Please cite this article in press as: Sciani JM, et al. Cytotoxic and antiproliferative effects of crude amphibian skin secretions on breast tumor cells. Biomed Prev Nutr (2012), http://dx.doi.org/10.1016/j.bionut.2012.11.001 ARTICLE IN PRESS G Model BIONUT-130; No. of Pages 9 Biomedicine & Preventive Nutrition xxx (2012) xxx–xxx Available online at www.sciencedirect.com Original article Cytotoxic and antiproliferative effects of crude amphibian skin secretions on breast tumor cells Juliana Mozer Sciani a,∗ , Paulo Luiz de-Sá-Júnior b , Adilson Kleber Ferreira a , Alexandre Pereira b , Marta M. Antoniazzi c , Carlos Jared c , Daniel Carvalho Pimenta a a Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, SP, Brazil b Laboratório de Genética, Instituto Butantan, São Paulo, SP, Brazil c Laboratório de Biologia Celular, Instituto Butantan, São Paulo, SP, Brazil a r t i c l e i n f o Article history: Received 25 October 2012 Accepted 6 November 2012 Keywords: Amphibia Skin secretion Breast cancer a b s t r a c t Amphibian skin secretions are rich sources of biologically active compounds, including antimicrobial peptides, alkaloids and steroids. Bufadienolidic steroids, for instance, may inhibit tumoral cells migration and proliferation. Besides, alkaloids possess known antitumoral activity, being both capable of being employed in cancer therapy. In this work, we evaluated the effects of crude skin secretion solutions of eight Brazilian amphibians, selected for their known high contents of steroids, alkaloids, peptides and proteins, over two breast cell lines. The extract of amphibians were tested over MCF-7 and MDA-MB-231 cells by MTT assay, Hoechst/PI staining and acridine orange, in order to verify cell death and apoptosis. Treated cells were also analyzed by flow cytometer. The skin secretions were also analyzed by HPLC, mass spectrometry and poliacrilamide gel electrophoresis. Skin secretion solutions of Rhinella crucifer, R. major, R. schneideri, R. margaritifer and Phyllomedusa hypocondrialis were able to induce cytotoxic effects and provoke morphological alterations in both cell lines, including apoptotic bodies and cell death, as assessed by acridine orange and Hoechst/PI staining; nevertheless, these skin secretion solutions were poorly active over human erythrocytes. Flow cytometry assays showed that Rhaebo guttatus, R. crucifer, R. margaritifer, R. major and P. hypocondrialis skin secretions were able to inhibit cell growth and cause induction of cell cycle in the G2/M phase. Moreover, P. hypocondrialis skin secretion solution increased the number of tumor cells in the apoptotic sub-G1 peak. Based on these data, these amphibian skin secretions present themselves as a potential source for new molecules active on cancer cells. © 2012 Elsevier Masson SAS. All rights reserved. 1. Introduction Frogs and their skin secretion properties may be found through- out several cultures and folklores around the world. This traditional knowledge has evolved into the concept that the amphibian skin may be a treasure-trove of biologically active compounds. A sur- vey of noxious or toxic secretions and the presence of biogenic amines, peptides, bufadienolides, tetrodotoxins and lipophilic alka- loids were described years ago [1,2]. Since that time, additional investigations have added hundreds of such compounds to the list of active substances isolated from amphibian skin, which play sev- eral roles, either in the maintenance of the homeostasis of the skin or in the chemical defense mechanisms of the animal. Not surpris- ingly, a large number of molecules in the amphibian skin secretion are antibiotics [2,3]. ∗ Corresponding author. Avenida Vital Brazil, 1500, São Paulo, SP, Brazil. CEP: 05503-900. Tel.: +55 11 3726 7222x2101; fax: +55 11 3726 7222x2018. E-mail address: jmsciani@butantan.gov.br (J.M. Sciani). The pharmaceutical industry already takes benefits from sev- eral therapeutically relevant compounds present in the amphibian skin. Bufadienolidic steroids, found in toad species of the genus Rhinella (formerly Bufo in the New World), were efficient, in vitro, against cancer cells proliferation [1,4,5]. Further investigation of this bufadienolides led to the isolation of marinobufagin, bufalin, telocinobufagin, hellebrigenin and cinobufagin [6–9], which have shown to be efficient against cancer cells in animal models and in migration of colon cancer cells (colon 26-L5 cell) [10], human leukemia cells (K562, U937, ML1 and HL-60) [11] and breast tumor cells (MCF-7) [4,12]. Moreover, some of these compounds have been demonstrated to possess high antitumoral activities, such as the impairment of angiogenesis, the reversal of multi-drugs resistance, the regulation of the immune response as well as the triggering of apoptosis [12,13]. On the other hand, frog dermatous glands synthesize and excrete a variety of mammalian-like hormones, neuropeptides and kinins, as well as opioid peptides and cytolytic antimicrobial pep- tides, all considered to be involved in the defense against mammal predation and/or microbial invasion/colonization of the naked frog 2210-5239/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.bionut.2012.11.001