Antiplasmin activity of natural occurring polyphenols Matteo Mozzicafreddo ⁎, Massimiliano Cuccioloni, Laura Bonfili, Anna Maria Eleuteri, Evandro Fioretti, Mauro Angeletti MCAB Department, University of Camerino, 62032, Camerino (MC), Italy ABSTRACT ARTICLE INFO Article history: Received 29 October 2007 Received in revised form 31 March 2008 Accepted 31 March 2008 Available online 16 April 2008 Keywords: Polyphenols Plasmin Plasminogen Biosensor E-cadherin Cancer cell The equilibrium between proteolytic enzymes and their cognate inhibitors is crucial in a number of physiological as well as pathological processes, including cancer, inflammatory processes and thrombosis. Therefore, both synthetic and natural small molecule inhibitors are object of extensive studies as drugs in the treatment of these pathologies. Two natural occurring polyphenolic compounds, representative of glycosylated and unglycosylated flavonoid structures, namely quercetin and rutin, were thereby tested as potential ligands of plasmin(ogen), a serine (pro)protease, whose role in tumor cell invasion and migration has been reported. Quercetin showed a ten folds higher affinity with plasmin with respect to rutin in terms of equilibrium dissociation constant, both compounds acting as in vitro moderate reversible inhibitors; additionally, quercetin and rutin prevented plasmin-incubated BB1 cells from releasing E-cadherin fragment to a different extent, respectively. Furthermore, a feasible mechanism of interaction was analyzed and discussed using a molecular modeling approach. © 2008 Elsevier B.V. All rights reserved. 1. Introduction The plasmin/plasminogen system (PPS) [1] is a proteolytic system, which is involved in many physiological as well as pathological processes. It includes several components, characterized by different structures and biological activities. Among them, the most important are the plasminogen, the plasminogen activators, with the two isoforms urokinase plasminogen activator (uPA) and tissue plasmino- gen activator (tPA), and the mature protease plasmin. In addition, the cell surface receptor for the urokinase plasminogen activator (uPAR), and the naturally occurring inhibitors, α-2 antiplasmin together with the plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2) play a fundamental role in the biological functions associated to the system [1,2]. PPS is responsible for the removal of fibrin from the circulation, and, together with matrix metalloproteinases, is also involved in other biological processes associated to proteolytic activities: e.g. ovulation [3–5], embryogenesis [6], intima proliferation [7], atherosclerosis [8], tumorigenesis, angiogenesis and obesity [9]. The role of this system in the tumor cell invasion and migration has been recently well documented [10–12] indicating that plasmin can act both directly (degrading proteins of the extracellular matrix and activating the plasminogen activators) and indirectly (through activation of the matrix metalloproteinase). Moreover, the PPS plays an important function in angiogenesis, a biological process that represents an absolute requirement for the growth of normal and neoplastic cells [13]. Moreover, this system regulates the proteolytic processing of cell surface molecules such as cadherins, the major cell– cell adhesion proteins [14]. Epidemiological and in vitro studies suggest a crucial role of the E- cadherin as tumor and invasion suppressor: in a variety of tumors [15–17], loss of E-cadherin expression has been associated with invasive tumor growth [18–21]. E-cadherin expression and function are controlled at multiple levels by a number of mechanisms, including transcriptional regulation, promoter methylation, gene mutation, tyrosine phosphorylation and cleavage of the extracellular domain by ectodomain shedding. Proteolytic ectodomain fragments of E-cadherin have been proposed to promote cancer cell invasion by interfering with E-cadherin function in cells containing intact E- cadherin/catenin complexes [22,23]. The biological activity of PPS is controlled by many natural inhibitors [2], which includes α-2 antiplasmin (the primary inhibitor of plasma), the plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2) and the protease nexin I that, at the same time, inhibits fibrinolysis (being active both on plasmin and uPA) and coagulation (being active on thrombin) [24]. Owing to the potential role of PPS in cancer cell invasiveness and metastasis the mechanisms of its regulation have attracted many researchers because of its potential role in cancer therapy. Among natural occurring inhibitors of proteases, flavonoids are a class of chemicals derived from the 2-phenyl-benzo-gamma-pyrane, which consists in two benzene rings joined by a third pyranic ring. Both their variety in nature and their biological activity depend upon Biochimica et Biophysica Acta 1784 (2008) 995–1001 ⁎ Corresponding author. Postal address: via Gentile III da Varano, 62032 Camerino (MC), Italy. Tel./fax: +39 737403247. E-mail address: matteo.mozzicafreddo@unicam.it (M. Mozzicafreddo). 1570-9639/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2008.03.016 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap