In vitro toxicity of camalexin derivatives in human cancer and non-cancer cells Martina Pilatova a,1 , Lenka Ivanova a,1 , Peter Kutschy b , Lenka Varinska a , Lydia Saxunova a , Maria Repovska b , Marek Sarissky a , Robert Seliga a , Ladislav Mirossay a , Jan Mojzis a,⇑ a Department of Pharmacology, Faculty of Medicine, Pavol Jozef Safarik University, Kosice, Slovak Republic b Department of Organic Chemistry, Institute of Chemical Sciences, Faculty of Science, Pavol Jozef Safarik University, Kosice, Slovak Republic article info Article history: Received 13 April 2012 Accepted 2 January 2013 Available online 20 January 2013 Dedicated to Assoc. Prof. Peter Kutschy Keywords: Camalexin Benzocamalexin Indole phytoalexins Cytotoxicity abstract The aim of the study was to investigate the cytotoxic activity of camalexin and its five synthetic deriva- tives in cancer and non-cancer cells. In cancer cells the benzocamalexin (BC) displayed the most potent activity with an IC 50 value of 23.3– 30.1 lmol/L. On the other hand, minimal toxicity (IC 50 > 100.0 lmol/L) in non-cancer cells was observed. Based on these results, BC was selected for further studies. Flow cytometric analysis revealed a BC-induced arrest of the cell cycle in the G2 phase associated with downregulation of a-tubulin, a1-tubulin, b5-tubulin expression. These findings suggest that the inhibi- tory effect of BC is mediated via inhibition of microtubule formation. Moreover, BC downregulated the expression of microtubule-related protein indicating the effect of this compound on microtubule assem- bly. After treatment with BC increase of the sub-G 1 DNA content fraction was noted which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by DNA fragmentation assay. More- over, quantitative real-time PCR showed that BC downregulated the expression of antiapoptotic genes Bcl-2 and Bcl-xL and upregulated the expression of proapoptotic Bax. Taken together, our study suggests that the blockade of cell cycle progression and initiation of apopto- sis may play an important role in the antiproliferative activity of BC in human cancer cells. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction In response to pathogen attack many plants elaborate a vast ar- ray of low molecular weight secondary metabolites called phytoal- exins. Phytoalexins include a chemically diverse group of compounds biosynthesized de novo by plants as a defense response to fungal and bacterial pathogens (Pedras et al., 2011). The concept of phytoalexin was introduced by Müller and Borger in 1940, and since then these compounds have been extensively studied not only with respect to their role in defense against pathogens and pests, but also with respect to their health-promoting effects (Bish- ayee, 2009; Chakraborty et al., 2010; Holland and O’Keefe, 2010; Müller and Borger, 1940; Patel et al., 2011; Wu et al., 2011). Indole phytoalexins represent a specific group of phytoalexins synthesized by plants of the family Brassicaceae (syn. Cruciferae). They are indole alkaloids, most of which contain a side chain or an- other heterocycle, containing a nitrogen atom and one or two sul- fur atoms. Until now, 44 cruciferous phytoalexins, i.e. metabolites pro- duced de novo have been reported (Pedras and Yaya, 2010). The chemical diversity of cruciferous phytoalexins suggest substan- tially different biological activities as was confirmed experimen- tally. Principally, they display antimicrobial activity against bacterial and fungal plant pathogens (for rev. see Pedras et al., 2011). However, some indole phytoalexins have also been shown to exhibit significant antiproliferative/anticancer activity (Mehta et al., 1995; Monde et al., 2005; Mezencev et al., 2009; Pilatova et al., 2005). Camalexin (2-(1H-indol-3-yl)thiazole) is an indole phytoalexin present in Arabidopsis and closely related plant species. It was first isolated from Arabidopsis thaliana and Camelina sativa leaves (Browne et al., 1991). Like many of the phytoalexins, camalexin is substituted with sulfur and nitrogen-containing side chain (Gla- wischnig, 2007). Although camalexin biosynthesis is induced by a great variety of plant pathogens, it is suggested that camalexin possesses primary antifungal action (Glawischnig, 2007). In addi- tion to its antifungal action, camalexin also exerts antiproliferative effects in human cancer cell lines. As documented by Moody and co-workers (1997) camalexin significantly inhibits growth of SKBr3 breast cancer cell line with IC 50 = 2.7 lmol. Recently, a proa- poptotic effect of camalexin in Jurkat leukemia cells was observed 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.01.006 ⇑ Corresponding author. Address: Department of Pharmacology, Faculty of Medicine, Pavol Jozef Safarik University, Trieda SNP 1, 04011 Kosice, Slovak Republic. Tel./fax: +421 55 6428524. E-mail address: jan.mojzis@upjs.sk (J. Mojzis). 1 These authors contributed equally to this work. Toxicology in Vitro 27 (2013) 939–944 Contents lists available at SciVerse ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit