The anti-cancer agent nemorosone is a new potent protonophoric mitochondrial uncoupler Gilberto L. Pardo-Andreu a,c, ⁎, Yanier Nuñez-Figueredo b , Valeria G. Tudella c , Osmany Cuesta-Rubio d , Fernando P. Rodrigues c , Cezar R. Pestana c , Sérgio A. Uyemura e , Andréia M. Leopoldino e , Luciane C. Alberici c , Carlos Curti c a Centro de Estudio para las Investigaciones y Evaluaciones Biológicas, Instituto de Farmacia y Alimentos, Universidad de La Habana, ave. 23 # 21425 e/ 214 and 222, La Coronela, La Lisa, CP 13600, Ciudad Habana, Cuba b Centro para las Investigaciones y Desarrollo de Medicamentos, Ave 26, No. 1605 Boyeros y Puentes Grandes, CP 10600, Ciudad Habana, Cuba c Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café s/n, 14040-903 Ribeirão Preto, SP, Brazil d Departamento de Química, Instituto de Farmacia y Alimentos, Universidad de La Habana, ave. 23 # 21425 e/ 214 and 222, La Coronela, La Lisa, CP 13600, Ciudad Habana, Cuba e Departamento de Analises Clinicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café s/n, 14040-903 Ribeirão Preto, SP, Brazil abstract article info Article history: Received 22 July 2010 received in revised form 1 October 2010 accepted 19 October 2010 Available online 31 October 2010 Keywords: Nemorosone Mitochondria Uncoupler Protonophore HepG2 cell death Cancer Nemorosone, a natural-occurring polycyclic polyprenylated acylphloroglucinol, has received increasing attention due to its strong in vitro anti-cancer action. Here, we have demonstrated the toxic effect of nemorosone (1–25 μM) on HepG2 cells by means of the MTT assay, as well as early mitochondrial membrane potential dissipation and ATP depletion in this cancer cell line. In mitochondria isolated from rat liver, nemorosone (50–500 nM) displayed a protonophoric uncoupling activity, showing potency comparable to the classic protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Nemorosone enhanced the succinate-supported state 4 respiration rate, dissipated mitochondrial membrane potential, released Ca 2+ from Ca 2+ -loaded mitochondria, decreased Ca 2+ uptake and depleted ATP. The protonophoric property of nemorosone was attested by the induction of mitochondrial swelling in hyposmotic K + -acetate medium in the presence of valinomycin. In addition, uncoupling concentrations of nemorosone in the presence of Ca 2+ plus ruthenium red induced the mitochondrial permeability transition process. Therefore, nemorosone is a new potent protonophoric mitochondrial uncoupler and this property is potentially involved in its toxicity on cancer cells. © 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved. 1. Introduction Nemorosone (1-benzoyl-4-hidroxy-8,8-dimethyl-3,5,7-tris (3-methyl-2-butenyl)-bicyclo-[3.3.1]-non-3-ene-2,9-dione; a tautomer) (Fig. 1) is a naturally occurring polycyclic polypreny- lated acylphloroglucinol, the major component of Clusia species resin (~50%) responsible for its antimicrobial activity (Ciochina and Grossman, 2006; de Oliveira et al., 1999). The brown Cuban propolis, whose main component is nemorosone, exerts a significant anti-proliferative action on MCF-7 human breast cancer cells (Popolo et al., 2009). Isolated nemorosone, in turn, elicits cytotoxic action in vitro against a panel of tumor cell lines including breast, colon, brain, ovary, liver, blood and lung carcinoma (Cuesta-Rubio et al., 2002; Diaz-Carballo et al., 2003, 2008a,b). Specifically, nemorosone's toxicity to neuroblastoma and leukemia parental cell lines has been ascribed to its ability to target the Akt/PKB signal transducer (Díaz-Carballo et al., 2008a,b). Mitochondrial events such as membrane potential dissipation, reactive oxygen species generation and release of caspase-activating proteins are implicated in cell necrosis and/or apoptosis (Kroemer and Reed, 2000). Therefore, compounds lipophilic enough to reach the mitochondrial membrane could induce cell death by means of mitochondrial mechanisms. Nemorosone appears to meet this criterion because its Log P value of 8.3 allows it with the ability to cross cellular and mitochondrial membranes. In this context, we have addressed in the present work the uncoupling property of nemor- osone and its toxicity to cancer cells. Studies were performed in both hepatic carcinoma (HepG2) cells and mitochondria isolated from rat liver; nemorosone's uncoupling potency was compared with that of the classic protonophore, carbonyl cyanide m-chlorophenyl hydra- zone (CCCP). The results demonstrate that, in addition to its toxicity Mitochondrion 11 (2011) 255–263 ⁎ Corresponding author. Centro de Estudio para las Investigaciones y Evaluaciones Biológicas, Instituto de Farmacia y Alimentos, Universidad de La Habana, ave. 23 # 21425 e/ 214 and 222, La Coronela, La Lisa, CP 13600, Ciudad Habana, Cuba. Tel.: +53 7 2719531; fax: +53 7 2736811. E-mail address: gilbertopardo@infomed.sld.cu (G.L. Pardo-Andreu). 1567-7249/$ – see front matter © 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2010.10.008 Contents lists available at ScienceDirect Mitochondrion journal homepage: www.elsevier.com/locate/mito