Functional Inhibition of Aquaporin-3 With a Gold-Based Compound Induces Blockage of Cell Proliferation ANA SERNA, 1 ANA GALÁN-COBO, 1 CLAUDIA RODRIGUES, 2 ISMAEL SÁNCHEZ-GOMAR, 1 JUAN JOSÉ TOLEDO-ARAL, 1 TERESA F. MOURA, 2,3 ANGELA CASINI, 4 GRAC ¸A SOVERAL, 2 ** AND MIRIAM ECHEVARRÍA 1,5 * 1 Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla (Departamento de Fisiología M edica y Biofísica), Seville, Spain 2 Instituto de Investigac ¸~ ao do Medicamento (iMed.ULisboa), Faculdade de Farm acia, Universidade de Lisboa, Lisboa, Portugal 3 Departamento de Química, FCT-UNL, Caparica, Portugal 4 Pharmacokinetics, Toxicology and Targeting, Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands 5 Centro de Investigaci  on Biom edica en Red sobre Enfermedades Respiratorias (CIBERES), Madrid, Spain AQP3 has been correlated with higher transport of glycerol, increment of ATP content, and larger proliferation capacity. Recently, we described the gold(III) complex Auphen as a very selective and potent inhibitor of AQP3’s glycerol permeability (P gly ). Here we evaluated Auphen effect on the proliferation of various mammalian cell lines differing in AQP3 expression level: no expression (PC12), moderate (NIH/3T3) or high (A431) endogenous expression, cells stably expressing AQP3 (PC12-AQP3), and human HEK293T cells transiently transfected (HEK-AQP3) for AQP3 expression. Proliferation was evaluated in the absence or presence of Auphen (5 mM) by counting number of viable cells and analyzing 5-bromo-2 0 -deoxyuridine (BrdU) incorporation. Auphen reduced 50% the proliferation in A431 and PC12-AQP3, 15% in HEK-AQP3 and had no effect in PC12-wt and NIH/3T3. Strong arrest in the S-G2/M phases of the cell cycle, supported by analysis of cyclins (A, B1, D1, E) levels, was observed in AQP3-expressing cells treated with Auphen. Flow-cytometry of propidium iodide incorporation and measurements of mitochondrial dehydrogenases activity confirmed absence of cytotoxic effect of the drug. Functional studies evidenced 50% inhibition of A431 P gly by Auphen, showing that the compound’s antiproliferative effect correlates with its ability to inhibit AQP3 P gly . Role of Cys-40 on AQP3 permeability blockage by Auphen was confirmed by analyzing the mutated protein (AQP3-Ser-40). Accordingly, cells transfected with mutated AQP3 gained resistance to the antiproliferative effect of Auphen. These results highlight an Auphen inhibitory effect on proliferation of cells expressing AQP3 and suggest a targeted therapeutic effect on carcinomas with large AQP3 expression. J. Cell. Physiol. 229: 1787–1801, 2014. © 2014 Wiley Periodicals, Inc. Aquaporins (AQPs) belong to a highly conserved group of membrane proteins involved in the transport of water and small solutes and with a variety of important physiological roles (Carbrey and Agre, 2009). The 13 human AQP isoforms (AQP0–12) are differentially expressed in many types of cells and tissues in the body and can be divided into two major groups: those strictly selective for water (called orthodox AQPs), and those that are also permeable to other small solutes including glycerol (called “aquaglyceroporins”). The latters include AQP3, AQP7, AQP9, and AQP10 isoforms (Takata et al., 2004). Phenotype analysis of AQP-null mice, as well as pathophysiological studies, suggested AQPs as drug targets (Verkman, 2009, 2011). Indeed, AQPs have recently been implicated in various diseases such as polycystic kidney disease, cataract, brain edema, gallstone disease, and nephrogenic diabetes insipidus, as well as in the development of obesity and cancer (Verkman, 2005; Verkman et al., 2008; Nico and Ribatti, 2010). Moreover, analysis of AQP involvement in the life-cycle of disease causing organisms suggests additional opportunities for pharmacological intervention in the treat- ment of human diseases (Beitz, 2005). All authors confirm that there are no conflicts of interest. Ana Gal  an-Cobo and Claudia Rodrigues contributed equally to this work. Contract grant sponsor: Instituto de Salud Carlos III; Contract grant number: Exp. PS09/00605. Contract grant sponsor: La Junta de Andalucía. Contract grant sponsor: Consejería de Innovaci  on Ciencia y Empresa; Contract grant number: P08-CTS-03574. Contract grant sponsor: Consejería de Salud; Contract grant number: PI0298-2010. *Correspondence to: Miriam Echevarría, Instituto de Biomedicina de Sevilla (IBIS), Av. Manuel Siurot s/n, Seville 41013, Spain. E-mail: irusta@us.es **Correspondence to: Grac ¸a Soveral, Instituto de Investigac ¸~ ao do Medicamento (iMed.ULisboa), Faculdade de Farm acia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. E-mail: gsoveral@ff.ul.pt Manuscript Received: 30 September 2013 Manuscript Accepted: 24 March 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 27 March 2014. DOI: 10.1002/jcp.24632 ORIGINAL RESEARCH ARTICLE 1787 Journal of Journal of Cellular Physiology Cellular Physiology © 2014 WILEY PERIODICALS, INC.