Results Effect of fucoidan on the growth of normal mouse fibroblasts The effect of fucoidan on the proliferation of normal mouse fibroblasts (L929 cells) was measured by MTT assay. As shown in Fig. 1, the cells viability of each fucoidan treatment group(50,100 and 200 mg/ml) was not statistically significant, compared with that of untreated controls (P.0.05). This result showed that fucoidan did not affect the viability of normal mouse fibroblasts. Fucoidan inhibited the growth of 4T1 cells In vitro assessment of the effects of different concentrations (50,100 and 200 mg/ml) of crude fucoidan on the growth of 4T1 cells was performed via measurement of cell viability using an MTT assay. The effect of various doses of fucoidan on 4T1 cells viability was shown in Fig. 2. 4T1 cells grown in control media displayed a continuous increase in viable cell number over the 3- day culture period. These results indicated that treatment with 50, 100 and 200 mg/ml fucoidan decreased the 4T1 cells viability in a time-dependent manner (P,0.05). After 3 days of culturing, 200 mg/ml fucoidan decreased viable 4T1 cell number by 85.3% as compared with those of untreated controls. Fucoidan induced apoptosis of 4T1 cells An apoptotic cell has special morphologic features such as cell shrinkage, chromatin condensation and margination as well as forming apoptotic bodies. In order to determine whether the fucoidan-inducing reduction in cell viability was attributable to the induction of apoptosis, we stained the 4T1 cells with Hoechst 33258 dye. The treatment of 4T1 cells with fucoidan resulted in the induction of chromatin condensation and fragmentation, which could be visualized as intense pycnotic bluish-white fluorescence within the cell nuclei. The 50,100,200 mg/ml fucoidan treated 4T1 cells that underwent apoptosis were calculated at 18.961.17%, 42.364.38% and 6767.52% respec- tively, while there were 7.460.29% of the apoptotic cells in the control cells (P,0.01) (Fig. 3A). We subsequently estimated the number of apoptotic cells by flow cytometry. The proportionof apoptotic increased in a concentration-dependent manner in cells that had been treated with fucoidan (Fig. 3B). When treated with Figure 1. Effect of fucoidan on the growth of normal mouse fibroblasts. The effect of fucoidan on the proliferation of normal mouse fibroblasts (L929 cells) was measured by MTT assay. The cells viability of each fucoidan treatment group(50,100 and 200mg/ml) was not statistically significant, compared with that of untreated controls (P.0.05). Data are shown as mean 6 S.D. Fucoidan did not affect the viability of normal mouse fibroblasts. doi:10.1371/journal.pone.0043483.g001 Anticancer Effect of Fucoidan on Breast Cancer Figure 2. Inhibitory effect of fucoidan on the growth of 4T1 Cells. The 4T1 cells were treated with 50, 100, and 200 mg/ml fucoidan separately for 1d, 2d and 3 d and measured for viability by MTT assay. Data are shown as mean 6 S.D. n = 5. *P,0.05, vs. Control, ** P,0.01. vs. Control. doi:10.1371/journal.pone.0043483.g002 fucoidan (50,100,200 mg/ml) for 48 h, the early apoptotic rates of 4T1 cells were 2461.34%, 4163.75% and 5965.27% respec- tively and significantly higher than that in control cells(11%60.89%) (P,0.05). Fucoidan altered the levels of the Bcl-2, Survivin, and phosphorylated ERKs To investigate the possible mechanism underlying the induction of apoptosis by fucoidan, we examined the expression of Bcl-2 and Bax in 4T1cells after treatment with fucoidan. Bcl-2 family proteins play critical roles in the regulation of apoptosis by either pro-apoptotic such as Bax, or anti-apoptotic such as Bcl-2. The ratio of the pro- and anti-apoptotic molecules was essential to apoptosis. The result of Western-blotting showed that fucoidan significantly decreased the expression of Bcl-2, did not modulate the expression of Bax (Fig. 4), but the ratio Bcl-2/Bax was decreased. This result demonstrated fucoidan has proapoptotic effects on 4T1 cells. Survivin is a unique member of the IAP (inhibitors of apoptosis) family. To examine whether fucoidan can affect Survivin expression, we analyzed the levels of survivin protein using Western blot. As shown in Fig. 4, fucoidan decreased the protein expression of survivin in 4T1 cells. Western blot analysis indicated phosphorylated ERKs expression was down-regulated when 4T1 cells were treated with fucoidan (50, 100, 200 mg/ml) for 48 h. Fucoidan induced cytochrome C release and up- regulated Caspase-3 protein expression In biological system, apoptosis may involve disruption of mitochondrial function through the abnormal expression of Bcl- 2 and/or Bax which induces the release of cytochrome C from mitochondria into the cytosol. Cytosolic cytochrome C can lead to the activation of caspases in the apoptosome and finally lead to the activation of caspase-3. Activation of caspase-3 subsequently leads to apoptosis. Therefore, we determined whether induction of apoptosis in 4T1 cells by fucoidan is associated with disruption of mitochondrial function and activation of caspases. Fucoidan treatment significantly increased levels of cytochrome C in the cytoplasm (Fig. 4). Activated or cleaved caspase-3 (17 kDa and 19 kDa) is crucial for the induction of apoptosis. The level of pre-caspase-3 decreased significantly after fucoidan treatment, while the level of caspase-3 cleavage protein increased after fucoidan treatment. PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43483