Rhodostomin inhibits thrombin-enhanced adhesion of ROS 17/2.8 cells through the blockade of avb3 integrin Rong-Sen Yang a, * , Huei-Shien Chiang b , Chih-Hsin Tang b , Chia-Shin Yeh b , Tur-Fu Huang b a Departments of Orthopaedics, College of Medicine, National Taiwan University, No. 7, Chung-Shan South Road, Taipei, Taiwan, ROC b Departments of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC Received 22 April 2005; revised 13 May 2005; accepted 31 May 2005 Available online 26 July 2005 Abstract Osteosarcoma is a very malignant bone tumor which has a high metastatic potential and usually lead to poor prognosis. The adhesion of tumor cells to the endothelium or extracellular matrix (ECM) is an essential step in the metastatic cascade. We investigated the effect of thrombin on the adhesion activity of the osteosarcoma cell line, ROS 17/2.8. Incubation with the low concentrations of thrombin (0.01–5 U/ml, 5 min to 24 h) elevated the adhesion activity of ROS 17/2.8 to both human umbilical vein endothelial cells (HUVEC) and extracellular matrix, with the peak effect at the concentration of 0.5 U/ml for 30 min at 37 8C. The ROS 17/2.8 cells responded to thrombin by a peak effect of increased adhesion to HUVEC (5.5 folds vs. control) and fibronectin (4.8 folds) after thrombin pretreatment (0.5 U/ml, 30 min, 37 8C). Pretreatment with monoclonal antibodies against b 3 integrins, including anti-avb3, 10E 5 and 7E 3 , effectively antagonized the thrombin-enhanced cell adhesion activity, whereas anti-a3b1 and anti-a5b1 did not antagonize the enhanced cell adhesion. Rhodostomin, an Arg-Gly-Asp (RGD)-containing snake venom peptide, and synthetic peptide RGDS also blocked the thrombin-enhanced ROS 17/2.8 cell adhesion. This study demonstrated that thrombin enhanced the cell adhesion of ROS 17/2.8 cells to HUVEC or ECM through an upregulation of b 3 integrins, and rhodostomin was a strong inhibitor on thrombin-enhanced cell adhesion, either to HUVEC or fibronectin substratum. q 2005 Elsevier Ltd. All rights reserved. Keywords: Thrombin; Metastasis; Osteosarcoma; Cell adhesion; Arg-Gly-Asp containing peptide. 1. Introduction The advance of surgery and adjuvant chemotherapy have improved the prognosis and survival of the osteosarcoma patients in the last decade. However, many cancer patients end up with lung metastasis in clinical practice. Therefore, investigation of the biology of tumor metastasis remains one of the most important issues. Tumor metastatic cascade consists of multiple successive steps, including adhesion of tumor cells at primary site, invasion into intravascular space, dissemination to distant sites, adhesion of tumor cells to vascular endothelium of distant tissues, extravasation and invasion into surrounding tissues, and finally formation of secondary tumor colonies (Mundy, 2002). The tumor cell adhesion to vasculature endothelium and/or extracellular matrix substratum is an important step during the tumor metastatic cascade (Masi et al., 1992; Miyasaka, 1995). The cell adhesion processes including cell–cell, cell–endo- thelium, and cell–extracellular matrix (ECM) interactions, Toxicon 46 (2005) 387–393 www.elsevier.com/locate/toxicon 0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2005.05.002 * Corresponding author. Tel.: C886 2 23123456x3958; fax: C886 2 23936577. E-mail address: yang@ha.mc.ntu.edu.tw (R.-S. Yang).