Mechanism of Action of Cytotoxic Cyclotides: Cycloviolacin O2 Disrupts Lipid Membranes Erika Svangård, † Robert Burman, † Sunithi Gunasekera, † Henrik Lo ¨vborg, ‡ Joachim Gullbo, ‡ and Ulf Go ¨ransson* ,† DiVision of Pharmacognosy, Department of Medicinal Chemistry, Biomedical Centre, Uppsala UniVersity, Uppsala, Sweden, and DiVision of Clinical Pharmacology, Department of Medical Sciences, UniVersity Hospital, Uppsala UniVersity, Uppsala, Sweden ReceiVed January 5, 2007 In recent years, the cyclotides have emerged as the largest family of naturally cyclized proteins. Cyclotides display potent cytotoxic activity that varies with the structure of the proteins, and combined with their unique structure, they represent novel cytotoxic agents. However, their mechanism of action is yet unknown. In this work we show that disruption of cell membranes plays a crucial role in the cytotoxic effect of the cyclotide cycloviolacin O2 (1), which has been isolated from Viola odorata. Cell viability and morphology studies on the human lymphoma cell line U-937 GTB showed that cells exposed to 1 displayed disintegrated cell membranes within 5 min. Functional studies on calcein- loaded HeLa cells and on liposomes showed rapid concentration-dependent release of their respective internal contents. The present results show that cyclotides have specific membrane-disrupting activity. Cyclotides are plant proteins characterized by an extraordinary structure: their N- and C-termini are joined by an ordinary peptide bond to form a continuously circular amide backbone. In addition, six of their approximately 30 amino acid residues are cysteines that form three disulfide bonds arranged in a cystine knot motif, as shown in Figure 1. These structural features are strictly conserved within this protein family, and they define the so-called cyclic cystine knot (CCK) motif, 1-3 which gives the cyclotides increased thermal, enzymatic, and chemical stability compared to linear peptides. 4,5 Today, more than 80 cyclotides have been isolated from the Violaceae and Rubiaceae plant families, and they form the largest known family of genetically encoded cyclic proteins. 6-10 They display a range of biological activities, including uterotonic, 11 hemolytic, 12,13 antimicrobial, 14 anti-HIV, 15 antifouling, 16 and in- secticidal 17 activities. Together with their remarkable stability this makes the CCK motif of great interest as a scaffold in protein engineering for potential pharmaceutical and agricultural applica- tions. 18,19 Despite the number of reported biological activities and the great interest in the cyclotides, the mechanism of action has not yet been established for any of these effects. Even if not all of the effects have been shown for a single cyclotide, it is conceivable that there might be a common mechanism of action for at least some of the effects. In fact, it has been suggested that a mechanism involving membrane interactions may explain their antimicrobial and hemolyt- ic activities based on their structural resemblance (with the exception of their circular backbone) to other antimicrobial peptides that have such a mechanism of action. 7,20-22 In our previous work we have shown that cyclotides represent a novel class of cytotoxic agents that display strong activity in a dose- dependent manner. 23,24 The cytotoxic activity was maintained throughout a cell line panel consisting of 10 human tumor cell lines, including solid tumor cells, and toward primary cultures of tumor cells from patients. In addition, the activity profile for cyclotides tested in the panel, which is designed to represent defined types of drug resistance, differs significantly from those of antitumor drugs in clinical use. This suggests a new mode of action. Sparked by these results and the fact that the tested cyclotides also display a definite structure-activity relationship, 23,24 we recently reported a detailed study of the most potent of the tested cyclotides, cyclo- violacin O2 (1), which has been isolated from Viola odorata L. (Violaceae). 25 In this study we showed that a glutamic acid residue, which is conserved in the cyclotide family, plays a crucial role for the cytotoxic activity and that at least one cationic residue is required to maintain activity at low cyclotide concentrations. In the present work we have used the same peptide, 1, to characterize the mode of action of the cytotoxic effect of this cyclotide toward human cancer cells. First, we have characterized the kinetics of the effect using the same fluorescent microculture cytotoxicity assay (FMCA) as was used for the cell line panel, 23,24 and by microscopy studies. Second, we describe the morphology of treated cells. Then, we have assessed specifically the membrane effects of the cyclotide using cells loaded with calcein and a liposome-based assay. Results and Discussion To characterize the mechanism of action of 1, we first established the kinetics of the cytotoxic effects using the FMCA and the U-937 GTB human lymphoma cell line. Cell survival was measured at 4, 8, 24, and 72 h. As shown in Figure 2, the concentration-response curves for the different time points essentially overlap, including the very steep shape of the curve between no and full effect. Following that overlap, the IC 50 values did not show any significant difference at the different time points. Thus, the results showed that 1 achieves its full effect within 4 h, and the similar shape of * Author to whom correspondence should be addressed. Tel: +46 18 471 50 31. Fax: +46 18 50 91 01. E-mail: ulf.goransson@fkog.uu.se. † Biomedical Centre. ‡ University Hospital. Figure 1. Sequence and schematic structure of cycloviolacin O2 (1). The solid black line represents the peptide backbone, and the sequence is shown by the one-letter codes for each amino acid. Note the unique features of the CCK motif: a cyclic backbone and three stabilizing disulfide bonds (in gray). These disulfides are arranged in a cystine knot; that is, two of the disulfides form a ring structure together with the backbone connecting the four cysteines, while the third disulfide is threaded through the ring. The arrow shows the direction of the genetic translation of the sequence. 643 J. Nat. Prod. 2007, 70, 643-647 10.1021/np070007v CCC: $37.00 © 2007 American Chemical Society and American Society of Pharmacognosy Published on Web 03/23/2007