF and other proinflammatory cytokines [27]. Metastasis is a rather complex process that occurs through a series of steps that include invasion, intravasation, transport through the circulatory system, arrest at a secondary site, and the extravasation and growth in a secondary organ [28]. Koyanagi S, at al [29] found that over-sulfated fucoidans clearly suppressed the neovascularization induced by Sarcoma 180 cells that had been implanted in mice. VEGF can promote tumor cell metastasis by triggering cell migration and invasion in an autocrine fashion. Focoidan suppressed the mitogenic and chemotactic actions of vascular VEGF165 by preventing VEGF165 from binding with its cell surface receptor. Fucoidan resulted in fewer lung metastases, which is at least partially relevant to the down-regulation of VEGF expression. Further study is required to provide more proof. In conclusion, fucoidan induced apoptosis, inhibited angiogen- esis and suppressed lung metastasis of breast cancer in vitro and in vivo. The possible mechanism might be that fucoidan inhibits expression of Bcl-2, Survivin, ERKs and VEGF and increases activation of Caspase-3. Fucoidan might be able to improve the survival of breast cancer patients by promoting apoptosis, interfering with angiogenesis and inhibiting lung metastasis. So PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43483