Anticancer Effect of Fucoidan on Breast Cancer Figure 9. Fucoidan decreased lung metastases of breast cancer. A) Representative photographs of lung metastases after treatment of fucoidan. Mice were euthanized 20 days after treatment. Lungs were harvested, put in a fixative (saturated picric acid: formaldehyde: acetic acid = 75:25:5) for 24 h, and then restore the color to the lung tissue by soaked in absolute alcohol. B) Photograph of Hematoxylin and eosin-stained sections in lung metastatic nodule (620 magnification). The deep blue dye cell mass was breast cancer metastases. C)The number of lung metastatic nodules (white) were counted. The results are presented as mean 6 SD (n = 7 tumors). The results are presented as mean 6 SD (n = 7 tumors). Compared with control mice, fucoidan resulted in significantly fewer lung metastases. **P,0.01 vs Control. doi:10.1371/journal.pone.0043483.g009 we strongly suggest that fucoidan be a potential therapeutic agent for the prevention and treatment of breast cancer. Materials and Methods Ethics Statement All experimental procedures were conducted in conformity with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No.85–23, revised 1985). The study protocol was approved by the Review Committee for the Use of Human or Animal Subjects of Medical College of Qingdao University. Fucoidan Crud fucoidan was purchased from Sigma (St. Louis, MO, U.S.A.). The fucoidan was dissolved in normal saline to 20 mg/ml and stored at 220uC until further use. Cell Culture The 4T1 mouse breast cancer cells were purchased from Shanghai Life Science of Chinese Academy of Sciences. 4T1 cells were routinely maintained in 1:1 (v/v) mixture of DMEM high glucose (Hyclone, Beijing, China)and 10% (vol/vol) fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, U.S.A.), 37uC in a tissue culture incubator with 5% CO2 and 98% relative humidity. The 4T1 cells were placed in 6-well plates and cultured as normal. The exponentially growing cells were used throughout the experiments. MTT Assay The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro- mide (MTT, Roche Diagnostics Corporation) assay was per- formed as previously described [30]. In brief, 4T1 cells were cultured in a 96-well plate at a density of 16105 cells per ml. The cells were then treated with 50, 100, and 200 mg/ml of fucoidan. After 1d, 2d and 3 d, the cells were treated with 20 ml MTT PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e43483