Anticancer Effect of Fucoidan on Breast Cancer Figure 3. Apoptosis induction after 48 h treatment with fucoidan in 4T1 cells. A) Morphology of 4T1 cells observed under fluorescence microscope (Hoechst332586400). DNA fragment and chromatin condensation were observed in fucoidan-treated cells, but few in untreated cells. 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% respectively, while there were7.460.29% of the apoptotic cells in the control cells (P,0.01). B) The apoptosis cells determined by FCM. 4T1 cells were stained with annexin V-FITC and propidium iodide (PI). The lower right quadrant (annexin V+/PI2) represents early apoptosis, and the upper right quadrant (annexin V+/PI+) represents late apoptosis and necrosis. The early apoptotic rates of 4T1 cells treated with fucoidan were 2461.34%, 4163.75% and 5965.27% respectively and significantly higher than that in control cells(1160.89%) (P,0.05). Data are a representative of three independent experiments showing similar results. 1: control; 2: fucoidan 50 mg/ml; 3: fucoidan 100 mg/ml; 4: fucoidan 200 mg/ml. doi:10.1371/journal.pone.0043483.g003 Figure 4. Expression of Bcl-2, Bax, Survivin, ERK, cytochrome C, Caspase-3 and VEGF in 4T1 cells after 48 h treatment of fucoidan detected by Western blot. Expression of the b-actin gene was used as the internal control. Data are mean6SD of one representative experiment performed in triplicate. Fucoidan significantly decreased the expression of Bcl-2, did not modulate the expression of Bax, but the ratio Bcl-2/Bax was decreased. Fucoidan also decreased the protein expression of Survivin and phosphorylated ERKs in 4T1 cells. Fucoidan treatment significantly increased levels of cytochrome C in the cytoplasm, whereas there was a significant corresponding decline of mitochondrial cytochrome C. Cytochrome oxidase (subunit II) was as sample loading control (cyt. oxid). The level of pro-caspase-3 decreased after fucoidan treatment, while the level of caspase-3 cleavage protein increased after fucoidan treatment. Fucoidan decreased VEGF expression in 4T1 cells. Lane 1: control cells; lanes 2–4: 4T1 cells treated with 50, 100 and 200 mg/ml fucoidan respectively. *P,0.05, vs. Control; **P,0.01, vs. Control. doi:10.1371/journal.pone.0043483.g004 PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43483