Anticancer Properties and Mechanisms of Fucoidan on Mouse Breast Cancer In Vitro and In Vivo Meilan Xue1, Yinlin Ge1*, Jinyu Zhang 1 , Qing Wang2, Lin Hou 1 , Yongchao Liu1, Lingling Sun 3 , Quan Li1 1 Department of Biochemistry and Molecular Biology, Medical College, Qingdao University, Qingdao, China, 2 Department of Ophthalmology, The Affiliated Hospital of Qingdao University, Qingdao, China, 3 Department of Pathology, The Affiliated Hospital of Qingdao University, Qingdao, China Abstract Background: Fucoidan is a sulfated polysaccharide derived from brown algae that has been reported to perform multiple biological activities, including antitumor activity. In this study, we examined the influence of crude fucoidan on mouse breast cancer in vitro and in vivo. Materials and Methods: In vitro, fluorescent staining, flow cytometry and Western blot were performed to analyze apoptosis and vascular endothelial growth factor (VEGF) expression of mouse breast cancer 4T1 cells. In vivo, therapy experiments were conducted on Babl/c mice bearing breast cancer. The tumor volume and weight were measured. The number of apoptotic cells and microvascular density (MVD) in tumor tissues were assessed by TUNEL and CD34 immunostaining. Immunohistochemical assays and ELISA assay were used to detect the expression of VEGF in tissues. Results: In vitro studies showed that crude fucoidan significantly decreased the viable number of 4T1 cells, induced apoptosis and down-regulated the expression of VEGF. The expression of Bcl-2 was decreased, and the ratio of Bcl-2 to Bax was significantly decreased. The expression of Survivin and phosphorylated extracellular signal regulated protein kinases (ERKs) was decreased. Cytochrome C was released from mitochondria into cytosol, and the cleaved Caspase-3 protein rose after fucoidan treatment. Intraperitoneal injection of fucoidan in breast cancer models reduced the tumor volume and weight. The enhanced antitumor efficacy was associated with decreased angiogenesis and increased induction of apoptosis. Conclusion: These findings indicated that crude fucoidan inhibited mouse breast cancer growth in vitro and in vivo. These data suggest that fucoidan may serve as a potential therapeutic agent for breast cancer. Citation: Xue M, Ge Y, Zhang J, Wang Q, Hou L, et al. (2012) Anticancer Properties and Mechanisms of Fucoidan on Mouse Breast Cancer In Vitro and In Vivo. PLoS ONE 7(8): e43483. doi:10.1371/journal.pone.0043483 Editor: Ming Tan, University of South Alabama, United States of America Received March 1, 2012; Accepted July 20, 2012; Published August 20, 2012 Copyright: 2012 Xue et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: geyinlin@126.com Introduction Fucoidan is a complex sulfated polysaccharide that is found in brown algae. The structures and compositions of fucoidan vary among different brown seaweed species, but generally the compound primarily consists of L-fucose and sulfate, along with small quantities of D-galactose, D-mannose, D-xylose, and uronic acid [1–3]. Recently, the diverse biological activities of fucoidan have been studied intensively; the putative bioactivities of fucoidan include antioxidant [4] and immunomodulatory, antivirus, antithrombotic and anticoagulant effects [5–8]. In particular, the antitumor activity has recently attracted consid- erable attention. There have also been a variety of studies addressing the anticarcinogenic effects of fucoidan. Fucoidan has been reported to enhance the activity of NK (natural killer) cells which is an important factor in anti-cancer activity [6]. In previous in vivo studies conducted using xenograft models, fucoidan has been reported to suppress the growth of Ehrlich ascites carcinoma [9,10] and Lewis lung adenocarcinoma, and has also been shown to inhibit the metastasis of Lewis lung adenocarcinoma [5] and 13762 MAT rat mammary adenocar- cinoma [11]. The findings of previous in vitro studies have demonstrated that fucoidan inhibits the growth of non-small-cell bronchopulmonary carcinoma NSCLCN6 cells [12] and human lymphoma HS-Sultan cells [13], and also inhibits the invasion of HT1080 human fibrosarcoma cells and the angiogenic activity of HeLa human uterine carcinoma cells [14]. Recently, fucoidan has been reported to induce apoptosis in several cancer cell lines [5,15], Fucoidan has also been shown to induce a substantial reduction in viable cell numbers and apoptosis of human lung carcinoma A549 cells as well as colon cancer HT- 29 and HCT116 cells in a dose-dependent manner [16,17]. But the mechanism is controversial because it is uncertain which cascade plays a pivotal role in the induction of apoptosis by fucoidan. However, to the best of our knowledge, the effects of crude fucoidan on the growth of breast cancer bearing mice and its underlying mechanisms have not yet to be determined in detail. Therefore we examined the effects of crude fucoidan extracted from F. vesicu-losus on the growth of breast cancer in vitro and in vivo, and to determine the mechanisms relevant to this effect. PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43483