View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Archivio istituzionale della ricerca - Università di Brescia European Journal of Pharmacology 691 (2012) 125–133 Contents lists available at SciVerse ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar Cardiovascular pharmacology Anti-angiogenic activity of the flavonoid precursor 4-hydroxychalcone Lenka Varinska a, Michiel van Wijhe b, Mirella Belleri c, Stefania Mitola c, Pal Perjesi d, Marco Presta c, Pieter Koolwijk b, Lenka Ivanova a, Jan Mojzis a,n a Department of Pharmacology, Faculty of Medicine, Pavol Jozef Safarik University, Trieda SNP 1, 04011 Kosice, Slovak Republic b Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center Amsterdam, Amsterdam, The Netherlands c Department of Biomedical Sciences and Biotechnology, Unit of General Pathology and Immunology, School of Medicine, University of Brescia, Brescia, Italy d Institute of Pharmaceutical Chemistry, Faculty of Medicine, University of Pecs, Pecs, Hungary article info abstract Article history: Angiogenesis, the growth of new blood vessels, is necessary for cancerous tumors to keep growing and Received 20 December 2011 spreading. Suppression of abnormal angiogenesis may provide therapeutic strategies for the treatment Received in revised form of angiogenesis-dependent disorders. In the present study, we describe the in vitro and in vivo anti- 23 April 2012 angiogenic activities of the flavonoid precursor 4-hydroxychalcone (Q797). This chalcone (22 mg/ml) Accepted 10 June 2012 suppressed several steps of angiogenesis, including endothelial cell proliferation, migration and tube Available online 18 June 2012 formation without showing any signs of cytotoxicity. Moreover, we found a selective effect on activated Keywords: endothelial cells, in particular with resting endothelial cells and the human epithelial tumor cell lines Angiogenesis (HeLa, MCF-7, A549). In addition, Q797 was able to modulate both vascular endothelial growth factor Chalcones (VEGF)- and basic fibroblast growth factor (FGF)- induced phosphorylation of extracellular signal- Endothelial cells regulated kinase (ERK)-1/-2 and Akt kinase. It did not influence the nuclear translocation of p65 subunit In vitro CAM assay of the nuclear factor-kB(NF-kB) when human endothelial cells were stimulated with tumor necrosis factor (TNF)-a. Taken together this indicates that the Q797-mediated inhibition of in vitro angiogenic features of endothelial cells is most likely caused by suppression of growth factor pathways. The potent inhibitory effect of Q797 on bFGF-driven neovascularization was also demonstrated in vivo using the chick chorioallantoic membrane (CAM) assay. In summary, this chalcone could serve as a new leading structure in the discovery of new potent synthetic angiogenesis inhibitors. & 2012 Elsevier B.V. All rights reserved. 1. Introduction activities including those that might be able to influence processes that are dysregulated during cancer development. They selectively Angiogenesis is the outgrowth of new blood vessels from induce apoptosis in various human carcinoma cell lines and they pre-existing vasculature. It is a necessary process in embryogen- are thought to be involved in the suppression of angiogenesis esis and wound healing but also plays a major role in several (Lamoral-Theys et al., 2010). pathologic processes such as tumor vascularisation, diabetic Many fruits and vegetables are also rich dietary sources of retinopathy, psoriasis and rheumatoid arthritis (Folkman 1995, flavonoid precursors, in particular the chalcones. These com- 2007). Therefore, newly designed anti-angiogenic agents have pounds could make a great contribution to the total daily intake potential for the treatment of a broad spectrum of diseases, of natural polyphenolics (Tomas-Barberan and Clifford, 2010), including cancer (Ribatti, 2009). since they appear to possess potentially beneficial effects against An increasing interest is devoted to naturally occurring cancer various diseases (de Campos-Buzzi et al., 2006; Sharma et al., chemo-preventive agents, including flavonoids, which are rich in 2009). Indeed, they are associated with several biologically active fruits, soybeans, vegetables, herbs, roots and leaves (Hollman and properties, including anti-inflammatory (Hsieh et al., 2000; Won Katan, 1999; Larsen et al., 2010; Yang et al., 2011). These poly- et al., 2005), anti-oxidative (Nerya et al., 2004), anti-fungal phenolic compounds display a remarkable spectrum of biological (Sivakumar et al., 2009a), anti-bacterial (Sivakumar et al., 2009b), chemo-preventive and anti-cancer activities (Ducki, 2007; Chua et al., 2010; Katsori and Hadjipavlou-Litina, 2009; Liu and Go, n Corresponding author. Tel./fax: þ421 55 6428524. 2006; Modzelewska et al., 2006). Although there are an increasing E-mail addresses: [email protected] (L. Varinska), number of articles relating the anti-cancer activities of chalcones, [email protected] (M. van Wijhe), [email protected] (M. Belleri), limited information is available about a possible anti-angiogenic [email protected] (S. Mitola), [email protected] (P. Perjesi), [email protected] (M. Presta), [email protected] (P. Koolwijk), activity of this class of compounds (Kang et al., 2010; Kim et al., [email protected] (L. Ivanova), [email protected] (J. Mojzis). 2010; Mojzis et al., 2008). 0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.06.017 126 L. Varinska et al. / European Journal of Pharmacology 691 (2012) 125–133 The structural features of chalcones presence of a reactive or methanol (Q764 and Q766). The purity of the compounds was enone moiety and its relative flexibility compared to other related checked by TLC using Merck silicagel 60 F254 alumina sheets. The natural products predispose the template to interactions with structure and stereochemistry of the compounds were character- diverse receptors and enzymes (Go et al., 2005), as well as their ized by elemental analysis, IR and NMR methods. The synthesized biological properties prompted us to investigate whether chal- Q797 and 4-hydroxyindanone had melting points in accord with cones and newly synthesized cyclic chalcone analogues modulate the literature values (Dimmock et al., 1998). angiogenesis in both in vitro and in vivo models. We present for the first time the evidence that one of the chalcones; 4-hydro- 2.2. Cell culture xychalcone (Q797) inhibits different steps of the angiogenesis process in vitro by affecting both vascular endothelial growth Human foreskin microvascular endothelial cells (hMVEC) and factor (VEGF) and basic fibroblast growth factor (bFGF) intracel- human umbilical vein endothelial cells (HUVEC) were isolated, lular signaling. In line with this in vitro data, Q797 also caused a cultured, and characterized as previously described (Defilippi significant anti-angiogenic activity in vivo. Q797 may represent et al., 1991; van Hinsbergh et al., 1987). Cells were cultured on the basis for the design of novel anti-angiogenic chalcone gelatine-coated dishes in cM199 (¼M199 medium supplemented derivatives. with 10% heat-inactivated human serum, 10% heat-inactivated new born calf serum, 3.75 mg/ml crude endothelial cell growth factor (ECGF), 5 U/ml heparin, 100 IU/ml penicillin, and 100 mg/ml 2. Materials and methods streptomycin) at 37 1C under 5% CO2/95% air atmosphere. Twenty-four hours prior to experiments endothelial cell cul- 2.1. Chemicals tures were refreshed with medium without crude endothelial cell growth factor and heparin. Medium 199 with 20 mM HEPES (M199), penicillin, streptomycin The human cervical adenocarcinoma cell line HeLa was cul- and newborn calf serum (NBCS) were obtained from Lonza (Verviers, tured in RPMI-1640 medium (PAA Laboratories, Pasching, Austria) Belgium). Tissue plastics were from Corning Life Science (Lowell, MA supplemented with 10% fetal bovine serum and antibiotics USA) and L-glutamine from Sigma–Aldrich (St. Louis, MO USA). (penicillin 100 U/ml and streptomycin 100 mg/ml; Invitrogen, Human serum (HS) was obtained from PAA (Pasching, Austria). Basic Carlsbad, CA USA). Human breast adenocarcinoma cell line MCF- fibroblast growth factor (bFGF) was purchased from Sigma–Aldrich 7 and human lung adenocarcinoma epithelial cell line A549 were (St. Louis, MO USA), vascular endothelial growth factor (VEGF-A) from cultured in D-MEM medium (Invitrogen, Carlsbad, CA USA) Biosource (Camarillo, CA USA) and tumor necrosis growth factor supplemented with 10% fetal bovine serum and antibiotics (TNF)-a was obtained from R&D Systems (Minneapolis, MN USA). (penicillin 100 U/ml and streptomycin 100 mg/ml). Matrigel basement membrane matrix was obtained from Becton Dickinson Labware (Bedford, MA USA). The chalcones: 4-hydroxy- 2.3. Formation of capillary like tubular structure in a 3-dimensional chalcone (Q797), 4-methylchalcone (Q171), 4-methoxychalcone matrix (Q126), 4-dimethylaminochalcone (Q192) and the cyclic chalcone analogues: (E)-2-(X-benzylidene)-1-indanones ((E)-2-(4-dimethyla- Human fibrin matrices were prepared in the wells of 96-wells minobenzylidene)-1-indanone (Q560), (E)-2-(4-methoxybenzyli- plates by addition of a 100 ml solution of a mixture of 0.1 U/ml dene)-1-indanone (Q397), (E)-2-(4-hydroxybenzylidene)-1-indan thrombin and 2 mg/ml plasma-derived, plasminogen-depleted one (Q764)), (E)-2-(2,4-dimethoxybenzylidene)-1-tetralone