Induction of Nucleolin Translocation by Acharan Sulfate in A549 Human Lung Adenocarcinoma Eun Ji Joo, 1 Hui Yang, 1 Youmie Park, 1 Nam Young Park, 1 Toshihiko Toida, 2 Robert J. Linhardt, 3 and Yeong Shik Kim 1 * 1 Natural Products Research Institute, College of Pharmacy, Seoul National University, 599 Gwanangno, Gwanak-gu, Seoul 151-742, Korea 2 Graduate School of Pharmaceutical Sciences, Chiba University, 263-8522 Chiba, Japan 3 Departments of Chemistry and Chemical Biology, Biology and Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, 110 8th Street, Troy, New York 12180 ABSTRACT Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, is a novel glycosaminoglycan, consisting primarily of the repeating disaccharide structure a-D-N-acetylglucosaminyl (1 ! 4) 2-sulfoiduronic acid. AS shows anti-tumor activity in vitro and in vivo. Despite this activity, AS is only weakly cytotoxic towards cancer cells. We examine the interactions between AS and cell-surface proteins in an effort to explain this anti-tumor activity. Using flow cytometry and affinity column chromatography, we confirm that AS has strong affinity to specific cell-surface proteins including nucleolin (NL) in A549 human lung adenocarcinomas. Surprisingly, we found the translocation of NL from nucleus to cytoplasm under the stimulation of AS (100 mg/ml) in vitro. Also, as NL exits the nucleus, the levels of growth factors such as bFGF and signaling cascade proteins, such as p38, p53, and pERK, are altered. These results suggest that the communication between AS and NL plays a critical role on signal transduction in tumor inhibition. J. Cell. Biochem. 110: 1272–1278, 2010. ß 2010 Wiley-Liss, Inc. KEY WORDS: ACHARAN SULFATE; NUCLEOLIN; TRANSLOCATION; A549 HUMAN LUNG ADENOCARCINOMA G lycosaminoglycans (GAGs) are known to play essential roles of normal cell and tumor cell behavior influencing proliferation, differentiation, migration, and adhesion through their binding to a large number of ligands and receptors regulating these processes [Capila and Linhardt, 2002]. GAGs are linear poly- saccharides consisting of repeating, negatively charged disacchar- ides of particular structures. In tumor biology, GAGs have a double- edged activity, both stimulating and inhibiting angiogenesis, tumor growth, and metastasis, depending on GAG structure and the types of cancer or animal models [Borsig et al., 2001; Sasisekharan et al., 2002]. Despite the ambiguous nature of these phenomena, GAGs such as heparin and heparan sulfate are considered a critical component of cell–cell communications at the cell membrane and in the extracellular matrix. A novel GAG, acharan sulfate (AS), was isolated from the body of the giant African snail Achatina fulica by our laboratory having the primary structure !4)-2-acetamido-2-deoxy-a-D- glucopyranose(1 ! 4)-2-sulfo-a-L-idopyranosyluronic acid (1 ! (GlcNAc-IdoA2SO  3 ) [Kim et al., 1996]. While related to heparin and heparan sulfate family, it is distinctly different from the vertebrate GAGs. In previous studies, AS showed anti-angiogenic activity in inflammation models [Ghosh et al., 2002], in vivo anticoagulant activity, anti-mitogenic activity on heparin-mediated basic fibroblast growth factor (bFGF) [Wang et al., 1997], and immunomodulating action [Shim et al., 2002]. AS also showed little cytotoxicity (0–200 mg/ml) on various cancer cells, inhibiting tumor growth in vivo through an anti-angiogenic effect [Lee et al., 2003]. To explain these effects, the specific proteins interacting with AS were explored using biotinylated A549 human lung adenocarci- nomas and AS affinity column. We had previously reported that cell-surface nucleolin (NL) in mouse Lewis lung carcinoma was an AS-binding protein and confirmed its inhibitory effect on tumor growth in vivo [Joo et al., 2005]. NL was first reported by Orrick et al. [1973], is known to function in the nucleus in rDNA transcription, rRNA maturation, ribosome assembly, and nucleolus formation [Chen et al., 1991; Ginisty et al., 1999; Srivastava and Pollard, 1999; Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 110:1272–1278 (2010) 1272 Grant sponsor: National Research Foundation of Korea; Grant number: 20090083533. *Correspondence to: Prof. Yeong Shik Kim, Natural Products Research Institute, College of Pharmacy, Seoul National University, 599 Gwanangno, Gwanakgu, Seoul 151-742, Republic of Korea. E-mail: kims@snu.ac.kr Received 15 January 2010; Accepted 2 April 2010  DOI 10.1002/jcb.22643  ß 2010 Wiley-Liss, Inc. Published online 17 May 2010 in Wiley InterScience (www.interscience.wiley.com).