Cytotoxic, Antitumour and Antimetastatic Activity of Two New Polyacetylenes Isolated from Vernonia scorpioides (Lam.) Pers Juliana B. Klein 1 , Alexandre E. Nowill 2 , Gilberto C. Franchi Jr 2 , Maique W. Biavatti 3 , Nara L.M. Quint~ ao 1 and Rilton A. de Freitas 4 1 P os-graduac ß~ ao em Ci^ encias Farmac^ euticas, Universidade do Vale do Itajaí, Itajaí, SC, Brazil, 2 Onco-Hematological Child Research Center, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil, 3 Department of Pharmaceutical Sciences, Federal University of Santa Catarina, Florianopolis, SC, Brazil and 4 Department of Chemistry, Federal University of Paran a, Curitiba, PR, Brazil (Received 13 May 2013; Accepted 6 June 2013) Abstract: Vernonia scorpioides (Lam.) Pers., popularly known as Enxuga, Erva-de-S~ ao Sim~ ao and Pirac a, has been used in folk medicine for its anti-inflammatory, wound healing and antimicrobial properties. Two polyacetylenes, 5-octa-2,4,6-triynyl-furan-2 (5H)-one (1) and 8′-hydroxy 3-4 dihydrovernoniyne (2), were isolated from the dichloromethane extract fraction of V. scorpio- ides. In this study, polyacetylene 1 demonstrated a more potent cytotoxic activity than 2 in the tumour cell lines examined, and cytotoxicity was found to be comparable to a commercial drug (p > 0.05) in melanoma cells. No significant cytotoxic effect was observed in normal cell lines. Furthermore, polyacetylene 1 induced an in vitro increase in caspase-3 activity in B16F10 cells. When polyacetylene 1 was administered intraperitoneally (i.p.) in mice, a reduction in solid tumour volume and metastasis was observed in mice injected with B16F10 cells. An increase in locomotor activity was also observed in mice with solid tumours, and an inhibition of mechanical hypersensitivity was observed in a mouse model of metastasis. Notably, no significant morpho- logical change was observed in several organs harvested from the treated mice. In conclusion, in vitro and in vivo anticancer activity of polyacetylene 1 was consistently observed and involved the induction of apoptosis by the activation of caspase-3. The anticancer activity demonstrated by polyacetylene 1, together with the absence of preliminary toxicological effects, represents a new and interesting option for the management of neoplastic disease. Vernonia scorpioides (Lam.) Pers. is a medicinal plant used in Brazil as an anti-inflammatory and antitumour agent, and it is also used to treat some skin conditions. This plant is com- monly found in Brazil, usually grows in poor deforested soils and is popularly known as Enxuga or Pirac a [1]. Although a few studies have examined its biological activity, new polyacetylenes and sesquiterpene lactones have recently been isolated and identified from V. scorpioides that exhibit cytotoxicity [2,3] and anti-inflammatory [4] activity. Polyacetylenes form a distinct group of chemically reactive natural products, and more than 1400 different types and related compounds have been isolated from higher plants [5]. Several studies have shown that polyacetylenes are a class of natural products with diverse biological activities, including anti-HIV (by the inhibition of sodium and potassium ATPase pump activity), immunosuppressant and antitumour activities [6,7]. Several plants contain active polyacetylene compounds with high cytotoxic activity [8–12]. For example, Panax ginseng L. contains several cytotoxic polyacetylenes in the lipophilic portion (petroleum ether), including falcarinol, panaxydol and panaxytriol, that have been shown to be 20 times more cyto- toxic in human gastric adenocarcinoma (MK-1) cells than in human fibroblast cells (MRC-5), suggesting that these com- pounds may be useful for the treatment of cancer [5,13]. Buskuhl et al. [2] have isolated a new polyacetylene from the dichloromethane (DCM) fraction of hydroalcoholic extract obtained from the flowers and leaves of Vernonia scorpioides, have investigated its potential cytotoxic effects in adenocarci- noma (HELA) and melanoma cells (B16F10) and have reported some interesting preliminary results. These promising data prompted us to study the cytotoxic effects of two Verno- nia polyacetylenes and to assess their apoptotic and antineo- plastic potential in several cell lines. In this manuscript, we evaluate the cytotoxic effects of polyacetylenes 1 and 2 isolated from the DCM and ethyl ace- tate (EtOAc) fractions of V. scorpioides in different solid tumour, leukaemia and lymphoma cell lines. Additionally, the cytotoxic effects of the most active polyacetylene (1) were evaluated for activity towards solid tumours and metastatic melanoma in C57BL/6 mice. Materials and Methods Chemicals. Roswell Park Memorial Institute medium 1640 (RPMI), Minimum Essential Medium (MEM), phosphate-buffered saline (PBS), foetal bovine serum (FBS), antibiotics (penicillin, streptomycin and neomycin), trypsin-EDTA, non-essential amino acids, sodium pyruvate and L-glutamine were purchased from Gibco (S~ ao Paulo, Brazil). The Caspase-3 Colorimetric Activity Assay Kit was purchased from Chemicon â International, a Serologicals Company (Temecula, USA). Acridine orange (AO), ethidium bromide (EB), MTT Author for correspondence: Nara Lins Meira Quint ~ ao, Programa de Mestrado em Ci^ encias Farmac^ euticas, Universidade do Vale do Itaja  õ, Rua Uruguai, n o 458, Bloco 27, CCS, sala 314, CEP 88302-202, Itaja  õ, SC, Brazil (fax (+55) 47 3341 7652, e-mails: nara.quintao@ univali.br or narafarmaco@yahoo.com.br). © 2013 Nordic Pharmacological Society. Published by John Wiley & Sons Ltd Basic & Clinical Pharmacology & Toxicology, 2013, 113, 307–315 Doi: 10.1111/bcpt.12098